Synergistic Killing Research Articles

Combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

Epstein-Barr virus (EBV) manipulates the ubiquitin-proteasome system and regulators of Bcl-2 family to enable the persistence of the virus and survival of the host cells through the expression of viral proteins in distinct latency patterns. We postulate that the combination of bortezomib (proteasome inhibitor) and venetoclax (Bcl-2 inhibitor) [bort/venetoclax] will cause synergistic killing of post-transplant lymphoproliferative disorder (PTLD) through targeting the pro-survival function of latent viral proteins such as latent membrane protein-1 (LMP-1) and EBV nuclear antigen-3C (EBNA-3C). Bort/venetoclax could synergistically kill spontaneous lymphoblastoid cell lines (sLCLs) derived from patients with PTLD and EBV-associated hemophagocytic lymphohistiocytosis by inducing DNA damage response, apoptosis and G1-S cell cycle arrest in a ROS-dependent manner. Bortezomib potently induced the expression of Noxa, a pro-apoptotic initiator and when combined with venetoclax, inhibited Mcl-1 and Bcl-2 simultaneously. Bortezomib prevented LMP-1 induced proteasomal degradation of IκBα leading to the suppression of the NF-κB signaling pathway. Bortezomib also rescued Bcl-6 from EBNA-3C mediated proteasomal degradation thus maintaining the repression of cyclin D1 and Bcl-2 causing G1-S arrest and apoptosis. Concurrently, venetoclax inhibited Bcl-2 upregulated by either LMP-1 or EBNA-3C. Bort/venetoclax decreased the expression of phosphorylated p65 and Bcl-2 at serine 70 thereby suppressing the NF-κB signaling pathway and promoting apoptosis, respectively. These data corroborated the marked suppression of the growth of xenograft of sLCL in SCID mice (p<0.001). Taken together, the combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

Development of a sequential release nanomaterial for co-delivery of hypoxia-induced tirapazamine and HIF-1α siRNA in cancer therapy

Unlike traditional drug carriers, sequential drug delivery systems can release different drugs in order, with the first released drug providing a prerequisite for the later released drug to maximize its function, thereby achieving stronger anti-tumor effects. Herein we constructed a temporal sequential system designated TPZ@MSN/HIF-1α siRNA@PDA@GOx (MTRPG) in which mesoporous silica nanoparticles were used as cores to load hypoxia induced chemotherapy drug tirapazamine (TPZ) and gene targeted nucleic acid drug HIF-1α siRNA, polydopamine (PDA) as acid –responsive coating as well as to realize photothermal therapy, and glucose oxidase (GOx) as the outermost layer to achieve starvation therapy and construct a deepened hypoxia to activate TPZ. Through in vitro and in vivo experiments, we demonstrated that the first released glucose oxidase catalyzed the oxidation of glucose, achieving starvation treatment while reducing the acidic environment and further exacerbating hypoxia in tumor cells. The reduced acidic conditions enabled the degradation of PDA, resulting in the release of loaded HIF-1α siRNA and TPZ. At the same time, PDA could also exert photothermal therapy under 808 nm near-infrared (808 nm NIR) laser irradiation. The later released hypoxia induced chemotherapy drug TPZ amplifies its anti-tumor activity under intensified hypoxia conditions. Meanwhile, the released HIF-1α siRNA interfered with the up-regulated HIF-1α induced by the deepened hypoxia condition, which caused hypoxia tolerance in tumors, reduced its expression activity, and achieved synergistic killing of tumor cells with chemotherapy. This work provides an effective multimodal synergistic therapy strategy to promote tumor therapeutic index, which may possess a promising future in clinical application.

Oncolytic Newcastle Disease Virus and Photodynamic Therapy as Dual Approach for Breast Cancer Treatment.

We hypothesized that attacking cancer cells by combining various modes of action can hinder them from taking the chance to evolve resistance to treatment. Incorporation of photodynamic therapy (PDT) with oncolytic virotherapy might be a promising dual approach to cancer treatment. NDV AMHA1 strain as virotherapy in integration with aminolaevulinic acid (ALA) using low power He-Ne laser as PDT in the existing work was examined against breast cancer cells derived from Iraqi cancer patients named (AMJ13). This combination was evaluated using Chou-Talalay analysis. The results showed an increased killing rate when using both 0.01 and 0.1 Multiplicity of infection (MOI) of the virus when combined with a dose of 6172.8 photons/gm (ph/gm) of PDT focused on cancer cells. integration of the attenuated NDV-AMHA1 strain with photodynamic therapy has a synergistic killing effect on breast cancer cells in vitro, suggesting that this strategy could have clinical application to overcome breast cancer.

