Strong Synergism Research Articles

EXTH-54. COMBINATORIAL DRUG SCREENING IDENTIFIES CARFILZOMIB AND ENZALUTAMIDE FOR TREATING AGGRESSIVE MENINGIOMAS

Abstract The goal of combinatorial therapy is to improve the effectiveness of treatment by targeting multiple aspects of cancer cells or overcoming resistance to single drugs. Therefore, we performed an automated combinatorial drug screening in meningioma cell lines and patient-derived tumor organoids (TOs). Three grade 3 meningioma cell lines (NCH93, IOMM-Lee, and KT21-MG1) were stably transduced with blue, green, and red fluorescence proteins. The cell lines were multiplexed into 384-well plates and treated in 5x5 dose-response (0-1000 nmol/l) matrices for 48h by the automated liquid handler Hamilton MicroLAB STAR®. The drug library consisted of 166 FDA-approved anticancer drugs. The viability of each cell line was demultiplexed by using fluorescence signals and CellTiterGlo. TOs were established from single cell suspensions of freshly resected meningioma tissue (n=20). The ZIP model was used to determine synergism. This drug screening effort generated 13,695 unique drug-drug combinations per cell line. Most synergistic combinations were observed in IOMM-Lee (1.14%), followed by KT21-MG1 (0.88%), and NCH93 (0.54%). The 110 most effective drug combinations were validated in wild-type meningioma cells. 75 drug combinations demonstrated a positive most synergistic area (MSA) score, indicating synergistic effects. Based on synergy scores and literature evidence, the 16 most promising combinations were selected for screening in TOs. The proteasome inhibitor carfilzomib in combination with the androgen receptor inhibitor enzalutamide exhibited the highest synergy in TOs. In 40% (n=8/20) of the cases, the combination showed strong synergism (MSA>10) resulting in an overall average MSA of 7.36, followed by carfilzomib and neratinib, and romidepsin and gemcitabine with average MSAs of 5.49, and 4.32, respectively. Furthermore, carfilzomib and enzalutamide induced apoptosis in TOs assessed by Caspase-3/7. This comprehensive combinatorial drug screening identified the synergistic combination of the proteasome inhibitor carfilzomib and androgen receptor inhibitor enzalutamide that can be an effective treatment option for selected meningioma patients.

Targeted nanoliposomes of oncogenic protein degraders: Significant inhibition of tumor in lung-cancer bearing mice

With 60 % of non-small cell lung cancer (NSCLC) expressing epidermal growth factor receptor (EGFR), it has been explored as an important therapeutic target for lung tumors. However, even the well-established EGFR inhibitors tend to promptly develop resistance over time. Moreover, strategies that could impede resistance development and be advantageous for both EGFR-Tyrosine kinase inhibitor (TKI)-sensitive and mutant NSCLC patients are constrained. Based on the critical relationship between EGFR, c-MYC, and Kirsten rat sarcoma virus (K-Ras), simultaneous degradation of EGFR and Bromodomain-containing protein 4 (BRD4) using “Proteolysis Targeting Chimeras (PROTACs)” could be a promising approach. PROTACs are emerging class of oncoprotein degraders but very challanging to deliver in vivo. Compared to individual IC50s, strong synergism was observed at 1:1 ratio of BPRO and EPRO in NSCLC cell lines with diverse mutation. Significant inhibition of cell growth with higher cellular apoptosis was observed in 2D and 3D-based cell assays in nanomolar concentrations. EGFR activation assay revealed 47.60 % EGFR non-expressing cells confirming EGFR-degrading potential of EPRO. A lung cancer specific nanoliposomal formulation of EGFR and BRD4-degrading PROTACs (EPRO and BPRO) was prepared and characetrized. Successful encapsulation of the two highly lipophilic molecules was achieved in EGFR-targeting nanoliposomal carriers (T-BEPRO) using a modified hydration technique. T-BEPRO revealed a particle size of 109.22 ± 0.266 nm with enhanced cellular uptake and activity. Remarkably, parenterally delivered T-BEPRO in tumor-bearing mice showed a substantially higher % tumor growth inhibition (TGI) of 77.6 % with long-lasting tumor inhibitory potential as opposed to individual drugs.

Hydrazide-Based Class I Selective HDAC Inhibitors Completely Reverse Chemoresistance Synergistically in Platinum-Resistant Solid Cancer Cells.

