Combination Index Values Research Articles

Hesperitin Synergistically Promotes the Senescence Induction of Pentagamavunone-1 in Luminal Breast Cancer Cells, T47D

Pentagamavunone-1 (PGV-1), a curcumin analog, is a promising anticancer candidate for several cancers that have been proven in vitro and in vivo. However, the efficacy of PGV-1 against breast cancer is subject to improvement to achieve a more suitable application. Here we propose hesperitin, a citrus flavonoid, to increase the anticancer potency of PGV-1 in luminal breast cancer cells. We use the T47D cell as the model to investigate the effect of co-administration of PGV-1 and hesperitin on cell cycle block, apoptosis modulation, and senescence phenomena. PGV-1 and hesperitin showed strong and weak cytotoxicity with an IC50 value of 2 µM and 100 µM, respectively. The co-treatment of PGV-1 and hesperitin resulted in strong synergistic effects with combination index (CI) value of ≤ 0.2. This combination caused apoptosis in correlation with cell cycle disruption in G2/M phase at 48 h. In particular, PGV-1 and hesperitin combination increased the incidence of cellular senescence significantly higher than the single treatment. Despite its senescence potentiation, hesperitin did not induce senescence in normal cells. Taken together, hesperitin may increase the anticancer potency of PGV-1 by modulating cell cycle arrest and apoptosis via the senescence mechanism.

Foretinib, a c-MET receptor tyrosine kinase inhibitor, tackles multidrug resistance in cancer cells by inhibiting ABCB1 and ABCG2 transporters

BackgroundABC transporter-mediated multidrug resistance (MDR) remains a major obstacle for cancer pharmacological treatment. Some tyrosine kinase inhibitors (TKIs) have been shown to reverse MDR. The present study was designed to evaluate for the first time whether foretinib, a multitargeted TKI, can circumvent ABCB1 and ABCG2-mediated MDR in treatment-resistant cancer models. MethodsAccumulation of fluorescent substrates of ABCB1 and ABCG2 in ABCB1-overexpressing MES-SA/DX5 and ABCG2-overexpressing MCF-7/MX and their parenteral cells was evaluated by flow cytometry. The growth inhibitory activity of single and combination therapy of foretinib and chemotherapeutic drugs on MDR cells was examined by MTT assay. Analysis of combined interaction effects was performed using CalcuSyn software. ResultsIt was firstly proved that foretinib increased the intracellular accumulation of rhodamine 123 and mitoxantrone in MES-SA/DX5 and MCF-7/MX cancer cells, with accumulation ratios of 12 and 2.2 at 25 μM concentration, respectively. However, it did not affect the accumulation of fluorescent substrates in the parental cells. Moreover, foretinib synergistically improved the cytotoxic effects of doxorubicin and mitoxantrone. The means of combination index (CI) values at fraction affected (Fa) values of 0.5, 0.75, and 0.9 were 0.64 ± 0.08 and 0.47 ± 0.09, in MES-SA/DX5 and MCF-7/MX cancer cells, respectively. In silico analysis also suggested that the drug-binding domain of ABCB1 and ABCG2 transporters could be considered as potential target for foretinib. ConclusionOverall, our results suggest that foretinib can target MDR-linked ABCB1 and ABCG2 transporters in clinical cancer therapy.

The interaction of radiotherapy and dual inhibition of BET and HAT/p300 in colorectal cancer.

146 Background: Colorectal cancer (CRC) is characterized by extensive DNA methylation at the promoters of tumor suppressor genes and enhanced activity of histone modifying proteins such as histone acetyltransferases (HATs) or histone readers such as bromodomain and extra-terminal domain (BET) proteins. BET inhibition has been associated with the reduced expression of DNA repair proteins and therefore can be used in combination with radiotherapy (RT) and chemotherapy. In the current study, we have evaluated the therapeutic potential of a clinical stage (phase 1) dual BET and CBP/p300 inhibitor, EP31670 (EP), in combination with RT. Methods: HCT116 CRC 2-D cultures were treated with EP 4 hours before RT. After pretreatment with EP (0, 0.1, 0.5, 1, 2, 5, and 10µM), cells were irradiated with various doses (0, 2, 4, 6, and 8Gy) of RT (300-kv x-rays). Crystal violet (CV) assay at 48 hours post RT was used to determine growth inhibition and viability between the various drug, RT, and combination groups. Synergy between treatments was evaluated by estimating the combination index (CI) values via mass-action law CompuSyn calculations using CV data. CI values of <1 from the growth inhibition assay suggested that EP synergized with RT for enhanced cytotoxicity in HCT116 CRC cells. Flow cytometry was used to assess DNA damage (γH2Ax), apoptosis and total death (Annexin V-PI assay), cell cycle analysis, and proliferation (EdU). Results: There was a significant dose-dependent increase in the residual DNA damage at 6 hours (8Gy + EP 0.5µM vs. 8Gy, p<0.0001) and 24 hours (8Gy + EP 0.5µM vs. 8Gy, p=0.002) for combination therapy over RT alone. Four-hour treatment with EP promoted G2 cell-cycle arrest (8Gy + EP 0.5µM vs. 8Gy, p<0.001) and significantly reduced the number of HCT cells in S-phase (8Gy + EP 0.5µM vs. 8Gy, p<0.001) and proliferation (EdU) of the HCT cells (8Gy + EP 0.5µM vs. 8Gy, p=0.0003) compared to RT alone. There was a significant increase in apoptotic cells the combination group (8Gy + EP 0.5µM vs. EP 0.5µM, p=0.008) when compared to the unirradiated cells. The combination group also showed significant increase in the total death compared to RT alone (8Gy + EP 0.5µM vs. 8Gy, p=0.03) at the G1-S checkpoint. Conclusions: EP31670 enhanced RT-induced growth arrest, reduced proliferation, and enhanced cell death in HCT116 CRC cells in synergy with the RT. Additional preclinical studies, in vivo studies, and eventual clinical studies can be conducted to further exploit this synergistic interaction.

POSS Engineering of Multifunctional Nanoplatforms for Chemo-Mild Photothermal Synergistic Therapy.