Estrogen-related receptor gamma (ERR[gamma]) as a therapeutic target for anaplastic thyroid cancer and the synergistic killing effect of DN200434 (ERR[gamma] inverse-agonist) and MK2206 (AKT inhibitor) in thyroid cancers

Estrogen-related receptor gamma (ERR[gamma]) as a therapeutic target for anaplastic thyroid cancer and the synergistic killing effect of DN200434 (ERR[gamma] inverse-agonist) and MK2206 (AKT inhibitor) in thyroid cancers

GTF2H5 Identified as a crucial synthetic lethal target to counteract chemoresistance in colorectal cancer

BackgroundSynthetic lethality (SL) emerges as a novel concept being explored to combat cancer progression and resistance to conventional therapy. Despite the efficacy of chemotherapy in select cases of colorectal cancer (CRC), a substantial proportion of patients encounter challenges, leading to an adverse prognosis of CRC patients. CRC-related SL genes offer a potential avenue for identifying therapeutic targets. MethodsCRC-related SL genes were obtained from the SynLethDB database. The bulk RNA sequencing data, mutation data, and clinical information for treated and untreated CRC patients were enrolled from the UCSC and GEO databases. The Tumor Immunology Single Cell Center database served as the repository for collecting and analyzing single-cell RNA sequencing data. The synergistic killing effect of SL genes and chemotherapeutic drugs on resistant cells was experimentally verified. ResultsIn the present study, pivotal SL genes associated with chemoresistance identified by using WGCNA and CRC patients categorized into two groups based on these genes. Variations between the groups were most pronounced in pathways associated with extracellular matrix remodeling. Further by integrating mutation data, five potential SL genes were discerned, which were highly expressed in the presence of TP53 or KRAS mutations, leading to a severely poor prognosis. Subsequent time series analysis revealed that the expression of GTF2H5 was gradually elevated at different stages of the transition from sensitive to resistant in CRC cells. Finally, it was preliminarily verified by experiments that GTF2H5 may play a key role in driving the drug-resistant transition within CRC cells. ConclusionsThe identification of SL genes that collaboratively interact with chemotherapeutic agents could provide new insights into solving the issue of chemotherapy resistance in CRC patients. And GTF2H5 wields a fundamental influence in inducing chemoresistance in CRC, which provided a potential therapeutic target for CRC.

Synergism of colistin and globular endolysins against multidrug-resistant gram-negative bacteria

Endolysins (lysins), a novel class of antibacterial agents derived from bacteriophages, efficiently lyse bacteria by degrading the peptidoglycan layer within the bacterial wall. Colistin, a classic peptide antibiotic with the ability to permeabilize the outer membrane, has recently shown great promise in synergizing with lysins against gram-negative bacteria. However, the exact mechanisms responsible for their synergy remain unclear. Here, we first demonstrated the synergistic bacterial killing of various lysin and colistin combinations. With a model lysin, LysAB2, we then confirmed that there is a threshold concentration of colistin causing sufficient permeabilization of the outer membrane for lysin to access the peptidoglycan layer and subsequently exert its lytic ability. The threshold colistin concentrations were found to range 0.2−0.8 μM for the tested bacteria, with the exact value largely depending on the density of lipopolysaccharides on the outer membrane. Beyond the threshold colistin level, LysAB2 could synergize with colistin at a concentration as low as 0.31 μM. Next, we proved for the first time that lysin-induced degradation of the peptidoglycan layer facilitated the disruption of cytoplasmic membrane by colistin, elevated the level of reactive oxygen species in bacterial cells, and boosted the killing effect of colistin. Additionally, the colistin-lysin combination could effectively eliminate established biofilms due to the biofilm dispersal ability of lysin. The in-vivo efficacy was preliminary confirmed in a Galleria mellonella infection model for combination with colistin doses (≥ 1.8 μg/larvae), which could reach beyond the threshold concentration, and a fixed LysAB2 dose (10 μg/larvae). In summary, our study provided the first experimental evidence unravelling the mechanisms behind the synergy of colistin and lysins. All these findings provided important insights in guiding the dosing strategy for applying this combination in future development.

Mixed alkyl/aryl phosphonates identify metabolic serine hydrolases as antimalarial targets

Malaria, caused by Plasmodium falciparum, remains a significant health burden. One major barrier for developing antimalarial drugs is the ability of the parasite to rapidly generate resistance. We previously demonstrated that salinipostin A (SalA), a natural product, potently kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism that results in a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a small library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent antiparasitic potencies that enabled the identification of therapeutically relevant targets. The active compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor orlistat and shows synergistic killing with orlistat. Our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are promising, synthetically tractable antimalarials.