In this work, we have synthesized a set of peptoid-based histone deacetylase inhibitors (HDACi) with a substituted hydrazide moiety as zinc-binding group. Subsequently, all compounds were evaluated in biochemical HDAC inhibition assays and for their antiproliferative activity against native and cisplatin-resistant cancer cell lines. The hydrazide derivatives with a propyl or butyl substituent (compounds 5 and 6) emerged as the most potent class I HDAC selective inhibitors (HDAC1-3). Further, compounds 5 and 6 outperformed entinostat in cytotoxicity assays and were able to reverse chemoresistance in cisplatin-resistant A2780 (ovarian) and Cal27 (head-neck) cancer cell lines. Moreover, the hydrazide derivatives 5 and 6 showed strong synergism with cisplatin (combination indices <0.2), again outperforming entinostat, and increased DNA damage, p21, and pro-apoptotic BIM expression, leading to caspase-mediated apoptosis and cell death. Thus, compounds 5 and 6 represent promising lead structures for developing new HDACi capable of reversing chemoresistance in cisplatin resistant cancer cells.

Assessing the cytotoxicity of phenolic and terpene fractions extracted from Iraqi Prunus arabicaagainst AMJ13 and SK-GT-4 human cancer cell lines.

Background: Breast and esophageal cancer are the most aggressive and prominent causes of death worldwide. In addition, these cancers showed resistance to current chemotherapy regimens with limited success rates and fatal outcomes. Recently many studies reported the significant cytotoxic effects of phenolic and terpene fractions extracted from various Prunus species against different cancer cell lines. As a result, it has a good chance to be tested as a complement or replacement for standard chemotherapies. Methods: The study aimed to evaluate the cytotoxicity of phenolic and terpene fractions extracted from Iraqi Prunus arabica on breast (AMJ13) and esophageal (SK-GT-4) cancer cell lines by using the MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide). Analysis using the Chou-Talalay method was performed to assess the synergistic effect between the extracted fractions and chemotherapeutic agent (docetaxel). Moreover, high-performance liquid chromatography (HPLC) analysis was conducted for the quantitative determination of different bioactive molecules of both phenolic and terpene fractions in the extract. Results: According to the findings, the treatment modalities significantly decreased cancer cell viability of AMJ13 and SK-GT-4 and had insignificant cytotoxicity on the normal cells (normal human fibroblast cell line) (all less than 50% cytotoxicity). Analysis with Chou-Talalay showed a strong synergism with docetaxel on both cancer cell lines (higher cytotoxicity even in low concentrations) and failed to induce cytotoxicity on the normal cells. Important flavonoid glycosides and terpenoids were detected by HPLC, in particularly, ferulic acid, catechin, chlorogenic acid, β-sitosterol, and campesterol. Conclusions: In conclusion, the extracted fractions selectively inhibited the proliferation of both cancer cell lines and showed minimal cytotoxicity on normal cells. These fractions could be naturally derived drugs for treating breast and esophageal cancers.

Rational design of Au-Bi bimetallic nanozyme for NIR-II laser mediated multifunctional combined tumor therapy

To maximize the therapeutic effects and minimize the adverse effects of synergistic tumor therapies, a multifunctional nanozyme Au-Bi/ZIF-8@DOX@HA (ABZ@DOX@HA) was designed and synthesized through the Au and Bi bimetallic doping of ZIF-8, loading of the DOX, and modifying with hyaluronic acid (HA). The ABZ@DOX@HA nanoparticles (NPs) could simulate the enzymatic activities of glucose oxidase (GOx) and peroxidase (POD). Upon irradiated by near-infrared region (NIR-II) laser, the strong synergism of the photothermal abilities of the loaded Au and Bi nanodots accelerated the collapse of the ABZ structure at the tumor site considerably and released Au, Bi nanodots and DOX. The results in vitro and in vivo proved that ABZ@DOX@HA nanozyme could effectively exert the combined tumor therapy of starvation treatment, photothermal therapy (PTT), chemodynamic therapy (CDT) and chemotherapy. The current research provides a new strategy to address the inherent challenges of easy clearance and short blood circulation of small-sized NPs during the treatment of tumors with nanomedicine, as well as the aggregation and oxidation of inorganic nanodots.

Synergistic Effects of Artesunate in Combination with Amphotericin B and Miltefosine against Leishmania infantum: Potential for Dose Reduction and Enhanced Therapeutic Strategies.

Leishmaniasis is a tropical infectious disease caused by Leishmania parasites. The disease can be spread by the bite of an infected sand fly. Currently, five chemotherapeutic drugs are available in leishmaniasis treatment. However, these drugs exhibit toxicity and serious adverse effects on infected individuals, necessitating alternative treatment strategies. One such strategy involves using combinations of existing antileishmanial drugs. In this study, we evaluated the interaction between artesunate (AS) and three antileishmanial drugs-amphotericin B (AmB), miltefosine (MF), and paromomycin (PM) against Leishmania infantum. This evaluation marks the first time such an assessment has been conducted. The Chou-Talalay combination index method was employed to analyze the drug interaction. The findings revealed that the interaction between AS and AmB ranged from antagonistic to synergistic, while the interaction between AS and MF showed moderate to strong synergism. In contrast, the interaction between AS and PM resulted in an antagonistic interaction, which differs from the combinations with AmB or MF. This study provides valuable insights for developing novel drug regimens for leishmaniasis treatment, emphasizing the potential of AS and its combination with existing antileishmanial drugs. Further research is necessary to optimize drug combinations and minimize adverse effects, leading to more effective therapeutic outcomes.

