Abstracts for Short Topic Presentations (STP) Session 3: Next Generation Cancer Therapeutics
Moderator: Vivek Gupta
Presentation 1
Next-Generation Antisense Strategy to Target AKT Genes for Breast Cancer Therapy
Natasha Shah, University of Saint Joseph, [email protected]
Purpose
AKT, or protein kinase B, is a serine-threonine kinase that regulates various biological functions in the body. In breast cancer, two isoforms of AKT—AKT1 and AKT2—are frequently mutated. Overexpression of AKT1 enhances tumor cell proliferation, growth, and the suppression of apoptosis, while overexpression of AKT2 increases tumor migration, invasion, and metastasis. To date, the simultaneous inhibition of AKT1 and AKT2 mRNA translation has not been achieved. We optimized an antisense oligonucleotide (ASO) design to target both AKT1 and AKT2 mRNA at the same time. Our ASO design incorporates chemical modifications such as phosphorothioate (PS) at alternate positions throughout the sequence and 2’-O-methyl (2’-O-Me) modifications at the terminal three positions on both ends, enhancing in vivo activity. Additionally, we have developed peptide nucleic acid (PNA)-based AKT inhibitors for comparative studies. The pH low insertion peptide (pHLIP) facilitates membrane insertion upon detecting the acidity of tumor cell surfaces. It has been utilized to deliver ASOs to tumor cells. Therefore, in this multi-disciplinary project, we are assessing pHLIP-conjugated PS-2’-O-Me AKT inhibitors and pHLIP-conjugated PNA AKT inhibitors to inhibit the translation of AKT mRNA as a clinically translatable strategy for treating breast cancer.
Methods
We utilized cell viability and gene expression studies to compare the efficacy of various AKT inhibitors in the MDA-MB-453 and MCF-7 breast cancer cell lines. In ongoing studies, we are assessing the effectiveness of the pHLIP-AKT inhibitor conjugates to achieve AKT1 and AKT2 knockdown at both mRNA and protein levels in multiple breast cancer cell lines.
Results
We observed decreased expression of AKT1 and AKT2 in the treatment group receiving the PS-2’-O-Me AKT inhibitor. Additionally, we noted a 50% reduction in cellular viability of MDA-MB-453 cells and a 30% reduction in cellular viability of MCF 7 following treatment with the PS-2’-O-Me AKT inhibitor.
Conclusion
Results presented in this study evaluated the efficacy of ASO-based AKT inhibitors in different breast cancer cell lines. We hope the results of our study will help establish a novel platform for RNA-based precision medicine for breast cancer therapy.
Presentation 2
Targeting the Genomic DNA of the Undruggable c-Myc Oncogene for Cancer Therapy
Ruchi Ruchi, University of Connecticut, [email protected]
Purpose
Several antisense (RNA targeting platforms) therapeutics have evolved through numerous chemical modifications. With these advancements, attention is now directed towards assessing these molecules to treat the fundamental causes of diseases by directly targeting genomic DNA. Malik et al. illustrated the application of γtcPNA-NLS to specifically target the homopurine region of the c-Myc oncogene within the nucleus. In the present study, we compare the therapeutic efficacy of γPNA1-NLS with the G-clamp modified γGcPNA2-NLS, to enhance clinical translation. We employed both PNA scaffolds to inhibit c-Myc transcription and facilitate gene silencing. These molecules were systematically evaluated for their binding efficiency to the target site. As a proof of concept, we performed cell culture-based studies to gauge their effectiveness. Our comparative analysis indicates the potential of targeting genomic DNA non-restrictedly, thereby presenting new opportunities for tackling previously undruggable oncogenes and advancing cancer therapy strategies.
Methods
We evaluated the binding efficiency of two peptide nucleic acids (PNAs) utilizing a dose-dependent gel shift assay, a cell culture-based amplicon assay, and Sanger sequencing. The expression of c-Myc was assessed in U2932 and A549 cell lines through quantitative polymerase chain reaction (qPCR) and Western blot analysis. Cytotoxic effects were examined via cell viability and clonogenic assays. To assess the potential for dual therapy, we combined low-dose PNA with the histone deacetylase (HDAC) inhibitor Romidepsin, measuring binding efficiency and protein expression expression.