Anti-Biofilm Action of Cineole and Hypericum perforatum to Combat Pneumonia-Causing Drug-Resistant P. aeruginosa.

Hospital-acquired antibiotic-resistant pneumonia is one of the major causes of mortality around the world that pose a catastrophic threat. Pseudomonas aeruginosa is one of the most significant opportunistic pathogens responsible for hospital-acquired pneumonia and gained resistance to the majority of conventional antibiotics. There is an urgent need for antibiotic alternatives to control drug-resistant pneumonia and other related respiratory infections. In the present study, we explored the antibacterial potential of cineole in combination with homeopathic medicines against biofilm-forming drug-resistant P. aeruginosa. Out of 26 selected and screened homeopathic medicines, Hypericum Perforatum (HyPer) was found to eradicate biofilm-forming drug-resistant P. aeruginosa most effectively when used in combination with cineole. Interestingly, the synergistic action of HyPer and cineole was also found to be similarly effective against planktonic cells of P. aeruginosa. Further, the potential synergistic killing mechanisms of cineole and HyPer were determined by analyzing zeta membrane potential, outer membrane permeability, and DNA release from P. aeruginosa cells upon treatment with cineole and HyPer. Additionally, molecular docking analysis revealed strong binding affinities of hypericin (an active ingredient of HyPer) with the PqsA (a quorum sensing protein) of P. aeruginosa. Overall, our findings revealed the potential synergistic action of cineole and HyPer against biofilm-forming drug-resistant P. aeruginosa. Cineole and HyPer could be used in combination with other bronchodilators as inhalers to control the biofilm-forming drug-resistant P. aeruginosa.

Salphen-Based Fe-N2O2@C Nanomaterial Applied in Synergistic Sonodynamic and Chemodynamic Therapy of Tumors

To synthesize a Salphen-based Fe-N2O2@C material with high peroxidase (POD)-mimicking activity and sonosensitivity for the synergistic sonodynamic (SDT) and chemodynamic (CDT) therapy of tumors. Fe-N2O2 was synthesized via the hydrothermal method, and Fe-N2O2@C was prepared by incorporating a ketjen black substrate. The morphology, structure, composition, enzyme mimic activity for reactive oxygen species (ROS) production, and sonosensitivity of the material were characterized. The ability and mechanism of Fe-N2O2@C to perform synergistic SDT and CDT killing of 4T1 mouse breast cancer cells were explored through in vitro experiments. The in vivo tumor-killing ability of Fe-N2O2@C combined with ultrasound irradiation was investigated using a subcutaneous 4T1 tumor-bearing mouse model. FFe-N2O2 and Fe-N2O2@C were both irregularly shaped nanospheres with average particle sizes of 25.9 nm and 36.2 nm, respectively. XRD, FTIR, and XPS analyses confirmed that both Fe-N2O2 and Fe-N2O2@C possessed a Salphen covalent organic framework structure with M-N2O2 coordination, and the ketjen black loading had no significant impact on this structure. Compared to Fe-N2O2, Fe-N2O2@C exhibited high POD-mimicking activity (with K m reduced from 19.32 to 5.82 mmol/L and v max increased from 2.51×10-8 to 8.92×10-8 mol/[L·s]) and sonosensitivity. Fe-N2O2@C in combination with ultrasound irradiation could produce a large amount of ROS within cells and a subsequent significant decrease in mitochondrial membrane potential, thereby inducing TEM-observable mitochondrial damage and causing cell apoptosis and death. In addition, in vivo experiments showed that Fe-N2O2@C in combination with ultrasound irradiation could effectively inhibit tumor growth in a 4T1 subcutaneous tumor-bearing mouse model without significant in vivo toxicity. In this study, we prepared a Salphen-based Fe-N2O2@C material with good biocompatibility, which can be used in combination with ultrasound irradiation to achieve SDT and CDT synergistic killing of tumor cells and inhibit tumor growth. This Salphen-based Fe-N2O2@C nanomaterial shows promising potential for multimodal tumor therapy.

NAPRT Silencing in FH-Deficient Renal Cell Carcinoma Confers Therapeutic Vulnerabilities via NAD+ Depletion.

Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is caused by loss of function mutations in fumarate hydratase (FH) and results in an aggressive subtype of renal cell carcinoma with limited treatment options. Loss of FH leads to accumulation of fumarate, an oncometabolite that disrupts multiple cellular processes and drives tumor progression. High levels of fumarate inhibit alpha ketoglutarate-dependent dioxygenases, including the ten-eleven translocation (TET) enzymes, and can lead to global DNA hypermethylation. Here, we report patterns of hypermethylation in FH-mutant cell lines and tumor samples are associated with the silencing of nicotinate phosphoribosyl transferase (NAPRT), a rate-limiting enzyme in the Preiss-Handler pathway of NAD+ biosynthesis, in a subset of HLRCC cases. NAPRT is hypermethylated at a CpG island in the promoter in cell line models and patient samples, resulting in loss of NAPRT expression. We find that FH-deficient RCC models with loss of NAPRT expression, as well as other oncometabolite-producing cancer models that silence NAPRT, are extremely sensitive to nicotinamide phosphoribosyl transferase inhibitors (NAMPTi). NAPRT silencing was also associated with synergistic tumor cell killing with PARP inhibitors and NAMPTis, which was associated with effects on PAR-mediated DNA repair. Overall, our findings indicate that NAPRT silencing can be targeted in oncometabolite-producing cancers and elucidates how oncometabolite-associated hypermethylation can impact diverse cellular processes and lead to therapeutically relevant vulnerabilities in cancer cells. Implications: NAPRT is a novel biomarker for targeting NAD+ metabolism in FH-deficient HLRCCs with NAMPTis alone and targeting DNA repair processes with the combination of NAMPTis and PARP inhibitors.

Cytokine-induced killer cells-mediated chlorin e6-loaded gold nanostars for targeted NIR imaging and immuno-photodynamic combination therapy for lung cancer

Recently, cytokine-induced killer (CIK) cells have a broad application prospect in the comprehensive diagnosis and treatment of tumors owing to their unique characteristics of killing and targeting malignant tumors. Herein, we report a facile strategy for synthesis of monodisperse gold nanostars (GNSs) based on PEGylation and co-loaded with the photosensitizer chlorin e6 (Ce6) to form GNSs-PEG@Ce6 NPs. Then employing CIK cells loading the as-prepared GNSs-PEG@Ce6 NPs to fabricate a CIK cells-based drug delivery system (GNSs-PEG@Ce6-CIK) for lung cancer. Among them, GNSs was functioned as transport media, Ce6 acted as the near-infrared (NIR) fluorescence imaging agent and photodynamic therapy (PDT), and CIK cells served as targeting vectors for immunotherapy, which can increase the efficiency of tumor enrichment and treatment effect. The results of cellular experiments demonstrated that GNSs-PEG@Ce6 NPs had good dispersibility, water solubility and low toxicity under physiological conditions, and the cultured CIK cells had strong anti-tumor properties. Subsequently, GNSs-PEG@Ce6-CIK could effectively inhibit the growth of A549 cells under the exposure of 633 nm laser, which showed stronger killing effect than that of GNSs-PEG@Ce6 NPs or CIK cells. In addition, they showed good tumor targeting and tumor synergistic killing activity in vivo. Therefore, GNSs-PEG@Ce6-CIK was constructed for targeted NIR fluorescence imaging, enhanced PDT and immunotherapy of lung cancer.

NIR-light controlled local delivery of nitric oxide based on self-targeting carbon dots

Nitric oxide (NO)-based gas therapy has received ever-increasing attention in treatment of many diseases, especially cancer. However, the selective organelle targeted delivery and controllable release of NO remains highly desirable. In this work, a sequentially targeting delivery nanoplatform has been constructed by encapsulating heat-responsive S-nitrosocysteine (the NO donor, SNC) conjugated mitochondria-targeting carbon dots (MitoCDs) and indocyanine green (ICG) within liposomes which are further modified with aptamer that can specifically bind to nucleolin overexpressed on the surface of several cancer cells. MitoCDs were prepared via one-step hydrothermal reaction using citric acid and m-diethylaminophenol as starting materials and used without any external targeting molecules. ICG molecules were employed as NIR-triggered photothermal agent followed by NO precisely controlled release in mitochondria. By combining gas and photothermal treatment, the nanocarrier produced a synergistic and superior killing effect on several cancer cell lines. And the cell growth inhibition of NCL-CSI@Lip on HEp-2 cells raised up to 72.39 %. Such an ‘all-in-one’ mitochondria-targeted NO nanocarrier may lead to new way for overcoming the problem of multidrug resistance in intracellular drug delivery.