Optimal formulation of bitter gourd and black galingale extract: Evaluation of effects on inflammation and oxidative stress-related genes

Reactive nitrogen and oxygen species (RNOS) are defined as highly reactive molecules that can be generated endogenously and exogenously. The overproduction of RNOS leads to inflammation, neurodegenerative diseases, diabetes, obesity, and cancer. Natural antioxidants derived from plants are molecules that can neutralize those RNOS. In this study, to increase the antioxidant capacity in neutralizing RNOS, an in vitro evaluation of a binary combination of bitter gourd (Momordica charantia L.) and black galingale (Kaempferia parviflora) extracts as potential natural antioxidant sources at different ratios was conducted. A combination at the ratio of 1:6 showed the highest total phenolic content (TPC) (336.48 ± 0.04 mg GAE/100g DW), while the ratios 1:6 (1511.73 ± 0.20 mg QE/100g DW) and 1:8 (1398.91 ± 0.28 mg QE/100g DW) exhibited the highest total flavonoid content (TFC) compared to the bitter gourd extract and other mixed ratios. The highest scavenging capacities in the DPPH, ABTS, and FRAP assays were detected at the ratios of 1:3 (IC50 9.1 ± 0.07 mg/mL; moderate synergism), 1:6 (IC50 1.07 ± 0.07 mg/mL; synergism), and 1:4 (IC50 2.44 ± 0.04 mg/mL; strong synergism), respectively. Based on the overall cumulative ability to scavenge free radicals with synergistic or additive antioxidant interactions and the presence of functional groups analyzed using FT-IR, the 1:6 ratio was determined as the best combination ratio to neutralize RNOS where 15 proposed beneficial bioactive compounds were detected. Additionally, in vivo studies showed that the increase of CAT mRNA expression and the suppression of NF-kB and CPT-1 mRNA expression at 250 mg/kg BW of dosage confirms that this methanolic mixed extract potentially can effectively act as both an anti-inflammation and antioxidant agent. This study demonstrates that a specific binary combination ratio of plant extracts can synergistically and additively increase their antioxidant capacity, even when utilized in small quantities.

Anti-fungal efficacy of Carvacrol against Candida glabrata clinical isolates of vulvovaginal candidiasis

Fungal infections affect over 1 billion people worldwide each year, including superficial infections like athlete's foot and more severe systemic infections. Fungal diseases are responsible for an estimated 1.5 million deaths annually, a figure comparable to or exceeding the mortality rate of diseases like malaria or tuberculosis. The limited arsenal of available antifungal drugs, coupled with the emergence of drug-resistant fungal strains, has increased this concern. Therefore, there is a significant need to explore alternative therapeutics to overcome fungal pathogens. Carvacrol, phenolic monoterpenoids, is present in essential oils of many plants and is known for its biological and pharmacological properties. In the present study, the efficacy of carvacrol was investigated against four Candida glabrata strains isolated from patients of vulvovaginal candidiasis, which have shown varying extents of susceptibility against fluconazole. Carvacrol, a phytoactive monoterpene phenol, has shown a minimum inhibitory concentration (MIC50) ranging from 75 to 125 µg/mL and minimum fungicidal concentration of 150 and 175 µg/mL for all clinical isolates, including wild-type strains. Carvacrol, in combination with fluconazole, has shown a strong synergism against wild type C. glabrata with a FIC index value of 0.156. Preliminary mechanistic investigations unveiled that exposure to carvacrol significantly reduced cell surface hydrophobicity and ergosterol content in all strains. In conclusion, carvacrol holds promising potential as an effective antifungal agent against C. glabrata, which is categorized as high priority in the first fungal pathogen priority list of the World Health Organisation released in 2022 for highlighting priority areas for action, including the development of effective therapeutic solution.

Functional genomics reveals an off-target dependency of drug synergy in gastric cancer therapy