Chemo-mild photothermal synergistic therapy can effectively inhibit tumor growth under mild hyperthermia, minimizing damage to nearby healthy tissues and skin while ensuring therapeutic efficacy. In this paper, we develop a multifunctional study based on polyhedral oligomeric sesquisiloxane (POSS) that exhibits a synergistic therapeutic effect through mild photothermal and chemotherapy treatments (POSS-SQ-DOX). The nanoplatform utilizes SQ-N as a photothermal agent (PTA) for mild photothermal, while doxorubicin (DOX) serves as the chemotherapeutic drug for chemotherapy. By incorporating POSS into the nanoplatform, we successfully prevent the aggregation of SQ-N in aqueous solutions, thus maintaining its excellent photothermal properties both in vitro and in vivo. Furthermore, the introduction of polyethylene glycol (PEG) significantly enhances cell permeability, which contributes to the remarkable therapeutic effect of POSS-SQ-DOX NPs. Our studies on the photothermal properties of POSS-SQ-DOX NPs demonstrate their high photothermal conversion efficiency (62.3%) and stability, confirming their suitability for use in mild photothermal therapy. A combination index value (CI = 0.72) verified the presence of a synergistic effect between these two treatments, indicating that POSS-SQ-DOX NPs exhibited significantly higher cell mortality (74.7%) and tumor inhibition rate (72.7%) compared to single chemotherapy and mild photothermal therapy. This observation highlights the synergistic therapeutic potential of POSS-SQ-DOX NPs. Furthermore, in vitro and in vivo toxicity tests suggest that the absence of cytotoxicity and excellent biocompatibility of POSS-SQ-DOX NPs provide a guarantee for clinical applications. Therefore, utilizing near-infrared light-triggering POSS-SQ-DOX NPs can serve as chemo-mild photothermal PTA, while functionalized POSS-SQ-DOX NPs hold great promise as a novel nanoplatform that may drive significant advancements in the field of chemo-mild photothermal therapy.

Intravenous injectable metformin-Cu(II)-EGCG coordination polymer nanoparticles for electrothermally enhanced dual-drug synergistic tumor therapy.

Intravenous injectable metformin-Cu(II)-EGCG infinite coordination polymer nanoparticles (metformin-Cu(II)-EGCG ICP NPs) have been synthesized, and an efficient strategy for synergistic tumor therapy by utilizing these nanoparticles in conjunction with micro-electrothermal needles (MENs) was proposed. These nanoparticles display exceptional uniformity with a diameter of 117.5 ± 53.3 nm, exhibit an extraordinary drug loading capacity of 90% and allow for precise control over the drug ratio within the range of 1 : 1 to 1 : 20 while maintaining excellent thermal stability. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and X-ray diffraction were employed to determine their chemical structure and coordination state. The combination index (CI) value of the metformin-Cu(II)-EGCG ICP NPs was calculated to be 0.19, surpassing that of the two individual drugs and metformin mixed with EGCG (0.98). Importantly, upon intravenous injection, metformin in nanoparticles demonstrated exceptional stability in the bloodstream, while both drugs were rapidly released within the acidic tumor microenvironment. Animal experiments showcased an impressive tumor inhibition rate of 100% within a mere 20-day time frame after the synergistic therapy with a lower dosage (5.0 mg kg-1 of nanoparticles), coupled with a minimal tumor recurrence rate of only 18.75% over a 60-day observation period. These findings indicate the promising prospects of these nanoparticles in tumor treatment.

SKI-349, a Sphingosine Kinases 1/2 Inhibitor, Suppresses Cell Viability, Invasion, and AKT/mTOR Signaling Pathway, and Shows Synergistic Cytotoxic Effects with Sorafenib in Hepatocellular Carcinoma.

SKI-349 is a novel sphingosine kinases (SPHK) inhibitor with anti-tumor effects. This study aimed to assess the effect of SKI-349 on cell biological behaviors, downstream pathways, and its synergistic effect with sorafenib in hepatocellular carcinoma (HCC). HCC cell lines (Huh7 and Hep3B) were treated with SKI-349 at concentrations of 1, 2, 4, or 8 μM. Then, SPHK1/2 activity, cell viability, proliferation, apoptosis, invasion, and protein expressions of phosphorylated-protein kinase B (p-AKT), AKT, phosphorylated-mammalian target of rapamycin (p-mTOR) and mTOR were detected. Combination index values of SKI-349 (0, 1, 2, 4, or 8 μM) and sorafenib (0, 2.5, 5, 10, or 20 μM) were calculated. SKI-349 decreased the relative SPHK1 and SPHK2 activity compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Meanwhile, SKI-349 reduced cell viability, 5-ethynyl-2'-deoxyuridine (EdU) positive cells, and invasive cells, while it increased apoptotic cells compared to blank control in a dose-dependent manner in Huh7 and Hep3B cell lines. Based on the western blot assay, SKI-349 decreased the ratio of p-AKT to AKT and that of p-mTOR to mTOR compared with blank control in a dose-dependent manner in the Huh7 and Hep3B cell lines. Additionally, SKI-349 combined with sorafenib declined cell viability with concentration gradient effects compared to SKI-349 sole treatment, and they had synergistic cytotoxic effects in Huh7 and Hep3B cell lines. SKI-349 suppresses SPHK1 and SPHK2 activity, cell viability, invasion, and AKT/mTOR signaling pathway, as well as exhibits a synergistic cytotoxic effect with sorafenib in HCC.

SYNERGISTIC EFFECT OF CURCUMA XANTHORRHIZA AND PHYSALIS ANGULATA EXTRACTS AS ANTIOXIDANTS AGAINST DPPH RADICALS

Background: As a single extract, Curcuma xanthorrhiza and Physalis angulata are known to have antioxidant activity. However, the interaction effect of antioxidants in suppressing DPPH (2,2-difenil-1-pikrilhidrazil) radicals that arise in these two combinations of plant extracts is not yet known. In this study, the combination of these two plants was investigated for their antioxidant properties and synergistic effects. Objective: This study aims to determine the effect of the combination of Curcuma xanthorrhiza and Physalis angulata as antioxidants and the interactions they cause. Methods: The rhizomes of Curcuma xanthorrhiza and the herb Physalis angulata were extracted separately with ethanol. After evaporation, the crude xtract was tested individually to obtain the IC50 value. Based on the IC50 value of each extract, 9 combination variations were made and the combination index (CI) value was calculated to determine the resulting interaction effect. Result: The antioxidant activity of Curcuma xanthorrhiza IC50 extract is 51.015 ppm, higher than Physalis angulata IC50 extract which is 90.488 ppm. The CI value observed in the combination of extracts in all combinations had a CI value <1. Conclusion: This research shows that the combination of Curcuma xanthorrhiza rhizome extract and Physalis angulata herb has synergistic antioxidant properties.
 Keywords: Combination; Synergis; Antioxidant; Combination Index