Results
Gel shift and amplicon assays indicate that γGcPNA2 has a superior binding affinity under both in vitro and cell culture conditions. Confocal imaging confirms efficient cellular uptake of γGcPNA2. Protein and viability assays demonstrate that γGcPNA2 exhibits greater efficacy compared to γPNA1 in both cell lines. Dual therapy with Romidepsin enables reduced γGcPNA2 dosing.
Conclusion
The study demonstrates that G-clamp modified γGcPNA2 is more effective than γPNA1 in inhibiting transcription and binding to the complementary sequence. A combination therapy using a lower dose of Romidepsin and the PNA enhances therapeutic potential, supporting the feasibility of dual therapy for c-Myc driven cancers.
Presentation 3
Carnitine-Tailored SNEDDS for Targeted Delivery of A1874 PROTAC in Glioblastoma via OCTN2 Receptor
Himaxi Patel, St. John's University, [email protected]
Purpose
Glioblastoma(GBM) is the most aggressive brain cancer, with a median survival of 12–15 months, accounting for nearly 50% of malignant brain tumors. Limitations of current medicine necessitate an investigation of novel therapeutic targets. A1874, a Proteolysis Targeting Chimera(PROTAC) that selectively degrades key oncoproteins of GBM i.e. BRD4 and MDM2 by recruiting them to an E3 ligase, offering a significant advantage over inhibitors. Being heterobifunctional, beyond the rule of 5 molecule(bRo5), it poses significant challenges in solubility, absorption and brain permeation. In GBM, the upregulation of Protein Kinase C(PKC) correlates with increased tumor cell proliferation, migration, and resistance to apoptosis contributing to GBM's aggressiveness. Herein, Palmitoyl-DL-carnitine chloride(PC) was used as a PKC inhibitor, providing cationic surface charge, aiding its stability further along with its potential to facilitate drug delivery across BBB through organic cation transporter 2(OCTN2). The objectives of the current research are: (1) Explore the anticancer activity of A1874 in GBM (2) Develop a brain cancer targeted lipid-based nanomedicine of A1874(PROnano).
Methods
Preformulation studies were carried out to estimate the solubility of A1874 in various aqueous buffers, oils, biocompatible organic solvents, and surfactants. Palmitoyl-DL-carnitine anchored self-nanoemulsifying drug delivery system was prepared and optimized for particle size, polydispersity index(PDI), and ζ-potential. Precipitation kinetics and solid-state characteristics were assessed. In vitro, the anticancer efficacy of A1874 and PROnano was evaluated in different GBM cell lines. Ongoing studies are in vitro drug release assay, permeability assay, and multicellular 3D spheroids model of brain cancer cells.
Results
A1874 exhibited negligible saturation solubility across various pH buffer solutions. Preconcentrate without PC and PROnano exhibited particle size/PDI and ζ-potential of 48.42 nm/0.134, -3.5mV, and 49.54 nm/0.205, +45.5mV respectively. The physical and chemical stability of PROnano was maintained for 24 hours. In vitro cytotoxicity studies demonstrated anticancer activity of A1874 in U-87MG and U-87MG/TR cells with IC50 value <0.6±1.56μM. However, for T-98G cells, the IC50 was found to be >10μM.
Conclusion
In conclusion, these findings demonstrate that A1874 exhibits notable anticancer activity in GBM cell lines. Additionally, PROnano could present potentially effective strategy for the targeted delivery of A1874 to brain cancer.
Presentation 4
Exploring the Efficacy of Inhaled Antimalarial Drug Against Non-Small Cell Lung Cancer
Meghana Mokashi, St. John's University, [email protected]
Purpose
Non-small cell lung cancer (NSCLC) remains the predominant subtype of lung malignancies, with limited therapeutic success due to late-stage diagnosis, chemoresistance, and systemic toxicities of conventional treatments. Drug repurposing presents a strategic approach to accelerating cancer therapy development. Amodiaquine (AQ), an FDA-approved antimalarial, exhibits anticancer activity through autophagy inhibition and apoptosis induction. However, its clinical translation is hindered by dose-limiting hepatotoxicity and poor pharmacokinetics. This study investigates the formulation and therapeutic efficacy of inhalable AQ-loaded liposomes to achieve localized pulmonary drug delivery, enhancing tumor-targeted cytotoxicity while minimizing systemic adverse effects.