BackgroundIntegrating molecular-targeted agents into combination chemotherapy is transformative for enhancing treatment outcomes in cancer. However, realizing the full potential of this approach requires a clear comprehension of the genetic dependencies underlying drug synergy. While the interactions between conventional chemotherapeutics are well-explored, the interplay of molecular-targeted agents with conventional chemotherapeutics remains a frontier in cancer treatment. Hence, we leveraged a powerful functional genomics approach to decode genomic dependencies that drive synergy in molecular-targeted agent/chemotherapeutic combinations in gastric adenocarcinoma, addressing a critical need in gastric cancer therapy.MethodsWe screened pharmacological interactions between fifteen molecular-targeted agent/conventional chemotherapeutic pairs in gastric adenocarcinoma cells, and examined the genome-scale genetic dependencies of synergy integrating genome-wide CRISPR screening with the shRNA-based signature assay. We validated the synergy in cell death using fluorescence-based and lysis-dependent inference of cell death kinetics assay, and validated the genetic dependencies by single-gene knockout experiments.ResultsOur combination screen identified SN-38/erlotinib as the drug pair with the strongest synergism. Functional genomics assays unveiled a genetic dependency signature of SN-38/erlotinib identical to SN-38. Remarkably, the enhanced cell death with improved kinetics induced by SN-38/erlotinib was attributed to erlotinib’s off-target effect, inhibiting ABCG2, rather than its on-target effect on EGFR.ConclusionIn the era of precision medicine, where emphasis on primary drug targets prevails, our research challenges this paradigm by showcasing a robust synergy underpinned by an off-target dependency. Further dissection of the intricate genetic dependencies that underlie synergy can pave the way to developing more effective combination strategies in gastric cancer therapy.

Synergistic anticancer effects of omega-3 fatty acids (EPA/DHA) and anticancer drug Doxorubicin against human lung adenocarcinoma

Single agent therapy in the treatment of cancer did not provide satisfactory clinical outcome in a long term due to drug resistance, cumulative toxicity and suboptimal efficacy. Therefore, combination therapies are apt to improve the quality of life in patients. In the present study, combinatorial effect of EPA and/or DHA with doxorubicin (Dox) was evaluated for their potential synergistic effect on human lung adenocarcinoma (A549) cells. The respective IC50 values obtained for EPA, DHA and Dox were 170, 140 and 2.5 μmol/L and thus EPA and DHA were separately combined with Dox at constant ratio of 68:1 and 56:1 respectively, depending on the ratio of their IC50 values. Different combinations of drugs revealed dose-dependent inhibition of cell viability and IC50 values were reduced to nearly 3-fold (58.75 μmol/L) and 4.5-fold (31.5 μmol/L) in EPA-Dox and DHA-Dox combination respectively. Combination index (CI) revealed strong synergism (CI < 0.5) in DHA-Dox (0.49) and moderate synergism (CI > 0.5) in EPA-Dox (0.79) at 0.5 Fa (Fraction affected), which is further confirmed by dose reduction index and isobologram. The mechanisms underlying synergistic interactions involve intracellular ROS generation and MMP depolarisation which causes changes in the cell morphology viz. Nuclear fragmentation/condensation, membrane blebbing and early/late apoptotic cells. Moreover, cell cycle arrest was observed at the S and G2/M phase, with more prominent effect in G2/M phase. No toxicity was imposed on non-cancerous HEK-293 T cells. These findings suggest that EPA-Dox and DHA-Dox could be explored further for animal studies and clinical trials for lung adenocarcinoma treatment.

Antimicrobial activity of chitosan and combination with antibiotics against mastitis-causing pathogens

Bovine mastitis (BM), primarily caused by bacterial pathogens infecting mammary glands, stands as the most prevalent disease in dairy cattle. Traditionally, antibiotics have been the primary choice of treatment, yet their overuse has led to widespread resistance and the presence of antibiotic residues in dairy products. Today, chitosan has emerged as a promising alternative in dairy farming. In this study, we systematically screened and assessed the antibacterial efficacy of five chitosan preparations of different viscosities and components. Additionally, we explored the synergistic antimicrobial potential of the most potent chitosan sample in combination with commonly employed antibiotics, including ampicillin, amoxicillin, oxacillin, and levofloxacin against four prevalent BM-causing pathogens: Staphylococcus epidermidis, Streptococcus agalactiae, Streptococcus uberis and Pseudomonas sp. Agar well diffusion, micro-dilution, and checkerboard techniques were applied to assess the antimicrobial activity and interaction effect. Results indicated that, at a concentration of 1%, low and medium viscosity samples (samples 1, 2, 3) exhibited relatively low activity, compared to very low viscosity ones (samples 4, 5). Notably, sample 5, a combination of chitosan sample 1 with orange and grapefruit essential oils, demonstrated the most potent antibacterial activity with a minimal inhibitory concentration (MIC) of 19.53 mg/L against S. agalactiae, S. uberis and S. epidermidis and 78.13 mg/L against Pseudomonas sp.. Furthermore, the combination of this chitosan sample and antibiotics exhibited some synergistic interactions against BM-causing pathogens, as indicated by the fractional inhibitory concentration (FIC) values ranging from ≥ 0.5 to ≤ 1. While these effects were notable, they did not reach the threshold for strong synergism (FIC &lt; 0.5). In summary, our study highlighted the high antibacterial activity of low viscosity chitosan, particularly in combination with essential oils. Although there were observed synergistic effects with antibiotics against BM-causing pathogens, the strength of these interactions was not robust enough to conclusively categorize them as strongly synergistic. Chitosan, however, emerges as a promising agent in the ongoing exploration of alternatives to antibiotics in the management of BM in dairy farming.