Synergistic role of thymoquinone and 5-fluorouracil in U-251MG glioblastoma cell line

Abstract Objectives Glioblastoma is a fast-growing and aggressive brain tumor. Despite the current treatment methods, such as chemical and surgical operations, the prognosis is still poor. Therefore, combined therapeutic strategies are proposed to maximize therapeutic efficacy and reduce toxicity. Thymoquinone has been shown to have neuroprotective effects in addition to its anti-cancer effects on different types of cancer. 5-Fluorouracil, on the other hand, is a cytotoxic chemotherapy agent used to treat cancer. As a synergistic combinational approach, this study aimed to examine the antiproliferative effects and production of reactive oxygen species in a glioblastoma cell line. Methods We have tested thymoquinone and 5-fluorouracil alone and in their combination to observe cellular growth with MTT assay. The combinational effects of the agents were determined by the CompuSYN software program. Cell proliferation was assayed with crystal violet assay. Reactive oxygen species production was analyzed by 2′,7′-dichlorodihydrofluorescein diacetate in glioblastoma cells. Results Thymoquinone and 5-fluorouracil inhibited cell growth of glioblastoma cells with half maximal inhibitory concentrations (IC50) of 45.93 and 14.02 µM for 48 h, respectively. At synergistic combinational concentrations, the crystal violet assay demonstrated that there is a positive correlation between combination index values and cell proliferation. Also, an increment in the production of reactive oxygen species was observed upon combinational treatments. Conclusions Our results indicate that the combinational strategy of these two agents reduced cell viability and proliferation in glioblastoma cells and showed strong synergistic anticancer efficiency.

In Vitro Evaluation of Cytotoxic and Antitumor Activities of The Tamoxifen and Doxorubicin Combination on MCF-7 and BT-474 Breast Cancer Cell Lines

The combination therapy of breast cancer has preferred for the patients to minimize possible side effects, drug resistance, recurrence and toxic effects. In this study, we aim to investigate the cytotoxic and antitumor activities the tamoxifen and doxorubicin combination in breast cancer cell lines, MCF-7 and BT-474. Tamoxifen (Tam) and doxorubicin (Dox) and their combination with different concentrations (0.625–20 μM Tam; 0.0625–2 μM Dox and 5 μM Tam+ 0.5/1.0/1.5 μM Dox combination were applied to MCF-7 and BT-474 cells for 48 hours. Afterthat, their cytotoxic activities were analyzed with MTT assay. Bcl-2, Mapt and Mrp1 are genes that induce cell proliferation, inhibit apoptosis and play role in drug resistance in cancer cells. To evaluate the antitumor activities of these genes in combination treatment, mRNA levels were analyzed by quantitative PCR. According to the MTT assay, it was determined that IC50 values as 17.26 μM and 16.65 μM for tamoxifen on MCF-7 and BT-474 breast cancer cell lines. IC50 values of doxorubicin in MCF-7 and BT-474 cells were 1.65 μM and 1.57 μM, respectively. It was found that the application of the combination drugs (15 μM tamoxifen and 1.5 μM doxorubicin) in MCF-7 and BT-474 cells have the lowest combination index values as 1.09 and 1.26, respectively. Therefore, the combination of 15 μM tamoxifen and 1.5 μM doxorubicin was selected and applied to both breast cancer cell lines for gene expression analysis. It was found that while Mrp1 and Mapt genes expressions were significantly upregulated, Bcl-2 gene expression was downregulated in MCF-7 cells. However, Bcl-2, Mrp1 and Mapt genes expressions in BT-474 cells were not significantly regulated. Altogether, these findings suggest that the combination of these two drugs may have a potential antagonistic interaction according combination index values.

Disruption of DNA-PK-Mediated Cgas Retention on Damaged Chromatin Potentiates Doxorubicin-Induced Cgas/Sting-Dependent Anti-Multiple Myeloma Activity

Multiple myeloma (MM) is an incurable plasma cell malignancy characterized by genomic instability. MM with multidrug resistance or extensive extramedullary plasmacytoma are difficult to manage and often treated with DNA damage-inducing chemotherapies, including topoisomerase II inhibitor doxorubicin. However, the efficacy of this treatment is still limited and requires further improvement. Targeting DNA damage response (DDR) may offer an opportunity for effective treatment of MM. In particular, in combination with DNA damage-inducing agents, this strategy has shown to improve chemo-radiotherapies in part by cGAS-STING-mediated innate immune response, which could be directly activated by an elevated level of cytosolic DNA due to DNA repair inhibition. However, as cGAS is primarily sequestered by chromatin in the nucleus, it remains unclear how cGAS is released from chromatin and translocated to the cytoplasm upon DNA damage, leading to cGAS-STING activation. Here, after testing the potential of several DDR inhibitors in sensitizing MM cells (MMCs) to doxorubicin, we found that inhibition of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key component of classical non-homologous end joining (c-NHEJ) repair, using DNA-PKcs inhibitor NU7441, synergistically enhanced doxorubicin-induced anti-MM effect by activation of cGAS-STING pathway, the combination index values at different combination doses were all less than 0.85. Knocking out either cGAS or STING in ARP-1 cells significantly reduced not only the transcription of IFNB1 and multiple ISGs but also the proportion of NU7441/doxorubicin-induced apoptotic cells. Stably overexpressing STING in U266 and RPMI-8226 cells significant upregulated the IFNB1 and ISGs levels, and made MMCs more susceptible to NU7441/doxorubicin-induced inhibition of cell proliferation. Despite a strong type I interferon (IFN) response induced by activation of cGAS-STING, apoptosis of MMCs caused by activated cGAS-STING was mediated in part by IRF3-NOXA-BCL2 axis, which is independent of type I IFN response. In addition to inducing intrinsic apoptosis of MMCs, activation of cGAS-STING signaling by NU7441/doxorubicin in MMCs also induced M1 polarization of macrophages (Mφs), which increased M1 marker CD86 of Mφs but decreased M2 marker CD163 and CD206, and reduced the IL-10 concentration secreted by Mφs, thus suppressing tumor-protecting potential of M2-like Mφs and improving bortezomib-induced apoptosis of MMCs. Mechanistic studies showed that the activation of cGAS-STING signaling was not due to the defective c-NHEJ following continuous DNA-PKcs inhibition, because NU7441 did not enhance the damaged DNA accumulation after doxorubicin induction detected by γH2AX immunoblot analysis and alkaline comet assay. Besides, deletion of XRCC4 in ARP-1 cells to directly impair c-NHEJ did not enhance doxorubicin-induced activation of cGAS-STING signaling, further excluding a role for impaired c-NHEJ. Instead, we found that cGAS was mainly localized in the nucleus and chromatin-bound in MMCs under mock treatment, and doxorubicin induced DNA damage and mobilized inactive cGAS into cytoplasm. Both mass spectrometry data and immunoblotting of cGAS immunocoprecipitates showed that cytoplasmic cGAS was associated with DNA-PK complex and nucleosomal histone H2A-H2B heterodimer, and DNA-PKcs inhibition promoted cGAS release from cytoplasmic chromatin fragments and increased the amount of free cytosolic cGAS and DNA, activating cGAS-STING. Disruption of cytoplasmic cGAS-nucleosome binding by mutating cGAS R236 or R255 sites to E abolished the effect of NU7441 on doxorubicin-induced activation of cGAS-STING signaling. Analysis of Datasets MMRF CoMMpass and GSE2658 showed that MM patients with higher DNA-PKcs expression showed poorer prognosis than those with lower expression. Additionally, the DNA-PKcs expression was elevated with MM disease progression according to Datasets GSE5900, GSE13591 and MMRF CoMMpass, these indicated a possible involvement of DNA-PKcs in MM progression and provided a basis for targeting DNA-PKcs in MM treatment. Taken together, our study suggests that DNA-PKcs may help maintain the cGAS sequestration in damaged chromatin. Inhibition of DNA-PKcs may consequently disrupt this sequestration, inducing activation of cGAS-STING signaling and improving the efficacy of doxorubicin in treating MM.

