Chou-Talalay Combination Index Research Articles

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.

Combination of FOLFOXIRI Drugs with Oncolytic Coxsackie B3 Virus PD-H Synergistically Induces Oncolysis in the Refractory Colorectal Cancer Cell Line Colo320.

FOLFOXIRI chemotherapy is a first-line therapy for advanced or metastatic colorectal cancer (CRC), yet its therapeutic efficacy remains limited. Immunostimulatory therapies like oncolytic viruses can complement chemotherapies by fostering the infiltration of the tumor by immune cells and enhancing drug cytotoxicity. In this study, we explored the effect of combining the FOLFOXIRI chemotherapeutic agents with the oncolytic coxsackievirus B3 (CVB3) PD-H in the CRC cell line Colo320. Additionally, we examined the impact of the drugs on the expression of microRNAs (miRs), which could be used to increase the safety of oncolytic CVB3 containing corresponding miR target sites (miR-TS). The measurement of cytotoxic activity using the Chou-Talalay combination index approach revealed that PD-H synergistically enhanced the cytotoxic activity of oxaliplatin (OX), 5-fluorouracil (5-FU) and SN-38. PD-H replication was not affected by OX and SN-38 but inhibited by high concentrations of 5-FU. MiR expression levels were not or only slightly elevated by the drugs or with drug/PD-H combinations on Colo320 cells. Moreover, the drug treatment did not increase the mutation rate of the miR-TS inserted into the PD-H genome. The results demonstrate that the combination of FOLFOXIRI drugs and PD-H may be a promising approach to enhance the therapeutic effect of FOLFOXIRI therapy in CRC.

Combating Acute Myeloid Leukemia via Sphingosine Kinase 1 Inhibitor-Nanomedicine Combination Therapy with Cytarabine or Venetoclax.

MP-A08 is a novel sphingosine kinase 1 (SPHK1) inhibitor with activity against acute myeloid leukemia (AML). A rationally designed liposome-based encapsulation and delivery system has been shown to overcome the physicochemical challenges of MP-A08 and enable its effective delivery for improved efficacy and survival of mice engrafted with human AML in preclinical models. To establish therapies that overcome AML's heterogeneous nature, here we explored the combination of MP-A08-loaded liposomes with both the standard chemotherapy, cytarabine, and the targeted therapy, venetoclax, against human AML cell lines. Cytarabine (over the dose range of 0.1-0.5 µM) in combination with MP-A08 liposomes showed significant synergistic effects (as confirmed by the Chou-Talalay Combination Index) against the chemosensitised human AML cell lines MV4-11 and OCI-AML3. Venetoclax (over the dose range of 0.5-250 nM) in combination with MP-A08 liposomes showed significant synergistic effects against the chemosensitised human AML cell lines, particularly in venetoclax-resistant human AML cells. This strong synergistic effect is due to multiple mechanisms of action, i.e., inhibiting MCL-1 through SPHK1 inhibition, leading to ceramide accumulation, activation of protein kinase R, ATF4 upregulation, and NOXA activation, ultimately resulting in MCL-1 degradation. These combination therapies warrant further consideration and investigation in the search for a more comprehensive treatment strategy for AML.

Synergistic effects of resveratrol with gemcitabine in pancreatic cancer chemotherapy by inhibiting the c-Met/PARP1 axis