Abstract PO-034: Targeting metabolic plasticity and flexibility via SIRT3 inhibition to restrain nucleotides availability in DLBCLs

Abstract Cancer cells need metabolic adaptations to survive in hostile environment such as low nutrient and drug treatment conditions. DLBCLs (Diffuse large B cell lymphoma) originate from a metabolic competitive/challenging environment of germinal center (GC) when B cells undergo positive selections against antigens. However, it is largely unknown whether and how metabolic flexibility (the ability to use different nutrients) and plasticity (the ability to process the same nutrient differently) contribute to DLBCLs. Our previous study identified that SIRT3, the only mitochondrial protein deacetylase, is critical to survival and proliferation of DLBCLs by promoting glutaminolysis. Our mitochondrial localized SIRT3 inhibitor, YC8-02, has pan-toxicity to all DLBCL cells tested regardless of different subtypes with somatic mutations. Here, we observed glucose as an alternative nutrient source to compensate in the TCA cycle in SIRT3 deficient cells. DLBCLs exhibited resistance to SIRT3 inhibition in high glucose condition, while SIRT3 inhibited DLBCLs highly depend on glucose metabolism. The combination of YC8-02 with glucose metabolism inhibitors induces synergistic killing to DLBCLs. As glucose is the main carbon source of cells, it is unknown how DLBCLs manage the flexibility of using glucose for different purposes. Using in vivo metabolic imaging (MALDI), we observed that, in contrast to normal GC B cells, lymphomas specifically use the glucose for de novo nucleotide synthesis. SIRT3 inhibition decreased nucleotide levels in DLBCL cells in vitro and in vivo. The metabolic flux tracing showed that DLBCL cells requires SIRT3 activity to activate a glucose/serine/one-carbon metabolism pathway for de novo nucleotide biosynthesis, and Inhibition of SIRT3 decreased the glucose and serine being used for nucleotides in DLBCLs. Serine as another potent source for nucleotides can rescue DLBCLs from SIRT3 inhibition. Furthermore, we validated that overexpression of enzymes, like MTHFD2, in one carbon metabolism pathway can rescue the proliferation in SIRT3 deficient cells. High nucleotides levels in medium also help DLBCLs survival in YC8-02 treatments. To overcome the metabolic flexibility on environmental nucleotides, we used inhibitors of nucleoside transporters (ENT1/2), dipyridamole and draflazine, which can synergize with YC8-02 to kill DLBCLs in vitro and eradicate systemic lymphomas in vivo. In summary, our results illustrate that DLBCLs with active SIRT3 exhibit metabolic plasticity by repurposing glucose to nucleotide biosynthesis, while lymphoma cells can also reply on metabolic flexibility to manage nutrient usage and gain extracellular nucleotides for survival under SIRT3 inhibition. Our combination treatment SIRT3 and ENT inhibitors can be a promising therapy for DLBCLs by restraining their metabolic plasticity and flexibility. Citation Format: Meng Li, Noel R Park, Chris Chin, Coraline Mlynarczyk, Hening Lin, Shawn Davidson, Ari M Melnick. Targeting metabolic plasticity and flexibility via SIRT3 inhibition to restrain nucleotides availability in DLBCLs [abstract]. In: Proceedings of the Fourth AACR International Meeting on Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2024 Jun 19-22; Philadelphia, PA. Philadelphia (PA): AACR; Blood Cancer Discov 2024;5(3_Suppl):Abstract nr PO-034.

Transcriptomic investigations of polymyxins and colistin/sulbactam combination against carbapenem-resistant Acinetobacter baumannii

Carbapenem-resistant Acinetobacter baumannii (CRAB) is a Priority 1 (Critical) pathogen urgently requiring new antibiotics. Polymyxins are a last-line option against CRAB-associated infections. This transcriptomic study utilized a CRAB strain to investigate mechanisms of bacterial killing with polymyxin B, colistin, colistin B, and colistin/sulbactam combination therapy. After 4 h of 2 mg/L polymyxin monotherapy, all polymyxins exhibited common transcriptomic responses which primarily involved disruption to amino acid and fatty acid metabolism. Of the three monotherapies, polymyxin B induced the greatest number of differentially expressed genes (DEGs), including for genes involved with fatty acid metabolism. Gene disturbances with colistin and colistin B were highly similar (89 % common genes for colistin B), though effects on gene expression were generally lower (0–1.5-fold in most cases) with colistin B. Colistin alone (2 mg/L) or combined with sulbactam (64 mg/L) resulted in rapid membrane disruption as early as 1 h. Transcriptomic analysis of this combination revealed that the effects were driven by colistin, which included disturbances in fatty acid synthesis and catabolism, and inhibition of nutrient uptake. Combination therapy produced substantially higher fold changes in 72 % of DEGs shared with monotherapy, leading to substantially greater reductions in fatty acid biosynthesis and increases in biofilm, cell wall, and phospholipid synthesis. This indicates synergistic bacterial killing with the colistin/sulbactam combination results from a systematic increase in perturbation of many genes associated with bacterial metabolism. These mechanistic insights enhance our understanding of bacterial responses to polymyxin mono- and combination therapy and will assist to optimize polymyxin use in patients.