Novel Pi3kδ Inhibitor Roginolisib Synergizes with the Bcl-2 Inhibitor Venetoclax in Hematological Malignancies

Roginolisib (IOA-244) is a first in class allosteric modulator and non-ATP competitive, selective PI3Kδ inhibitor currently in a Phase 1 clinical study in lymphoma and solid tumors. Initial structural and biochemical studies identified unique chemical structure, excellent selectivity, and excellent PK properties for roginolisib as compared to earlier PI3Kδ inhibitors, while the clinical trial has demonstrated a favorable safety profile. Consistent with prior PI3Kδ inhibitors, roginolisib inhibits the in vitro growth of lymphoma cells. In contrast to other PI3Kδ inhibitors, roginolisib activity is correlated with the expression levels of PIK3CD, suggesting specific and on target cancer cell-intrinsic effects (PMID: 37066023). To identify rational combination strategies, initial pharmacological screening of 474 compounds in combination with roginolisib was carried out using a MTT assay in the lymphoma cell lines HH (cutaneous T cell lymphoma, CTCL) and SP-53 (mantle cell lymphoma, MCL). Initial screening identified that roginolisib synergized with the bcl-2 inhibitor venetoclax in the HH cell line with a combination index value (CI) of 0.003 by the Chou-Talalay method. To further validate this finding, the combination of roginolisib with venetoclax or with another bcl-2 inhibitor S55746 was tested in a broad range of lymphoma cell lines including GRANTA519, JVM2, SP49 (MCL); FARAGE, TMD8 (diffuse large B cell lymphoma); MEC1 (chronic lymphocytic leukemia); MJ (cutaneous T cell lymphoma); and YT (NK lymphoma). The combination showed synergism in all the cell lines (CI 0.81 - 0.05) except SP49 (CI- 1.3). Similarly, the combination of roginolisib with S55746 showed synergism in most of the cell lines (CI- 0.46 - 0.089), additive effect in SP49 (CI- 1) and antagonistic effect in GRANTA519 (CI- 1.3) and FARAGE (CI- 2.17). To assess the tumor-intrinsic effect of roginolisib in patient derived CLL cells, PBMCs isolated from CLL patients responding to prior BTK inhibitor (i) (n=4) or progressed on BTKi (n=6) were tested to check the effect of roginolisib on BCR signalling. Cells were treated with increasing doses of roginolisib from 0.625 μM to 5 μM in the presence of anti-IgM. Other BCRi such as idelalisib, duvelisib and acalabrutinib were used at 5 μM concentrations as positive controls. Roginolisib showed a dose dependent suppression of the activity of BCR downstream kinases such as AKT and ERK. The suppression of phosphorylation of AKT and ERK were comparable with other BCRi tested. Additionally, the apoptosis analysis by annexin/7AAD showed comparable activity of roginolisib with idelalisib, duvelisib or acalabrutinib at 5 μM concentration. To investigate the possible synergy of roginolisib with venetoclax in CLL patient samples who are BTKi responders (n=6) or BTKi progressors (n=8), apoptosis analysis was carried out in patient PBMCs. Viability of CLL cells following 48h of ex vivo treatment with increasing concentrations of roginolisib (0.625 to 5 μM) and venetoclax (1 to 8 nM) alone or in combination was tested. Both the BTKi responders and progressors showed strong synergy with the combination, with slightly better cell killing activity in BTKi progressors ( p=0.007) than responders ( p=0.019). To understand the mechanism by which roginolisib increases the sensitivity to venetoclax, changes in the expression levels of various Bcl-2 family proteins that have been implicated in venetoclax resistance such as Mcl-1, Bcl-xL, Bcl-2, A1 and BIM were measured by immunoblotting. Changes in expression were evaluated in PBMCs from both the BTKi responders (n=8) and progressors (n=7) after 24h treatment with 0.625 and 5 μM concentrations of roginolisib. A significant dose dependent downregulation of Mcl-1 was observed compared to DMSO treated cells in both BTKi responders ( p=0.0033) and progressors ( p= 0.0025). In addition, a significant increase in proapoptotic BIM EL was also observed at both the 0.625 μM ( p=0.0054) and 5 μM ( p=0.0194) concentrations compared to DMSO. In summary, the novel PI3Kδ selective inhibitor roginolisib shows tumor cell killing on various hematologic malignancies including CLL cells from patients with disease progression on BTK inhibitors. In addition, roginolisib synergized with venetoclax in lymphoma cell lines and CLL patient samples. Our data support extending this combination strategy to clinical trials in hematological malignancies.