Objective: Pancreatic cancer is a highly malignant tumor of the digestive tract with a dismal prognosis. A key challenge of pancreatic cancer is its resistance to chemotherapy. The c-Met/PARP1 axis plays an important role in the therapeutic resistance of breast cancer and hepatocellular carcinoma. Therefore, this study aims to explore potential therapeutic targets for improving chemotherapy for pancreatic cancer and the underlying mechanisms. Methods: Gemcitabine-resistant pancreatic cancer cell lines were constructed by our laboratory using a continuous low-concentration gemcitabine induction method. The proliferation and apoptosis of combination therapy were examined using flow cytometry and comet assay. Synergistic effects of two drugs were determined by Chou-Talalay's combination index (CI). The interactions between proteins were predicted using the AutoDock model and detected through Coimmunoprecipitation assays. Results: The combination of resveratrol and gemcitabine inhibited proliferation and promoted apoptosis of pancreatic cancer cells. We found that c-Met and PARP1 were highly expressed in gemcitabine-resistant pancreatic cancer cells. However, resveratrol inhibited their expression and improved the effectiveness of gemcitabine-induced DNA damage. In addition, our data demonstrated that resveratrol and gemcitabine had synergistic effects (CI < 1). Furthermore, the protein interactions between c-Met and PARP1 were attenuated after the early stage of resveratrol intervention. Using AutoDock models, we predicted the potential binding sites where resveratrol could impact the protein interaction between c-Met (tyrosine kinase domain) and PARP1 (CAT domain). Conclusion: Our results suggested that the synergistic effects of resveratrol with gemcitabine depend on the c-Met/PAPR1 axis. Using resveratrol as a combined chemotherapy agent may have clinical benefits for patients with refractory pancreatic cancer.

Abstract C139: The IRAK4 inhibitor emavusertib (CA-4948) synergizes with second generation BTK inhibitors acalabrutinib and zanubrutinib in MYD88-L265P mutated lymphoma cell lines

Abstract Background. IRAK4 is a protein kinase downstream the Toll-like receptor (TLR), activated through the adaptor protein MYD88, crucial for the signaling cascade that leads to NF-κB-mediated cytokine and survival factor expression. Somatic mutations in the gene coding for the adaptor protein MYD88 are common in lymphoid tumors. In particular, the MYD88-L265P mutations, known to sustain NF-κB activation, is specifically observed in a fraction of the activated B cell (ABC) diffuse large B-cell lymphomas (DLBCL), in the vast majority of lymphoplasmacytic lymphoma (LPL), and in rare marginal zone lymphomas (MZLs). So far, no success has been achieved to directly inhibit MYD88. Emavusertib (CA-4948) is an IRAK4 inhibitor, which has shown preclinical activity in ABC DLBCL and MZL models, especially in cases bearing the MYD88-L265P. We and others have shown synergism when combining emavusertib with the first generation BTK inhibitor ibrutinib. Clinical trials are on-going exploring emavusertib as single agent or in combination in patients with hematological cancers or solid tumors. Since ibrutinib is associated with possible toxicities that can outbalance its clinical efficacy, here, we have tested the combination of emavusertib with acalabrutinib and zanubrutinib, two 2nd generation BTK inhibitors that have shown improved safety profile in clinical trials. Methods. Cell lines were exposed to five increasing concentrations of each compound as single agent or in combination for 72 h, followed by MTT assay. The beneficial effect of the combinations compared to the single agents was considered both as synergism according to the Chou-Talalay combination index and as potency and efficacy according to the MuSyC algorithm. Results. Cell lines bearing the MYD88-L265P mutation and derived from ABC DLBCL (OCI-Ly-10, TMD8, HBL1) or MZL/LPL (Karpas-1718) were exposed to emavusertib, acalabrutinib or zanubrutinib as single agent or in combination. Synergism, defined as a median Chou-Talalay index < 0.9, was observed for both the combination of emavusertib plus acalabrutinib and for the combination of emavusertib plus zanubrutinib in all four cell lines. Based on the MuSyC algorithm, the synergistic activity of emavusertib was mainly due to an improved efficacy (i.e., maximal effect) rather than improved potency (i.e., minimal active dose) of the BTK inhibitors. Conclusions. In MYD88-L265P mutated lymphoma cell lines, synergism is observed combining the IRAK4 inhibitor emavusertib with the 2nd generation BTK inhibitors acalabrutinib and zanubrutinib. Our data sustain further clinical exploration of the combination of IRAK4 and BTK inhibitors for lymphoma patients. Citation Format: Francesca Guidetti, Alberto J. Arribas, Eleonora Cannas, Emanuele Zucca, Anastasios Stathis, Reinhard von Roemeling, Francesco Bertoni. The IRAK4 inhibitor emavusertib (CA-4948) synergizes with second generation BTK inhibitors acalabrutinib and zanubrutinib in MYD88-L265P mutated lymphoma cell lines [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr C139.