Methods
AQ liposomes were formulated via thin-film hydration and characterized for size, polydispersity, zeta potential, and drug encapsulation. Stability studies were conducted at 4°C and 25°C. The aerodynamic performance of AQ liposomes was assessed using NGI. Cellular uptake was studied using fluorescence microscopy. In vitro cytotoxicity, colony formation, migration inhibition, and 3D spheroid assays were performed on A549 and H460 NSCLC cell lines. Apoptotic potential was determined via caspase-3 activation.
Results
AQ liposomes exhibited a particle size of ~114 nm, and encapsulation efficiency of 82.2%. Stability assessment confirmed physicochemical integrity over two months at 4°C and 25°C. AQ liposomes demonstrated efficient pulmonary deposition with a mass median aerodynamic diameter (MMAD) of 3.12 µm and a high fine particle fraction (~88%), indicating suitability for deep lung deposition. Cellular internalization studies revealed a 120-fold enhancement in AQ-liposome uptake compared to free AQ. Cytotoxicity assays in A549 and H460 NSCLC cell lines demonstrated a 2.6-fold reduction in IC50 values with AQ liposomes versus free drug. Colony formation and scratch assays confirmed significant inhibition of tumor proliferation and migration. In 3D spheroid models, AQ liposomes exhibited superior tumor penetration, leading to dose-dependent spheroid disintegration upon multiple administrations. Apoptotic potential was validated by caspase-3 activation, demonstrating enhanced programmed cell death in AQ-liposome-treated cells.
Conclusion
The inhalable AQ-loaded liposomes present a novel, targeted strategy for NSCLC therapy, effectively enhancing drug bioavailability, reducing systemic toxicity, and improving anticancer efficacy. This localized delivery system offers a promising translational pathway for AQ repurposing in lung cancer treatment. Future in vivo validation will be imperative for clinical progression.
Moderator: Vivek Gupta
Presentation 1
Next-Generation Antisense Strategy to Target AKT Genes for Breast Cancer Therapy
Natasha Shah, University of Saint Joseph, [email protected]
Purpose
AKT, or protein kinase B, is a serine-threonine kinase that regulates various biological functions in the body. In breast cancer, two isoforms of AKT—AKT1 and AKT2—are frequently mutated. Overexpression of AKT1 enhances tumor cell proliferation, growth, and the suppression of apoptosis, while overexpression of AKT2 increases tumor migration, invasion, and metastasis. To date, the simultaneous inhibition of AKT1 and AKT2 mRNA translation has not been achieved. We optimized an antisense oligonucleotide (ASO) design to target both AKT1 and AKT2 mRNA at the same time. Our ASO design incorporates chemical modifications such as phosphorothioate (PS) at alternate positions throughout the sequence and 2’-O-methyl (2’-O-Me) modifications at the terminal three positions on both ends, enhancing in vivo activity. Additionally, we have developed peptide nucleic acid (PNA)-based AKT inhibitors for comparative studies. The pH low insertion peptide (pHLIP) facilitates membrane insertion upon detecting the acidity of tumor cell surfaces. It has been utilized to deliver ASOs to tumor cells. Therefore, in this multi-disciplinary project, we are assessing pHLIP-conjugated PS-2’-O-Me AKT inhibitors and pHLIP-conjugated PNA AKT inhibitors to inhibit the translation of AKT mRNA as a clinically translatable strategy for treating breast cancer.
Methods
We utilized cell viability and gene expression studies to compare the efficacy of various AKT inhibitors in the MDA-MB-453 and MCF-7 breast cancer cell lines. In ongoing studies, we are assessing the effectiveness of the pHLIP-AKT inhibitor conjugates to achieve AKT1 and AKT2 knockdown at both mRNA and protein levels in multiple breast cancer cell lines.