Targeting HUWE1 in Multiple Myeloma Leads to Impaired Replicative Stress Response and Enhanced Sensitivity to Bortezomib

Introduction Despite continuous advances in therapies over the last few decades, Multiple Myeloma (MM) remains an incurable malignancy with almost all patients developing relapsed/refractory disease. This emphasizes the need for novel approaches that can improve the efficacy of current standard of care treatments. Previous work from our lab and others has identified dysregulation of the E3 ligase HUWE1 in MM and highlighted it's potential as a therapeutic target. Through its interaction with a diverse set of substrates, HUWE1 is implicated in many key cellular processes including stress responses and DNA replication and repair pathways and is being increasingly recognised as a major regulator of MYC activity. In this study, we uncover a novel role for HUWE1 in DNA replication and repair and assess the combination of HUWE1 and proteasome inhibition as a potential therapeutic strategy in MM. Methods MM cell lines were transfected with SMARTvector Inducible Human HUWE1 shRNA or a non-targeting control (NTC) shRNA (Dharmacon, USA). Replicative stress was induced by treatment with 2 mM Hydroxyurea (HU) and assessed using immunofluorescence staining and iPOND (isolation of proteins on nascent DNA) analysis. Viability was assessed using CellTiter® Glo and combination indices (CI) calculated using CompuSyn software. Commercially available HUWE1 inhibitor BI8622 (MedChemExpress) and novel HUWE1 inhibitors synthesised in house were used throughout the study. Results HUWE1 knockdown and/or inhibition in MM cell lines led to an accumulation of cells in S phase, consistent with previous studies indicating that HUWE1 is required for effective DNA replication. Using proteomic profiling and co-immunoprecipitation, we identified and validated Replication Protein A, 70 kDa (RPA70) as a novel substrate of HUWE1. RPA70 is a subunit of the heterotrimeric RPA complex which plays a critical role in the recruitment of DNA repair proteins. RPA70 is the first subunit to translocate to sites of DNA damage where it binds to DNA and recruits the other subunits (RPA32 and RPA14) forming the complex, thus allowing for initiation of DNA repair. We observed significantly less K63-linked ubiquitination of RPA70 (p=0.00213) in the absence of HUWE1, with an associated decrease in both phosphorylation (p=0.0064) and localisation of RPA subunit RPA32 to DNA following treatment with hydroxyurea (HU) to induce replicative stress. Subsequent iPOND analysis determined that this is associated with reduced recruitment of repair proteins, including RAD51, to DNA. Moreover, induced replicative stress in HUWE1 depleted/inhibited cells resulted in significantly higher levels of DNA damage at 6hrs (p=0.00421) and 24hrs (p=0.0219) post HU treatment. A recent study demonstrated that HUWE1 ubiquitination of MYC promotes the formation of MYC multimers that protect stalled replication forks to limit the formation of double strand breaks (DSB) (Nature 2022;612:148-155). In line with this we also observe decreased ubiquitination of MYC in the HUWE1 knockdown cell line compared to the NTC following HU treatment. Finally, we assessed the efficacy of combining HUWE1 inhibitors with bortezomib which is known to impair DSB repair. Dual inhibition of HUWE1 and proteasome activity resulted in synergistic effects (CI values < 1) and a corresponding increase in DNA damage compared to HUWE1 inhibitor or bortezomib alone as measured by levels of ƴH2AX. Conclusion We have identified RPA as a novel substrate for HUWE1 and demonstrate that targeting HUWE1 results in increased replication stress and a dampened DNA repair capacity, underpinned by reduced recruitment of repair proteins. This work outlines a clear role for HUWE1 in genome stability in MM cells and highlights that HUWE1 inhibitors represent a novel anti-myeloma strategy that acts in synergy with bortezomib to exacerbate DNA damage.

Photodynamic Therapy with an Association of Methylene Blue and Toluidine Blue Promoted a Synergic Effect against Oral Squamous Cell Carcinoma

Among the most malignant cancers, oral squamous cell carcinoma (OSCC) stands out as the most common malignant head and neck tumor. Despite advances in the field of treatment, the prognosis of patients with OSCC remains poor. Aiming to overcome the limitations of the currently existing therapies against OSCC, the present work aims to investigate the potential of photodynamic therapy (PDT) with phenothiazine derivatives used alone or in combination. The incorporation of methylene blue (MB) and toluidine blue (TB) was evaluated in OSCC cell lines (HSC-3 and SCC-9) and a nontumor cell line (Hfib). Both compounds exhibited concentration and time-dependent incorporation, with higher rates observed in tumor cells. Regarding dark-phase cytotoxic activity, SCC-9 cells were the most sensitive cell line with an IC50 value of 362.6 µM and 41.4 µM for MB and TB, respectively. Using PDT, all lineages showed greater sensitivity, presenting lower IC50 values when compared to the dark phase values. The combination index values of 0.69 (dark phase) and 0.73 (clear phase) associated with concave isobolograms, in both phases, revealed that MB and TB have synergistic effects when combined against SCC-9 cells. These findings suggest that MB or TB assisted with PDT holds promise for OSCC treatment.

Evaluation of synergism in essential oils against the cattle tick Rhipicephalus microplus in Burkina Faso

The cattle tick Rhipicephalus microplus affects animal production economically by reducing weight gain and milk production and causing diseases, such as babesiosis and anaplasmosis. Using synthetic acaricides to reduce their incidence has caused the emergence of resistant tick populations. The present study aimed to assess the in vitro acaricidal activity of combinations of essential oils (EOs) from Ocimum americanum, Ocimum gratissimum, and Lippia multiflora against R. microplus larvae. In fact, numerous biological properties have been reported on EOs from these three plants, including acaricidal properties. Hence, a larval immersion test was performed using a population of R. microplus resistant to synthetic acaricides used in Burkina Faso. Results revealed that EO from O. gratissimum was the most effective on R. microplus larvae with LC50 and LC90 values at 10.36 and 15.51 mg/mL, respectively. For EO combinations, the most significant synergistic effect was obtained by combination 6 (1/3 O. americanum + 2/3 O. gratissimum +1/6 L. multiflora), with a combination index value of 0.44. All combinations presented dose reduction index >1, indicating a favorable dose reduction. According to the literature, this is the first study to determine the combination effect of EOs from the abovementioned plants in controlling R. microplus activity in vitro. Thus, the combination of these EOs is an alternative to control the resistant populations of invasive cattle ticks.