Broad synergistic antiviral efficacy between a novel elite controller-derived dipeptide and antiretrovirals against drug-resistant HIV-1.

The naturally occurring dipeptide Tryptophylglycine (WG) is enhanced in human immunodeficiency virus (HIV-1) infected Elite Controllers (EC). We have shown that this dipeptide has an anti-HIV-1 effect and evaluated now its synergistic antiretroviral activity, in combination with current antiretrovirals against multi-drug resistant HIV-1 isolates. Drug selectivity assay with WG-am and ARVs agains HIV-1 resistant isolates were carried out. Subsequently, two methods, Chou-Talalay's Combination Index (CI) and ZIP synergy score (SS), were used to quantify the synergism. WG-am had a moderate/strong synergism with the four tested antiretrovirals: raltegravir, tenofovir, efavirenz, darunavir. WG-am:TDF had strong synergism at ED50, ED75, ED90 (CI: <0.2) in isolates resistant to protease inhibitors or integrase strand inhibitors (INSTI), and a slightly less synergism in isolates resistant to non-nucleoside or nucleotide reverse transcriptase inhibitors. WG-am combined with each of the four drugs inhibited all drug-resistant isolates with over 95% reduction at maximum concentration tested. The highest selectivity indexes (CC50/ED50) were in INSTI-resistant isolates. Our data suggest that WG, identified as occurring and enhanced in Elite Controllers has a potential to become a future treatment option in patients with HIV-1 strains resistant to any of the four major categories of anti-HIV-1 compounds.

Enhanced transfer hydrogenation of 2-heptanone to 2-heptanol over synergistic Co/ZnO catalysts

2-Heptanol is an important secondary alcohol, applied in food, pharmaceutical and plastic industries. However, current industrial synthetic methods require energy intensive hydrogenation of 2-heptanone using homogeneous noble catalysts in alkaline medium. In this work, we presented a systematic study on enhanced transfer hydrogenation of 2-heptanone to 2-heptanol using non-noble Co/ZnO catalysts without alkaline promoters. The strong synergism of Co and ZnO contributes to formation of amorphous metallic Co species and electronically reconfigured CoO species, leading to a 20-fold activity enhancement at 180 °C and a TOF of 89.4 h−1 over Co0.09/ZnO-500 catalysts. Structure-dependency studies further revealed that, the lattice strain between Co and ZnO induced the generation of multi-phased Co/CoO/Co:ZnO interfaces, facilitating tandem H2 generation, spillover and activation of C=O bond (of 2-heptanone molecule), respectively. The insights of molecular catalysis on interface of amorphous Co/ZnO for enhanced C=O bond activation, provide fundamental understanding for transfer hydrogenation of various other ketones into valuable alcohols.

Synergistic corrosion inhibition of rubber seed extract with KI on cold rolled steel in sulfuric acid solution

BackgroundPlant-based inhibitor has emerged an important topic owing to the combing merits of eco-friendly, low cost, rich natural resources as well as easy preparation. Rubber seed is the agricultural waste, and is mainly composed of oil and fatty acids those contain many polar oxygen atoms, and so it can be extracted to prepare the potential corrosion inhibitor. MethodsRubber seed extract (RSE) is prepared using reflux extraction method with the extracting solvent of ethanol aqueous solution. The synergistic inhibition effect of RSE and potassium iodide (KI) on the corrosion of cold rolled steel (CRS) in 0.50 M H2SO4 solution is firstly studied by weight loss, potentiodynamic polarization (PDP) curves and electrochemical impedance spectroscopy (EIS) methods. CRS surface was thoroughly characterized using metallographic microscope, scanning electron microscope (SEM), atomic force microscope (AFM), X-ray photoelectron spectroscopy (XPS) and contact angle measurements. FTIR, UV–vis and liquid chromatography mass spectrometry (LC-MS) compositional analysis of RSE were used to analyze the efficient components in RSE. Significant findingsThe results indicate that individual RSE or KI cannot function as a successful inhibitor to prevent CRS corrosion, but the mixture of RSE/KI performs better at inhibiting, with a maximal inhibition efficacy of up to 96.4% of 200 mg L−1 RSE/100 mg L−1 KI. The synergistic coefficient of the mixture is larger than 1, indicating that a true synergism. RSE/KI can drastically retard both cathodic and anodic processes, and interfacial capacitance falls as the charge transfer resistance of CRS/solution rises. It exhibits remarkable inhibition properties when the capacitor arc grows. There is only a small corrosion response in the inhibited media, as evidenced by the variations in electrical conductivity and ultraviolet spectrum before and after immersion. The hydrophilicity and surface roughness of CRS surface is dropped as a result of the introduction of RSE/KI. A series of metallurgical microscopy, AFM, SEM or contact angle tests indicate a synergistic inhibition effect is more pronounced for RSE/KI. XPS results clearly demonstrate that elements including Fe, S, N, and I are bonded to the surface of CRS. The primary ingredients of oleic acid, polyphenols, phenylpropanoids can also produce a strong synergism with KI.