α-Ketoglutarate downregulates thiosulphate metabolism to enhance antibiotic killing

Potentiation of the effects of currently available antibiotics is urgently required to tackle the rising antibiotics resistance. The pyruvate (P) cycle has been shown to play a critical role in mediating aminoglycoside antibiotic killing, but the mechanism remains unexplored. In this study, we investigated the effects of intermediate metabolites of the P cycle regarding the potentiation of gentamicin. We found that α-ketoglutarate (α-KG) has the best synergy with gentamicin compared to the other metabolites. This synergistic killing effect was more effective with aminoglycosides than other types of antibiotics, and it was effective against various types of bacterial pathogens. Using fish and mouse infection models, we confirmed that the synergistic killing effect occurred in vivo. Furthermore, functional proteomics showed that α-KG downregulated thiosulphate metabolism. Upregulation of thiosulphate metabolism by exogenous thiosulphate counteracted the killing effect of gentamicin. The role of thiosulphate metabolism in antibiotic resistance was further confirmed using thiosulphate reductase knockout mutants. These mutants were more sensitive to gentamicin killing, and less tolerant to antibiotics compared to their parental strain. Thus, our study highlights a strategy for potentiating antibiotic killing by using a metabolite that reduces antibiotic resistance.

Abstract PO2-16-01: Molecular correlates of drug response to guide therapy in triple-negative breast cancer

Abstract TNBC accounts for 10-20% of total breast cancer cases in the US and is more aggressive, higher grade, and has a poorer prognosis than other forms of breast cancer. TNBC is more likely to metastasize within 3-5 years than other forms of breast cancer and has a median survival of 17.6-21.3 months after metastasis. Advances in immunotherapy are promising, but recent trial results of immunotherapy-chemo combination have resulted in a 3-year overall survival rate under 36% in a PD-L1 based selected cohort. Thus, there is an unmet need for innovations that lead to new treatment options and improve outcomes for patients with TNBC in a personalized manner beyond only one or a few markers. In our recently published study (PMID: 31655920), we tested the dose response for a panel of 78 investigational drugs and plotted this readout against molecular characterization to identify statistically significant DNA, RNA, and protein predictors of drug efficacy in TNBC. We have expanded on this published study by identifying correlates from 3D cell culture. According to the literature, 3D culture systems are more representative of in vivo activity. We have increased our sample number (now 27 cell lines and 8 ex vivo PDXs) and expanded our drug library to 387 compounds, selected for their diverse mechanisms and promise in other cancers. Following screening, we prioritized 35 compounds based on their elevated activity and variance of response. From these 35 prioritized compounds, we identified molecular correlates of drug response for 27 compounds (77% of prioritized compounds yielding molecular correlates compared to 50% in our previously published study). Focusing on a known active drug in TNBC that is approved for patients with metastatic TNBC, we identified multi-omic (protein expression/activity and RNA expression) molecular correlates of response to a chemotherapeutic- SN-38, the payload of Sacituzumab and the active metabolite of irinotecan. We utilized a sub-panel of RNA correlates to characterize treatment-naive PDXs and predict response to SN-38. We then tested SN-38 response ex vivo for 5 PDXs to show that we could accurately identify sensitive/resistant models to SN-38 therapy. From significant protein correlates, we identified that the autophagy and AKT pathways were elevated in models with poor response to SN-38. Co-targeting the autophagy or AKT pathway with SN-38 was an effective strategy to identify rational, synergistic therapeutic combinations at therapeutically relevant levels (effective dose (ED) 50 and ED75). We evaluated irinotecan in vivo using a cell line with sensitivity in our 3D assay, demonstrating strong activity in vivo to validate that our screening assay accurately predicts in vivo response. We have further applied this methodology to targeted inhibitors with potential activity in TNBC. We have identified an ATR inhibitor, berzosertib, that targets the DNA damage response (DDR) pathway and correlates with protein expression and activity of Aurora kinases (AKs). Furthermore, combining the pan-AK inhibitor danusertib with berzosertib results in synergistic killing of TNBC cells. As a future direction, we are evaluating this combination in vivo utilizing an ethnically diverse patient-derived xenograft library grown in mice to evaluate this novel combination in a mock clinical trial and to identify molecular correlates of response and resistance that could be used to guide future human trials. Citation Format: Nathan Merrill, Nathalie Vandecan, Athena Apfel, Habib Serhan, Peter Ulintz, Liwei Bao, Xu Cheng, Aki Morikawa, Sofia Merajver, Matthew Soellner. Molecular correlates of drug response to guide therapy in triple-negative breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-16-01.