Combining Inotuzumab Ozogamicin (IO) with PARP Inhibitors, Olaparib and Talazoparib, Exerts Synergistic Cytotoxicity By Inhibiting the Repair of IO-Induced DNA Strand Breaks in IO-Resistant ALL Cells

[Introduction]Inotuzumab ozogamicin (IO) is highly effective in the treatment of relapsed/refractory CD22-positive B-acute lymphoblastic leukemia (B-ALL). IO exerts the antileukemic effect via DNA single- and double-strand breaks induced by calicheamicin. Although IO has been reported to show more than 80% response rate, the duration of remission is only 4.6 months, suggesting the early acquisition of drug resistance. Thus, it is crucial to elucidate the mechanism of resistance to IO and overcome it. Polyadenosine diphosphate-ribose polymerase (PARP) inhibitors, such as olaparib and talazoparib, show the antitumor effect by inhibiting DNA repair. This study was conducted to enhance the antitumor effect of IO by inhibiting IO-induced DNA damage repair using PARP inhibitors. The study attempted to overcome the resistance of B-ALL cell lines developed to be IO-resistant. [Methods]Ph-negative, CD22-positive B-ALL Reh cell line was used to determine the cytotoxicity of IO and PARP inhibitors. Inotuzumab ozogamicin was kindly provided by Pfizer. Six IO-resistant cell lines were newly established by long-term exposure of Reh cells to IO, followed by the limiting dilution. FACS analysis evaluated CD22 expression. WTS assay and Annexin-V positivity were used to determine the antileukemic effects. The Comet assay assessed the DNA repair function. The DNA microarray compared gene expression between Reh and IO-resistant Reh clones. [Results] The growth inhibition effect of IO was significantly augmented by the addition of the minimally toxic concentration of olaparib or talazoparib in Reh cells. The combination index values were 0.19 and 0.42, respectively, demonstrating a strong synergism. Combining IO with olaparib or talazoparib increased IO-induced apoptosis compared to single-agent IO. The apoptosis rates induced by 20 ng/mL IO with minimally toxic concentrations of olaparib or talazoparib were 51.7% and 66.1%, respectively, showing significantly higher apoptosis rates than IO alone (23.5%). Assessment of DNA strand breaks using the Comet assay showed that the recovery of IO-induced DNA damage was inhibited in the presence of PARP inhibitors, and DNA strand breaks persisted. Next, six IO-resistant Reh (Reh-IO-R) cell lines were established as described above. The expressions of CD22 were more than 60%. All 6 cell lines had more than 60-fold resistance to IO with the 50%-inhibitory concentration (IC 50) values 474.3～782.3 ng/mL, compared with that of Reh cells (2.1 ng/mL). The IC 50 values of olaparib or talazoparib were similar to Reh and Reh-IO-R cell lines. When IO was combined with olaparib or talazoparib, the growth of Reh-IO-R cells was inhibited, with median IC 50 values were 359.4[290.3-406.2] and 179.1[59.3-240.5] ng/mL, respectively. This combination also remarkably increased apoptosis induction in Reh-IO-R cells. The apoptosis rate of Reh cells at 100 ng/mL IO alone was 77%, whereas the apoptosis rate of 6 Reh-IO-R cell lines averaged 5.9% [5.7-6.6%]. The median percentage of apoptosis in Reh-IO-R cell lines was 12.0% by IO + olaparib and 17.1% by IO + talazoparib. Furthermore, synergism was obtained in Reh-IO-R cell lines with the combination of 1,000 ng/mL IO with olaparib or talazoparib. The median percentage of apoptotic cell death was 24.2% by IO 1,000 ng/mL alone and 45.3% by the IO+olaparib, and 56.8% by the IO+talazoparib. These results suggest that the potentiating effect of PARP inhibitors would be attributed to more DNA cleavage occurring. The expression of genes ( ATM, ATR, DNA-PK, CHK1/2, PARP, RAD, ERCC1, BRCA1/2 Ku70/80) related to DNA repair was similar between Reh and Reh-IO-R cells. Of note, ABCB1 (p-gp) was overexpressed in Reh-IO-R cells. These results suggest that the expression of p-gp was one of the mechanisms of the resistance to IO, and the combination of IO and PARP inhibitors was effective to a certain extent in the Reh-IO-R cells expressing p-gp. This would be because the PARP inhibitors were still effective in inhibiting the DNA repair in Reh-IO-R cells where the DNA repair function was not augmented. [Conclusion]The combination of IO with PARP inhibitors synergized the antileukemic effect of IO by inhibiting DNA strand break repair in CD22-positive ALL cell lines and IO-resistant ALL cells, suggesting that these results may contribute to overcoming the IO resistance mechanisms.

Targeting BCL-XL with a Novel VHL-Based BCL-XL Degrader DT-2216 in Pre-Clinical JAK2-Mutant AML Post-MPN Models