Combined Inhibition of Protein Kinase CSNK2 and BET Proteins As a Novel Therapeutic Strategy for Mantle Cell Lymphoma

Currently available therapeutic approaches for Mantle Cell Lymphoma (MCL), including the Bruton Tyrosine Kinase (BTK) inhibitors ibrutinib, acalabrutinib and zanubrutinib, are not curative. Therefore, the discovery of novel molecules amenable of drug targeting is a research priority in the MCL field. Bromodomain-containing protein 4 (BRD4) is a member of the Bromodomain (B) and extra terminal (ET) (BET) protein family that regulate chromatin modifications and assembly of transcriptional complexes. Inhibition of BET by available compounds have shown promising anticancer effect. In MCL, BET inhibitors, such as INCB054329 or JQ-1, have been shown to increase apoptosis through downregulation of the AKT-mTOR, ERK, and other B Cell Receptor (BCR)-triggered cascades. We and others have previously demonstrated that the S/T kinase CSNK2 supports chronic BCR activation in MCL by upregulating the AKT/PI3K and NF-κB pathways. Inactivation of CSNK2 results in efficacious induction of MCL cell death. Notably, BRD4 activity is regulated by CSNK2 and double inhibition of BRD4 and CSNK2 has shown significant activity in other tumor models. The most effective and tested CSNK2 chemical inhibitor is CX4945 (silmitasertib), but very recently a novel compound, SGC-CK2, has been developed. No data are available on the effects of SGC-CK2 on MCL cells. In this work, we have evaluated the effects of the combined inhibition of CSNK2 and BET proteins on MCL cell growth and survival signaling pathways. CSNK2 inhibitors employed were CX4945 and SGC-CK2. CSNK2 gene silencing was achieved with the generation of anti- CSNK2 short hairpin-RNA IPTG-inducible MCL cell clones. BET inhibitors used were JQ-1 and INCB054329. Cell survival, apoptosis and proliferation were investigated by AnnexinV/PI staining and FACS analysis, by Western blot (WB) detection of PARP cleavage and Mcl1 expression and by other cell proliferation assays. The synergic action of BET and CK2 inhibitors was assessed by the Chou-Talalay combination index method. CK2 expression and survival-related signaling components were analyzed by WB, qRT-PCR and protein expression assays. CX4945 and the novel inhibitor SGC-CK2 displayed a potent anti-tumor activity on MCL cells as judged by the effective induction of apoptosis and proliferation arrest. A synergistic effect of CSNK2 and BET inhibitors was observed in all the MCL cell lines tested, (Jeko-1, Rec-1, including Granta-519 that are known to be much less sensitive to ibrutinib) and with all the inhibitors tested. The same results were obtained in the CSNK2 gene silencing models. Preliminary data showed that the cooperative cytotoxic effect of CK2 inhibition and BET inhibition on MCL apoptosis was observed also in MCL patient derived B cells, but not in healthy B lymphocytes form buffy coat. CSNK2 inactivation was associated with strong down-regulation of c-Myc and Mcl-1 proteins and with hampered activation of NF-κB and PI3K/AKT-dependent signaling. Surprisingly, BET protein inhibition caused increased Mcl-1 protein expression and augmented Ser 529 p65-NF-kB phosphorylation in Granta-519 and Rec-1 cells. Remarkably, CK2 inactivation led to a robust reduction of the BET inhibitor-induced increase of Mcl1, and NF-kB Ser 529 phosphorylation, thus counteracting BET-inhibitors-evoked compensatory pathways that could favor apoptosis resistance. Therefore, combined CSNK2 and BET proteins inhibition could represent an innovative strategy for chemotherapy and BTKi-resistant MCL.

Obinutuzumab-Based Drug-Free Macromolecular Therapeutics Synergizes with Topoisomerase Inhibitors.