Results
We observed decreased expression of AKT1 and AKT2 in the treatment group receiving the PS-2’-O-Me AKT inhibitor. Additionally, we noted a 50% reduction in cellular viability of MDA-MB-453 cells and a 30% reduction in cellular viability of MCF 7 following treatment with the PS-2’-O-Me AKT inhibitor.
Conclusion
Results presented in this study evaluated the efficacy of ASO-based AKT inhibitors in different breast cancer cell lines. We hope the results of our study will help establish a novel platform for RNA-based precision medicine for breast cancer therapy.
Presentation 2
Targeting the Genomic DNA of the Undruggable c-Myc Oncogene for Cancer Therapy
Ruchi Ruchi, University of Connecticut, [email protected]
Purpose
Several antisense (RNA targeting platforms) therapeutics have evolved through numerous chemical modifications. With these advancements, attention is now directed towards assessing these molecules to treat the fundamental causes of diseases by directly targeting genomic DNA. Malik et al. illustrated the application of γtcPNA-NLS to specifically target the homopurine region of the c-Myc oncogene within the nucleus. In the present study, we compare the therapeutic efficacy of γPNA1-NLS with the G-clamp modified γGcPNA2-NLS, to enhance clinical translation. We employed both PNA scaffolds to inhibit c-Myc transcription and facilitate gene silencing. These molecules were systematically evaluated for their binding efficiency to the target site. As a proof of concept, we performed cell culture-based studies to gauge their effectiveness. Our comparative analysis indicates the potential of targeting genomic DNA non-restrictedly, thereby presenting new opportunities for tackling previously undruggable oncogenes and advancing cancer therapy strategies.
Methods
We evaluated the binding efficiency of two peptide nucleic acids (PNAs) utilizing a dose-dependent gel shift assay, a cell culture-based amplicon assay, and Sanger sequencing. The expression of c-Myc was assessed in U2932 and A549 cell lines through quantitative polymerase chain reaction (qPCR) and Western blot analysis. Cytotoxic effects were examined via cell viability and clonogenic assays. To assess the potential for dual therapy, we combined low-dose PNA with the histone deacetylase (HDAC) inhibitor Romidepsin, measuring binding efficiency and protein expression expression.
Results
Gel shift and amplicon assays indicate that γGcPNA2 has a superior binding affinity under both in vitro and cell culture conditions. Confocal imaging confirms efficient cellular uptake of γGcPNA2. Protein and viability assays demonstrate that γGcPNA2 exhibits greater efficacy compared to γPNA1 in both cell lines. Dual therapy with Romidepsin enables reduced γGcPNA2 dosing.
Conclusion
The study demonstrates that G-clamp modified γGcPNA2 is more effective than γPNA1 in inhibiting transcription and binding to the complementary sequence. A combination therapy using a lower dose of Romidepsin and the PNA enhances therapeutic potential, supporting the feasibility of dual therapy for c-Myc driven cancers.
Presentation 3
Carnitine-Tailored SNEDDS for Targeted Delivery of A1874 PROTAC in Glioblastoma via OCTN2 Receptor
Himaxi Patel, St. John's University, [email protected]
Purpose
Glioblastoma(GBM) is the most aggressive brain cancer, with a median survival of 12–15 months, accounting for nearly 50% of malignant brain tumors. Limitations of current medicine necessitate an investigation of novel therapeutic targets. A1874, a Proteolysis Targeting Chimera(PROTAC) that selectively degrades key oncoproteins of GBM i.e. BRD4 and MDM2 by recruiting them to an E3 ligase, offering a significant advantage over inhibitors. Being heterobifunctional, beyond the rule of 5 molecule(bRo5), it poses significant challenges in solubility, absorption and brain permeation. In GBM, the upregulation of Protein Kinase C(PKC) correlates with increased tumor cell proliferation, migration, and resistance to apoptosis contributing to GBM's aggressiveness. Herein, Palmitoyl-DL-carnitine chloride(PC) was used as a PKC inhibitor, providing cationic surface charge, aiding its stability further along with its potential to facilitate drug delivery across BBB through organic cation transporter 2(OCTN2). The objectives of the current research are: (1) Explore the anticancer activity of A1874 in GBM (2) Develop a brain cancer targeted lipid-based nanomedicine of A1874(PROnano).