INTRODUCTION Outcomes of acute Myeloid Leukemia (AML) progressed from JAK2-mutant ( JAK2-mut) myeloproliferative neoplasms (MPNs) (post-MPN AML) are dismal, with limited responsiveness to cytotoxic therapies, hypomethylating agents, BCL-2 inhibitor or JAK2 inhibitor, with a median overall survival of less than 1 year ( Dunbar, et al. Blood, 2020). BCL-xL has been found to be overexpressed in cells from JAK2-mut MPN patients ( Petiti et al. J Cell Mol Med, 2020), thus making it a potential therapeutic target. However, the clinical utility of navitoclax, a BCL-xL and BCL-2 dual inhibitor, as demonstrated in combination with JAK2 inhibitor ruxolitinib in patients with myelofibrosis ( Harrison et al. J Clin Oncol, 2022), has been notable for an on-target thrombocytopenia due to the essential role of BCL-xL in platelet survival. In contrast, DT2216, a VHL-recruiting BCL-xL PROTAC, exhibits potent degradation of BCL-xL in leukemia cells while sparing platelets due to minimal expression of VHL in platelets ( Khan et.al Nat. Med, 2019). Consequently, DT2216 may represent an attractive strategy to target BCL-xL, reducing the apoptotic threshold and increasing responsiveness to chemotherapy selectively in tumor cells. In this study, we evaluated the pre-clinical efficacy of DT2216 in combination with ruxolitinib, 5-azacytidine (AZA), or MCL-1 inhibitor S63845 in JAK2-mut AML models. METHODS To validate the dependency of BCL2L1 (gene encoding BCL-xL) on JAK2-mut AML, we analyzed genome-wide CRISPR screen data, RNA-seq data from the DepMap portal and Beat AML 1.0 cohort. To assess the efficacy of DT2216-based treatments, including DT2216 alone, DT2216/AZA combination, DT2216/ruxolitinib combination, and DT2216/S63845 combination, we conducted the Cell Titer Glo (CTG) assay using JAK2-mut AML cell lines and primary leukemia samples. Since most JAK2-mut AML patients are resistant to ruxolitinib, we included ruxolitinib-resistant (ruxo-re) cell lines. The combination index (CI) values obtained at the ED 50, ED 75, and ED 90 were used to determine synergism (CI <1) between the agents, with lower values indicating stronger synergism. Clonogenic assays with three distinct JAK2-mut AML patient samples were performed using DT2216 and/or AZA to assess the anti-leukemia effects on hematopoietic stem and progenitor cells (HSPCs). Western blot was used to determine BCL-xL degradation and measure the expression of BCL-2 and MCL-1 after drug treatments. RESULTS The analysis of BCL2L1 gene expression (p<0.05, JAK2-mut AML cell lines versus other AML cell lines; p<0.01, post-MPN AML versus de novo AML) and BCL2L1 CRISPR gene effect (p<0.05, JAK2-mut AML cell lines versus other AML cell lines) confirmed the role of BCL-xL as a target in JAK2-mut AML. DT2216 as a single agent significantly reduced cell viability in JAK2-mut AML cell lines, with an average IC 50 of 1.61 μM in parental cell lines (range 0.74 - 2.35 μM) and an average IC 50 of 5.37 μM in ruxo-re cell lines (range 1.29 - 8.99 μM) at 72 hours. Notably, DT2216 also markedly reduced cell viability in primary samples (n=3), with an average IC 50 value of 1.21 μM (range 0.41 - 2.59 μM) at 24 hours. Moreover, the average CIs of DT2216/S68425 combination, DT2216/AZA and DT2216/ruxolitinib combination were 0.13 (range 0.07 - 0.17), 0.17 (range 0.61 - 0.22), 0.36 (range 0.14 - 0.54), respectively at 72 hours. Clonogenic assays in three primary JAK2-mut AML samples demonstrated a 69% (range 58.3 - 77.5%) reduction of colony count when treated with DT2216 alone (50 nM) (p<0.001, versus DMSO group) and 76% (range 70.8 - 79.8%) reduction when treated with the combination of DT2216 (50 nM) with AZA (100 nM) (p<0.0001, versus DMSO group) (Fig. 1A). Western blot analysis demonstrated efficient degradation of the BCL-xL protein, with average reductions of 78.5% (range: 70 - 90%), 82.75% (range: 68 - 96%) (Fig. 1B), and 86.75% (range: 83 - 97%) after 24 hours of treatment with DT2216 alone or in combination with AZA or ruxolitinib in both the parental and ruxo-re SET2 and HEL cell lines. CONCLUSIONS These findings highlight the promising efficacy of DT2216 in JAK2-mut AML, as evidenced by reduced cell viability, on-target degradation of BCL-xL, and synergistic anti-leukemia effects when combined with AZA, ruxolitinib or MCL-1 inhibitor. These results provide valuable insights into future therapeutic strategies for the treatment of JAK2-mut AML, particularly in the context of post-MPN AML.

Venetoclax Resistance Results in Broad Resistance to Majority of Anti-MM Agents Due to the Suppression of Apoptosis but Can be Overcome By BCMA-Targeted Immunotherapy

Venetoclax represents the first example of personalized medicine in multiple myeloma (MM), as it has meaningful clinical activity in patients harboring the t(11;14) translocation. This subgroup of patients generally displays upregulated BCL-2 expression and/or a higher BCL-2/BCL-X L ratio, reflecting an imbalance between cell survival (promoted by BCL-2) and cell death (promoted by BCL-X L). The Bellini study demonstrated significantly superior response rate and progression-free survival (PFS) with combination of Venetoclax and Bortezomib in relapsed myeloma, however, the overall survival in the arm with venetoclax was inferior. Part of the decrease in overall survival (OS) was considered to be related with increased incidence of infections, however, OS is also significantly impacted by response to subsequent therapies. Here, we investigated whether acquired venetoclax resistance could lead to global resistance to multiple subsequent anti-MM therapies and in turn can explain the decreased OS. We exposed venetoclax-sensitive myeloma cells (KMS27 and KMS12PE) to high-dose venetoclax treatment and generated monoclonal drug-tolerant expanded persister (DTEP) clones from single cell with a three- to ten-fold increase in IC50 compared to the parental cells (4 single cell clones for each cell line). Interestingly, the venetoclax resistance evolved without genomic alterations, as the parental and resistant cells did not show marked differences in mutational frequencies or copy number variations, including the absence of genomic alterations in the BCL-2 gene, such as the Gly101Val mutation, 1q21 gain or 8p loss. Consistent with previous literature in other disease models, these resistant clones also exhibited reduced mitochondrial priming and had increased protein expression of anti-apoptotic regulators, including MCL-1, BCL-X L, and BCL-W, thus overcoming the venetoclax-induced inhibition of the anti-apoptotic regulator BCL-2, resulting in the sequestration of the pro-apoptotic ligand BIM. We next evaluated if the altered mitochondrial priming observed in the resistant cells was also responsible for a broader resistance to anti-cancer agents. We observed that venetoclax-resistant clones were resistant to most standard-of-care anti-MM agents including alkylating agents (Melphalan, cyclophosphamide, bendamustine), proteasome inhibitors (bortezomib and carfilzomib) or immunomodulatory drugs (lenalidomide and pomalidomide) than parental cells, suggesting a broad resistance to anti-cancer agents possibly due to reduced apoptotic signaling. This indicates that switching to these agents after the acquisition of venetoclax resistance may not be efficacious. Since these data suggested a functional substitution between BCL-2 family members, we next evaluated if MCL1 or BCL-XL represent a co-dependency in resistant cells that can be therapeutically targeted to overcome resistance. Simultaneous inhibition of MCL1 (via S63845) or BCL-XL (via A155463) and BCL2 (via venetoclax) increased BIM release and enhanced cell death in the resistant clones compared to single agents, with combination index (CI) values < 0.3 in all doses tested. We further investigated the efficacy of immunotherapeutic approaches to overcome venetoclax resistance as these immunotherapies do not rely exclusively on traditional apoptotic signaling to mediate cell death. We observed that both antibody-dependent cellular cytotoxicity (ADCC) induced by Daratumumab and BCMA targeting CAR-T cells induced comparable cell death in venetoclax-resistant clones and parental cells. Moreover, myeloma cells from a patient with t(11;14) MM progressing on venetoclax, showed significant cytotoxicity to anti-BCMA CAR T cells in vitro and achieved a deep response subsequently when treated with Cilta-cel. In conclusion, we report that resistance to venetoclax confers broad resistance to standard-of-care myeloma drugs and that immunotherapies, especially CAR-T cells and CD38 monoclonal antibody, may remain effective, thus conferring potential benefits in managing acquired resistance to venetoclax.