Chlorin Conjugates in Photodynamic Chemotherapy for Triple-Negative Breast Cancer.

Breast cancer (BC) is the most common type of cancer in women and the number of new cases in the US is still increasing each year. Triple-negative breast cancer (TNBC), which comprises 15-20% of all breast cancer, is a heterogeneous disease and is considered the most aggressive type of breast cancer due to the lack of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expressions for treatments. Traditional chemotherapy is the standard protocol for the treatment of TNBC. Toxicity and multidrug resistance are major drawbacks to chemotherapy. The lack of molecular targets and poor prognosis for TNBC prompts an urgent need to discover novel therapeutic strategies to improve clinical outcomes and quality of life for patients. Photodynamic therapy (PDT) or light treatment is a binary anti-cancer procedure that uses a photosensitizer (PS) that, upon light activation, produces cytotoxic oxygen species, destroying tumor cells. PDT is minimally invasive and can be repeated a few times without accumulating significant toxicity in the surrounding tissues. The primary goal of this study was to investigate in vitro photodynamic chemotherapy as a ternary combination therapy using our synthesized photosensitizers (chlorin-vitamin conjugates and their corresponding indium complexes) co-treated with known chemotherapeutic agents (taxol, doxorubicin, cisplatin, fluorouracil, or methotrexate) in the presence of light and determine the optimum conditions as a pre-clinical study of an enhanced tumoricidal effect against TNBC. Our results indicated that the best combination for an effective chemophotodynamic effect involves a ternary treatment of the indium complex of the chlorin-lipoic acid conjugate (InCLA) co-treated with taxol, which exhibited strong synergism at the nanomolar concentration when combined in the presence of visible light irradiation. Other ternary combinations containing taxol with a synergistic anti-tumor effect against TNBC include chlorin-pantothenic acid (CPA) and chlorin-biotin (CBTN) conjugates. Several other ternary combinations containing InCLA, CBTN, and CPA with either cisplatin, fluorouracil, or methotrexate were identified to generate a synergistic or additive effect. The light dosage remained constant, but the dosages of photosensitizers and chemotherapy drugs were varied to obtain the lowest possible concentration for the desired effect. The synergistic, additive or antagonistic effects of the drug combinations were determined based on the Chou-Talalay method, with InCLA-taxol having the lowest combination index (CI) of 0.25. Fluorescence and transmission electron microscopy (TEM) images provided evidence of apoptosis as the preferred mode of cell death. Our study demonstrated the combination of PDT and chemotherapy as a potential treatment option for TNBC patients.

Antibiotic Augmentation of Thermal Eradication of Staphylococcus epidermidis Biofilm Infections.

Staphylococcus epidermidis is a major contributor to bacterial infections on medical implants, currently treated by surgical removal of the device and the surrounding infected tissue at considerable morbidity and expense. In situ hyperthermia is being investigated as a non-invasive means of mitigating these bacterial biofilm infections, but minimizing damage to the surrounding tissue requires augmenting the thermal shock with other approaches such as antibiotics and discerning the minimum shock required to eliminate the biofilm. S. epidermidis biofilms were systematically shocked at a variety of temperatures (50-80 °C) and durations (1-10 min) to characterize their thermal susceptibility and compare it to other common nosocomial pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa. Biofilms were also exposed to three classes of antibiotics (ciprofloxacin, tobramycin and erythromycin) separately at concentrations ranging from 0 to 128 μg mL-1 to evaluate their impact on the efficacy of thermal shock and the subsequent potential regrowth of the biofilm. S. epidermidis biofilms were shown to be more thermally susceptible to hyperthermia than other common bacterial pathogens. All three antibiotics substantially decreased the duration and/or temperature needed to eliminate the biofilms, though this augmentation did not meet the criteria of synergism immediately following thermal shock. Subsequent reincubation, however, revealed strong synergism on a longer timescale.