Methods
Preformulation studies were carried out to estimate the solubility of A1874 in various aqueous buffers, oils, biocompatible organic solvents, and surfactants. Palmitoyl-DL-carnitine anchored self-nanoemulsifying drug delivery system was prepared and optimized for particle size, polydispersity index(PDI), and ζ-potential. Precipitation kinetics and solid-state characteristics were assessed. In vitro, the anticancer efficacy of A1874 and PROnano was evaluated in different GBM cell lines. Ongoing studies are in vitro drug release assay, permeability assay, and multicellular 3D spheroids model of brain cancer cells.
Results
A1874 exhibited negligible saturation solubility across various pH buffer solutions. Preconcentrate without PC and PROnano exhibited particle size/PDI and ζ-potential of 48.42 nm/0.134, -3.5mV, and 49.54 nm/0.205, +45.5mV respectively. The physical and chemical stability of PROnano was maintained for 24 hours. In vitro cytotoxicity studies demonstrated anticancer activity of A1874 in U-87MG and U-87MG/TR cells with IC50 value <0.6±1.56μM. However, for T-98G cells, the IC50 was found to be >10μM.
Conclusion
In conclusion, these findings demonstrate that A1874 exhibits notable anticancer activity in GBM cell lines. Additionally, PROnano could present potentially effective strategy for the targeted delivery of A1874 to brain cancer.
Presentation 4
Exploring the Efficacy of Inhaled Antimalarial Drug Against Non-Small Cell Lung Cancer
Meghana Mokashi, St. John's University, [email protected]
Purpose
Non-small cell lung cancer (NSCLC) remains the predominant subtype of lung malignancies, with limited therapeutic success due to late-stage diagnosis, chemoresistance, and systemic toxicities of conventional treatments. Drug repurposing presents a strategic approach to accelerating cancer therapy development. Amodiaquine (AQ), an FDA-approved antimalarial, exhibits anticancer activity through autophagy inhibition and apoptosis induction. However, its clinical translation is hindered by dose-limiting hepatotoxicity and poor pharmacokinetics. This study investigates the formulation and therapeutic efficacy of inhalable AQ-loaded liposomes to achieve localized pulmonary drug delivery, enhancing tumor-targeted cytotoxicity while minimizing systemic adverse effects.
Methods
AQ liposomes were formulated via thin-film hydration and characterized for size, polydispersity, zeta potential, and drug encapsulation. Stability studies were conducted at 4°C and 25°C. The aerodynamic performance of AQ liposomes was assessed using NGI. Cellular uptake was studied using fluorescence microscopy. In vitro cytotoxicity, colony formation, migration inhibition, and 3D spheroid assays were performed on A549 and H460 NSCLC cell lines. Apoptotic potential was determined via caspase-3 activation.
Results
AQ liposomes exhibited a particle size of ~114 nm, and encapsulation efficiency of 82.2%. Stability assessment confirmed physicochemical integrity over two months at 4°C and 25°C. AQ liposomes demonstrated efficient pulmonary deposition with a mass median aerodynamic diameter (MMAD) of 3.12 µm and a high fine particle fraction (~88%), indicating suitability for deep lung deposition. Cellular internalization studies revealed a 120-fold enhancement in AQ-liposome uptake compared to free AQ. Cytotoxicity assays in A549 and H460 NSCLC cell lines demonstrated a 2.6-fold reduction in IC50 values with AQ liposomes versus free drug. Colony formation and scratch assays confirmed significant inhibition of tumor proliferation and migration. In 3D spheroid models, AQ liposomes exhibited superior tumor penetration, leading to dose-dependent spheroid disintegration upon multiple administrations. Apoptotic potential was validated by caspase-3 activation, demonstrating enhanced programmed cell death in AQ-liposome-treated cells.
Conclusion
The inhalable AQ-loaded liposomes present a novel, targeted strategy for NSCLC therapy, effectively enhancing drug bioavailability, reducing systemic toxicity, and improving anticancer efficacy. This localized delivery system offers a promising translational pathway for AQ repurposing in lung cancer treatment. Future in vivo validation will be imperative for clinical progression.