Drug-free macromolecular therapeutics (DFMT) utilizes modified monoclonal antibodies (or antibody fragments) to generate antigen-crosslinking-induced apoptosis in target cells. DFMT is a two-component system containing a morpholino oligonucleotide (MORF1) modified antibody (Ab-MORF1) and human serum albumin conjugated with multiple copies of complementary morpholino oligonucleotide (MORF2), (HSA-(MORF2)x ). The two components recognize each other via the Watson-Crick base pairing complementation of their respective MORFs. One HSA-(MORF2)x molecule can hybridize with multiple Ab-MORF1 molecules on the cell surface, thus serving as the therapeutic crosslink-inducing mechanism of action. Herein, various anti-neoplastic agents in combination with the anti-CD20 Obinutuzumab (OBN)-based DFMT system are examined. Three different classes of chemotherapies are examined: DNA alkylating agents; proliferation pathway inhibitors; and DNA replication inhibitors. Chou-Talalay combination index mathematics is utilized to determine which drugs engaged synergistically with OBN-based DFMT. It is determined that OBN-based DFMT synergizes with topoisomerase inhibitors and DNA nucleotide analogs but is antagonistic with proliferation pathway inhibitors. Cell mechanism experiments are performed to analyze points of synergism or antagonism by investigating Ca2+ influx, mitochondrial health, lysosomal stability, and cell cycle arrest. Finally, the synergistic drug combinatorial effects of OBN-based DFMT with etoposide in vivo are demonstrated using a human xenograft non-Hodgkin's lymphoma mouse model.

Targeting sphingolipid metabolism with the sphingosine kinase inhibitor SKI-II overcomes hypoxia-induced chemotherapy resistance in glioblastoma cells: effects on cell death, self-renewal, and invasion

BackgroundGlioblastoma patients commonly develop resistance to temozolomide chemotherapy. Hypoxia, which supports chemotherapy resistance, favors the expansion of glioblastoma stem cells (GSC), contributing to tumor relapse. Because of a deregulated sphingolipid metabolism, glioblastoma tissues contain high levels of the pro-survival sphingosine-1-phosphate and low levels of the pro-apoptotic ceramide. The latter can be metabolized to sphingosine-1-phosphate by sphingosine kinase (SK) 1 that is overexpressed in glioblastoma. The small molecule SKI-II inhibits SK and dihydroceramide desaturase 1, which converts dihydroceramide to ceramide. We previously reported that SKI-II combined with temozolomide induces caspase-dependent cell death, preceded by dihydrosphingolipids accumulation and autophagy in normoxia. In the present study, we investigated the effects of a low-dose combination of temozolomide and SKI-II under normoxia and hypoxia in glioblastoma cells and patient-derived GCSs.MethodsDrug synergism was analyzed with the Chou-Talalay Combination Index method. Dose–effect curves of each drug were determined with the Sulforhodamine B colorimetric assay. Cell death mechanisms and autophagy were analyzed by immunofluorescence, flow cytometry and western blot; sphingolipid metabolism alterations by mass spectrometry and gene expression analysis. GSCs self-renewal capacity was determined using extreme limiting dilution assays and invasion of glioblastoma cells using a 3D spheroid model.ResultsTemozolomide resistance of glioblastoma cells was increased under hypoxia. However, combination of temozolomide (48 µM) with SKI-II (2.66 µM) synergistically inhibited glioblastoma cell growth and potentiated glioblastoma cell death relative to single treatments under hypoxia. This low-dose combination did not induce dihydrosphingolipids accumulation, but a decrease in ceramide and its metabolites. It induced oxidative and endoplasmic reticulum stress and triggered caspase-independent cell death. It impaired the self-renewal capacity of temozolomide-resistant GSCs, especially under hypoxia. Furthermore, it decreased invasion of glioblastoma cell spheroids.ConclusionsThis in vitro study provides novel insights on the links between sphingolipid metabolism and invasion, a hallmark of cancer, and cancer stem cells, key drivers of cancer. It demonstrates the therapeutic potential of approaches that combine modulation of sphingolipid metabolism with first-line agent temozolomide in overcoming tumor growth and relapse by reducing hypoxia-induced resistance to chemotherapy and by targeting both differentiated and stem glioblastoma cells.