Abstract 7153: PHI-101 synergizes with chemotherapy and venetoclax in preclinical models of acute myeloid leukemia

Abstract FLT3 mutations are one of the most frequent genetic alterations in AML, resulting in poor prognosis. Although the development of FLT3 tyrosine kinase inhibitors (TKIs) provided new treatment options, patients with FLT3-mutant AML still suffer from high relapse rates when treated with FLT3 TKI alone. Accumulating evidence supports a combination treatment strategy to lower the recurrence rate. We have previously shown that PHI-101 demonstrated potent activity in various FLT3 mutant AML cell lines when used alone. Here we explore the use of PHI-101 in combination with other therapeutics in preclinical models of AML. We investigated the preclinical efficacy of the combination of PHI-101 with FDA-approved AML therapeutic agents in FLT3 mutant (MV4-11, Molm13, Molm14) and wild-type (THP-1) AML cell lines. Cell growth inhibition was assessed by CCK-8 and/or CellTiter-Glo assays. The Chou-Talalay method was used to calculate the combination index (CI) to test for synergy. Western blotting analysis was performed to explore the potential mechanisms underlying the combination effects. AML xenograft mouse models were used to assess the in vivo efficacy of PHI-101 combination therapy. Statistical comparisons included unpaired t-tests or one-way ANOVA. Combination treatment of PHI-101 with venetoclax or azacytidine showed strong synergistic effects on growth inhibition against both FLT3-ITD mutant (CI&amp;lt;0.3) and wild-type AML cells (CI &amp;lt;0.6). It is noteworthy that the FLT3-ITD homozygous cell line (MV4-11) showed especially strong synergism (CI &amp;lt;0.1). In the xenograft mouse model experiments, 14-day treatment with either PHI-101 alone or in combination with venetoclax or azacytidine showed significant suppression of tumor growth compared to venetoclax or azacytidine alone (in average tumor growth inhibition (TGI) 88.3% vs 8.6%)(p&amp;lt;0.05). In particular, PHI-101 3 mg/kg/day (mpk) with venetoclax 80mpk resulted in a stronger anti-tumor effect (TGI 94.2%) compared to PHI-101 (TGI 87.4%) or venetoclax alone (-9.6% of TGI) (p&amp;lt;0.05) even 21 days after stopping the treatment. In addition, PHI-101 treatment showed a survival advantage in AML animal models, and the combination with venetoclax led to significantly longer survival compared to the control groups. Our preclinical data showed that the combination of PHI-101 with venetoclax or azacytidine resulted in a highly effective anti-leukemic response against AML cells by inducing DNA damage, BCL-2 inhibition, and MCL-1 degradation. Additionally, treatment of mice with low-dose PHI-101 inhibited tumor regrowth and further potentiated venetoclax or azacytidine response in vivo. Our results suggest that the combination of PHI-101 and venetoclax or azacitidine may have the advantage of improving clinical responses in FLT3-ITD AML patients and offers a potential therapeutic option to treat patients with newly diagnosed or relapsed/refractory FLT3-mutant AML. Citation Format: Hee-sun Hwang, Gi-Jun Sung, Kyung-Ah Kim, Ky-Youb Nam, Kyu-Tae Kim, June H. Han, Jeong-Hyeok Yoon, Bao Nguyen, Li Li, Tessa Seale, Donald Small. PHI-101 synergizes with chemotherapy and venetoclax in preclinical models of acute myeloid leukemia [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 7153.

Abstract 534: Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2

Abstract Background: Copanlisib is a PI3K inhibitor with selectivity against PI3Kδ and PI3Kα and is approved for treating patients with follicular lymphoma and marginal zone lymphoma (MZL). Preclinical studies have demonstrated strong synergism of copanlisib and BCL2 inhibitor venetoclax in different lymphoma models, and the combination is under clinical evaluation. Understanding the resistance mechanisms could help the design of improved therapies. Hence, we generated MZL cell lines resistant to the copanlisib/venetoclax combination. Methods: All cells were tested for cell identity and Mycoplasma status. Resistance was confirmed stable after 3-weeks of drug-free culture. Multi-drug resistance phenotype was ruled out by confirming sensitivity to vincristine. Targeted agents already in the clinics were combined with copanlisib/venetoclax. Cells underwent transcriptome and methylation profiling, targeted DNA sequencing, and immunophenotypic and immunoblotting analyses. Results: We developed SSK41-derived models with resistance to copanlisib (n.=1) or venetoclax (n.=1) as single agents and to copanlisib/venetoclax (n.=2) by long IC50 or by upfront IC90 exposure. The single-drug-resistant cells showed only partial resistance to copanlisib/venetoclax. In contrast, the cells that developed exposing cells to copanlisib/venetoclax IC90 exhibited a more substantial decrease in sensitivity to the combination. Copanlisib/venetoclax induced apoptosis and cell cycle arrest in PAR but not in resistant cells. Sensitivity to other PI3K, BCL2, and BTK inhibitors was decreased in all resistant lines. The response to MCL1-i, mTOR-i, and epigenetic agents (decitabine, HDAC, BET, and EZH2 inhibitors) was maintained. Triple combinations of copanlisib/venetoclax with the AURKA inhibitor alisertib, PLK inhibitor rigosertib, or the multikinase inhibitor amcasertib were active in both parental and resistant cells. No acquired mutations were observed via targeted DNA sequencing designed to cover various coding genomic regions known to be recurrently mutated in mature B-cell neoplasms, including BCL2, nor via RNA-Seq for PIK3CA/B/G/D genes. Integration of RNA and methylation profiles showed deregulation of genes involved in type-I IFN, chemokines (CCL25, CXCL13), apoptosis regulation, and JAK-STAT signaling. Resistant cells upregulated CD19 expression compared to parental cells. Immunoblotting showed the inability of copanlisib/venetoclax to block BCR signaling in resistant cells. Conclusions: We created four novel MZL-derived models of secondary resistance to PI3K and BCL2 inhibitors, which will help clarify possible modalities to overcome resistance. Novel potential targets were already identified, such as AURKA or PLK, to be further exploited. Citation Format: Alberto J. Arribas, Luciano Cascione, Eleonora Cannas, Helen Bellerjeau, Federica Fuzio, Aleix Noguera, Andrea Rinaldi, Manel Esteller, Emanuele Zucca, Andrea Alimonti, Davide Rossi, Anastasios Stathis, Francesco Bertoni. Novel marginal zone lymphoma models of resistance to the dual inhibition of PI3K and BCL2 [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 534.