Penilaian Indeks Kinerja Sistem Irigasi (Studi Kasus Daerah Irigasi Cirompang Kabupaten Garut Jawa Barat)

Irrigation is an effort to provide, regulate and distribute irrigation water. The Cirompang Irrigation Area is located in Bungbulang District, Garut Regency, West Java Province, with a functional area of 847 Ha, covering the service area of 4 (four) villages, namely Cihikeu Village, Bungbulang Village, Margalaksana Village and Hanjuang Village, Bungbulang District. The location of the Cirompang Irrigation Area Weir is in the village of Cintarasa, Samarang sub-district. The availability of water in the upstream, downstream and main network areas during the planting season I to planting season III is felt to be sufficient but in the middle areas the availability of water is reduced or it can be said to be non-existent due to reduced water potential and there are damaged irrigation networks and accumulation of sedimentation which results in obstruction of water flow. The Cirompang secondary canal experienced moderate to moderate damage, there was accumulation of mud or sedimentation which affected the flow of water and besides that there was also a lack of personal O&M officers in the field such as irrigation officers, weir operation officers, water gate officers and workers who have so far relied on participation. water user farmers (P3A/GP3A). The research method was carried out by direct observation in the field, interviews and secondary data analysis. Calculation of the real need for operation and maintenance is needed to determine the O&M funding and priority recommendations proposed to increase the value of irrigation system performance appraisal (IKSI). The results of the IKSI assessment for the Cirompang irrigation area, the combined index value is in the bad criteria, namely with a weight value of 48.14%, meaning that the irrigation network requires rehabilitation of the irrigation system and needs immediate treatment. 117,626,150.00 In addition, O&M financing in Cirompang for the last 3 years is still far from ideal conditions, namely an average of only 10% of AKNOP. Efforts to increase the water discharge, it is suggested for the Public Works and Spatial Planning Office of Garut Regency is to increase the budget allocation, empower the participation of water-using farmers and rehabilitate irrigation networks.

Predicting Antioxidant Synergism via Artificial Intelligence and Benchtop Data.

Lipid oxidation is a major issue affecting products containing unsaturated fatty acids as ingredients or components, leading to the formation of low molecular weight species with diverse functional groups that impart off-odors and off-flavors. Aiming to control this process, antioxidants are commonly added to these products, often deployed as combinations of two or more compounds, a strategy that allows for lowering the amount used while boosting the total antioxidant capacity of the formulation. While this approach allows for minimizing the potential organoleptic and toxic effects of these compounds, predicting how these mixtures of antioxidants will behave has traditionally been one of the most challenging tasks, often leading to simple additive, antagonistic, or synergistic effects. Approaches to understanding these interactions have been predominantly empirically driven but thus far, inefficient and unable to account for the complexity and multifaceted nature of antioxidant responses. To address this current gap in knowledge, we describe the use of an artificial intelligence model based on deep learning architecture to predict the type of interaction (synergistic, additive, and antagonistic) of antioxidant combinations. Here, each mixture was associated with a combination index value (CI) and used as input for our model, which was challenged against a test (n = 140) data set. Despite the encouraging preliminary results, this algorithm failed to provide accurate predictions of oxidation experiments performed in-house using binary mixtures of phenolic antioxidants and a lard sample. To overcome this problem, the AI algorithm was then enhanced with various amounts of experimental data (antioxidant power data assessed by the TBARS assay), demonstrating the importance of having chemically relevant experimental data to enhance the model's performance and provide suitable predictions with statistical relevance. We believe the proposed method could be used as an auxiliary tool in benchmark analysis routines, offering a novel strategy to enable broader and more rational predictions related to the behavior of antioxidant mixtures.

Phytochemical profiles, antioxidant activities, and synergistic antiproliferative effects of blueberry and apple peel extracts.

Blueberries and apples exhibit favorable bioactivity and health benefits due to their rich phytochemicals. Natural phytochemicals exist in complex forms, but there are few reports on whether have additive, synergistic or antagonistic effects between different phytochemicals. The aim of the present investigation was to elucidate the synergistic effects of blueberry extract (BE) and apple peel extract (APE) together in inhibiting the proliferation of HepG2 liver cancer cells. Meanwhile, phytochemical characterization of BE and APE were detected by HPLC, and total antioxidant activity was determined via cellular antioxidant activity (CAA) assay, oxygen radical absorption capacity (ORAC) assay and peroxy radical scavenging capacity (PSC) assay. The results showed that BE and APE were rich in phytochemicals and had potent antioxidant activities, which synergistically inhibited cell proliferation. In the bilateral combination, the dose reduction index (DRI) value increased 2-fold, and the combination index (CI) value at 95% inhibition was less than 1. Additionally, BE+APE supplementation could promote the expression levels of p53 and c-myc genes. In conclusion, the BE and APE had strong antioxidant activity and exhibited synergistic inhibition against HepG2 cells proliferation. This can provide a theoretical basis for the synergistic effect of different phytochemicals in health care. This article is protected by copyright. All rights reserved.