Abstract 3370: Anti-tumor activity of Debio 0123 in combination with sacituzumab govitecan in preclinical models of breast cancer

Abstract Background: Debio 0123 is a WEE1 inhibitor that has previously shown significant preclinical efficacy in combination with DNA damaging agents and is currently being investigated in phase 1 clinical studies either as a monotherapy or in combination with different standard therapies. Sacituzumab govitecan (SG) is a TROP2 directed antibody-drug-conjugate (ADC) carrying an SN38 payload that has recently gained approval in TNBC and ER+ve breast cancer after demonstrating improvements in survival versus standard therapies. Despite these promising data some patients still do not respond to SG treatment, develop resistance or experience toxic side effects. Herein we present preclinical data that demonstrates Debio 0123 improves tumor response to SG in breast cancer models and provides sustained complete responses and anti-metastatic activity. Methods: Synergy between Debio 0123 and SG was evaluated in breast cancer cell lines, with varying levels of TROP2 expression, in vitro using standard cytotoxicity assay in a matrix combination format. Efficacy of Debio 0123 and SG as monotherapies or in combination was further assessed in vivo, in both TROP2high and TROP2low subcutaneous breast cancer xenograft models at different doses. Results: In vitro, Debio 0123 in combination with SG demonstrated synergistic tumor cell killing in all 4 breast cancer cell lines tested, at all concentrations. In vivo, TROP2high MDA-MB-468 breast cancer xenograft tumors treated with Debio 0123 at 30mg/kg QD resulted in minimal anti-tumor activity as a monotherapy but completely regressed 100% of the tumors in combination with SG. Furthermore, 40% of these complete regressions were sustained for up to 100 days post-treatment initiation. These data were then further replicated at a reduced dose of SG. In the TROP2low MDA-MB-231luc model, Debio 0123 significantly improved response to SG resulting in a 2-fold improvement in tumor growth inhibition. Additionally, combination treatment significantly inhibited metastasis of these tumors to the lung and lymph nodes as monitored by bioluminescence. Collectively, these results lead to a significant improvement in animal survival as a combination compared to either treatment as a monotherapy. Conclusion: These results demonstrate that Debio 0123 synergizes with, and significantly improves breast cancer tumor cell response to, SG regardless of TROP2 expression levels (both in vitro and in vivo), leading to complete regressions and inhibition of metastasis. Collectively these data support Debio 0123 in combination with SG could be a potential therapeutic approach for improving responses in patients with breast cancer. Citation Format: Luke Piggott, Esteban Rodrigo Imedio, Francesco Staehli. Anti-tumor activity of Debio 0123 in combination with sacituzumab govitecan in preclinical models of 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 3370.

Abstract 4563: Ferroptotic mechanism of drug induced toxicity of PARPi and AurKi in prostate cancer

Abstract BACKGROUND: Cancer is typically treated with a combination of drugs rather than monotherapy stratagems, but identification of synergistic drug combinations for the treatment of cancers is challenging. Here we present the combination of PARP and Aurora Kinase inhibitors synergize through induction of the ferroptosis pathway of cell death. METHODS: We identified these drugs through an unbiased screen of enriched drug class response correlations. We then validated a synergistic cell killing effect of multiple drugs of each class by in vitro drug screening. Next, we performed reversal experiments using a ferroptosis inhibitor and lipid peroxide staining to confirm ferroptosis is a relevant cell death pathway. We performed Western immunoblot analysis in cells overexpressing putative Aurora Kinase phospho-substrates in the relevant pathway of interest, and performed site directed mutagenesis experiments to show the relevant amino acid altered by kinase activity. We also performed a series of qPCR and co-IP experiments to show PARPi effect on the antioxidant signaling pathway. RESULTS: We validated and identified synergy in a prostate cancer specific context. Next, we found Talazoparib and Alisertib induced ferroptosis after synergy was reduced with a ferroptosis inhibitor and lipid peroxides increased accumulation. We found a novel mechanism action for Alisertib, wherein Aurora Kinase inhibitors can alter the phospho-status and activity of the iron reduction enzyme Steap3.We also found that PARP inhibitors can affect the Nrf2-driven antioxidant signaling pathway on a transcriptional level. CONCLUSION: Aurora Kinase inhibitors and PARP inhibitors for the treatment of prostate cancer was identified and was validated as synergistic, which revealed new functional roles of both drugs in the context of cell killing. These findings represent a new clinical combination of drugs in prostate cancer and reveal a novel role for this drug class in ferroptosis. Citation Format: August John, Kuntian Luo, Zhenkun Lou, Liewei Wang. Ferroptotic mechanism of drug induced toxicity of PARPi and AurKi in prostate 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 4563.