The Rhizosphere Functional Microbial Community: A Key Driver of Phosphorus Utilization Efficiency in Karst Forest Plants

Microorganisms play a pivotal role in transforming and making phosphorus (P) available in soil through various mechanisms. However, their specific contributions to alleviating P limitation and enhancing P utilization efficiency in plants within the context of a P-deficient karst ecosystem remains unclear. In this study, eco-stoichiometric methods were employed to evaluate the P utilization efficiency of plants grown in the surveyed karst forest located in Guizhou Province, China. Metagenomic sequencing was utilized to further explore the functional genes and microorganisms involved in soil P cycling. The N:P ratio for 18 out of the 20 surveyed plants exceeded 16, indicating widespread P limitation in karst plants. Among them, plants with high P utilization efficiencies (Nandina domestica Thunb.; Mahonia bodinieri Gagnep.; Pyracantha fortuneana (Maxim.) Li) exhibited higher relative abundances of genes involved in soil P cycling compared to plants with low P utilization efficiencies (Tirpitzia sinensis (Hemsl.) Hallier f.; Albizia kalkora (Roxb.) Prain; Morella rubra Lour.), indicating greater potentials within their rhizosphere microbiomes for soil P transformation. The relative abundance of these functional genes had a significant and positive effect on plant P utilization efficiencies. Structural equation modeling further indicated that microbial P cycling gene abundance directly drove the increase in plant P utilization efficiencies. Specifically, genes involved in soil organic P mineralization (G6PD, suhB, phoD, ppx) and the P uptake and transform system (pstS, pstA, pstB, pstC) contributed to the enhancement of plant P utilization efficiencies. Soil microbial communities involved in P cycling were predominately attributed to Proteobacteria (45.16%–60.02%), Actinobacteria (9.45%–25.23%), and Acidobacteria (5.90%–9.85%), although their contributions varied among different plants. The rhizosphere functional microbial community can thus alleviate P limitation in karst plants, thereby enhancing plant P utilization efficiencies. This study investigated the strong synergism between karst plants and rhizosphere microorganisms and their associated underlying mechanisms from genetic and microbial perspectives.

Time-dependent hormesis transfer from five high-frequency personal care product components to mixtures

There is potential for personal care products (PCPs) components and mixtures to induce hormesis. How hormesis is related to time and transmitted from components to mixtures are not clear. In this paper, we conducted determination of components in 16 PCP products and then ran frequent itemset mining on the component data. Five high-frequency components (HFCs), betaine (BET), 1,3-butanediol (BUT), ethylenediaminetetraacetic acid disodium salt (EDTA), glycerol (GLO), and phenoxyethanol (POE), and 14 mixtures were identified. For each mixture system, one mixture ray with the actual mixture ratios in the products was selected. Time-dependent microplate toxicity analysis was used to test the luminescence inhibition toxicity of five HFCs and 14 mixture rays to Vibrio qinghaiensis sp.-Q67 at 12 concentration gradients and eight exposure times. It is showed that BET, EDTA, POE, and 13 mixture rays containing at least one J-type component showed time-dependent hormesis. Characteristic parameters used to describe hormesis revealed that the absolute value of the maximum stimulatory effect (|Emin|) generally increased with time. Notably, mixtures composed of POE and S-type components showed greater |Emin| than POE alone at the same time. Importantly, the maximum stimulatory effective concentration, NOEC/the zero effective concentration point, and EC50 remained relatively stable. Nine hormesis transmission phenomena were observed in different mixture rays. While all mixtures primarily exhibited additive action, varying degrees of synergism and antagonism were noted in binary mixtures, with no strong synergism or antagonism observed in ternary and quaternary mixtures. These findings offer valuable insights for the screening of HFCs and their mixtures, as well as the study of hormesis transmission in personal care products.