Abstract 3132: A novel dual drug antibody-drug conjugate targeting hTrop2 has synergetic anti-tumor activity in solid tumor models

Abstract Antibody-drug conjugates (ADCs) are emerging therapeutic agents for targeted cancer treatment with remarkable clinical success. However, tumor heterogeneity and microenvironment complexity are still the major factors contributing to drug resistance, recurrence, and metastasis, which cannot be resolved with a mono therapy strategy. We’ve developed a novel dual drug ADC (BiADC), BR113, by conjugating an anti-hTrop2 antibody with a drug linker of a Topoisomerase I inhibitor payload, and another drug linker of an immune stimulator. Our studies show that the homogeneous ADC containing two distinct payloads is a new promising drug class, with two different anti-tumor MOAs, synergistic antigen-specific cell killing potency, desirable pharmacokinetic profiles, and minimal toxicity at therapeutic doses. In the cancer cells and human PBMC co-culture assay, BR113 exerts significantly more potent killing on cancer cells than two individual single-drug ADCs and the combination of two single-drug ADCs, which suggested a synergy on anti-tumor effects of this dual-drug ADC. In the xenograft mouse model studies, BR113 exhibits greater treatment effect and survival benefit than the combo of two single-drug ADCs at various doses. This robust superior anti-tumor activity is further confirmed in a hTrop2-MC38 syngeneic model with a prolonged anti-tumor protection against the tumor re-challenge. Finally, the novel BiADC, BR113, is well tolerated in hTop2 humanized mice after repeated administration. Our findings highlight the therapeutic potential of the BiADC format for treating refractory advanced solid tumors. The outstanding anti-tumor activity across various CDX tumor models and its favorable safety profile in mice support further evaluation of the innovative BiADC, BR113. Citation Format: Jie Zhu, Xiaobei Zhao, Eileen Li, Hong Xu, Lei Nie, Gang Chen, Haibing Wang. A novel dual drug antibody-drug conjugate targeting hTrop2 has synergetic anti-tumor activity in solid tumor models [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 3132.

Abstract 152: Exploring the impact of heat shock protein 90 on the antitumor efficacy and stemness in medulloblastoma cells

Abstract Background: Medulloblastoma (MB) is the most common children’s malignant brain tumor. Although MB can be treated with conventional chemotherapies, chemoresistance has been frequently developed and thereby hampers their therapeutic effectiveness. The preliminary reports of our research showed that some heat shock proteins have higher levels in MB cells. Among these heat shock proteins, HSP70 and HSP90 expressions are particularly high in MB CD133+ cancer stem cells (CSCs), which arouses our attention and interest. Numerous studies demonstrated that heat shock proteins are prominent in some chemotherapy-resistant cancer cells and CSCs. As known HSP90 and its client proteins are required for the maintenance of self-renewal/stemness and regulation of cell cycle, synergistic killing of cancer cells by simultaneous inhibition of HSP90 expression and activity may be a considerable good therapeutic strategy for treating MB. The purpose of this study is to verify the regulatory function of HSP90 in chemoresistance and stemness of MB. Materials and Methods: The levels of HSP90, its client proteins (P13K/pAKT, Erk, NFκB, and c-Myc), and stemness makers were detected using immunoblotting and immunoprecipitation after the treatment of HSP90 inhibitor (17-AAG) or knockdown of HSP90 by siRNA in MB and MB CSCs. The antitumor effects of HSPs inhibitors or knockdown of HSPs on these cells were evaluated by MTT assay, Annexin V/PI analysis and cell cycle analysis. Results: Our findings revealed elevated expression of HSP90 and its target client proteins in MB cells compared to normal astrocytes. Silencing or inhibiting HSP90 expression induced cell cycle arrest in MB cells. Notably, CD133+ MB CSCs exhibited higher HSP90 levels than parental MB cells. Inhibition of HSP90 led to reduced sphere formation and expression of stemness markers in CD133+ MB CSCs. Treatment with 17-AAG demonstrated antitumor activity in CD133+ MB CSCs, and a synergistic effect was observed when combining conventional chemotherapeutic agents with 17-AAG in MB cells Conclusion: These results illustrated that inhibition of HSP90 could eliminate CSCs, and thereby overcome resistance to chemotherapeutic agents in MB. Chemotherapies in combination with HSP90 blockade may be an emerging MB therapy in the future. Citation Format: Shu-Mei Chen, Hsieng-Yun Huang, Chien-Kai Wang, Yi-Yuan Yang, Sy-Jye Leu, Ying-Ying Li. Exploring the impact of heat shock protein 90 on the antitumor efficacy and stemness in medulloblastoma cells [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 152.