Abstract 492: Pharmacological inhibition of CXCR4 increases the anti-tumor activity of conventional and targeted therapies in B cell lymphoma models

Abstract Introduction: CXCR4 is expressed in different B cell lymphoid neoplasms, and its levels are upregulated by BCR/PI3K blockade. High expression has been associated with resistance to BCR/PI3K inhibitors and gene mutations with reduced sensitivity to BTK inhibitors. We assessed whether its pharmacological inhibition with the potent CXCR4 inhibitor SPX5551 (POL5551) increases the antitumor activity of anti-lymphoma agents in lymphoma models, including some with secondary resistance to PI3K/BTK inhibitors. Methods: Antiproliferative activity was measured in mantle cell lymphoma (MCL, n=10), chronic lymphocytic leukemia (CLL,2), and marginal zone lymphoma (MZL, 2 + 4 derivatives with secondary resistance to PI3K and BTK inhibitors) cell lines exposed (72h) to increasing doses of compounds as single and in combination. Drugs included ibrutinib, copanlisib, idelalisib. The beneficial effect of the combinations versus the single agents was considered both as synergism (Chou-Talalay combination index), and as potency and efficacy (MuSyC algorithm). Mechanisms of action were studied by RNA-Seq, cell cycle and apoptosis assays, and immunofluorescence. Results: Combination of SPX5551 with conventional or targeted therapies was beneficial, either additive or synergistic, across all tested lymphomas. The combination of SPX5551 and ibrutinib was superior to single treatments in MCL, MZL or CLL, with stronger benefit in MZL and MCL than CLL. SPX5551/copanlisib showed synergism in both MCL and MZL, and SPX5551 addition overcame resistance in MZL with secondary resistance to idelalisib, copanlisib or ibrutinib. These results were confirmed using the clinically available drug candidate balixafortide (SPX6326, POL6326).RNA-Seq in the MCL REC1 model showed a much bigger impact of the SPX5551+ibrutinib on transcriptome than single agents: 2100 transcripts differentially expressed in the combination group vs DMSO (P<0.01), 662 (ibrutinib), 260 (SPX5551). Downmodulated transcripts by the combination belonged to cytokine-mediated, TNF, NFkB, TLR, JAK-STAT pathways, MYC targets. Compared to single agents, the combo had a stronger downregulation of, among others, TNF, EGR2/3, CCL3/4, CXCL10, CXCR3, CD83, DUSP2, BIRC3, MIR17HG, MYC. Upregulated genes by SPX5551+ibrutinib involved apoptosis and p53. SPX5551 addition to ibrutinib further increased cells in subG0 phase and induced apoptosis compared to single agents. Consistent with changes observed by transcriptomic profiling, combination downmodulated pAKT, RELA and RELB protein levels compared to single treatments. Conclusions: Results suggest that a combination with the CXCR4 inhibitor SPX5551 adds beneficial effect to BCR inhibitors and might overcome resistance to PI3K/BTK inhibitors. Adding SPX551 to the treatment of patients with B cell lymphoma warrants further exploration. Citation Format: Laura Barnabei, Alberto J. Arribas, Luciano Cascione, Francesca Guidetti, Filippo Spriano, Chiara Tarantelli, Andrea Rinaldi, Anastasios Stathis, Davide Rossi, Emanuele Zucca, Johann Zimmermann, Francesco Bertoni. Pharmacological inhibition of CXCR4 increases the anti-tumor activity of conventional and targeted therapies in B cell lymphoma models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 492.

Targeting Glutaminolysis to Treat Multiple Myeloma: An In Vitro Evaluation of Glutaminase Inhibitors Telaglenastat and Epigallocatechin-3-gallate.

Cancer is associated with metabolic changes from increased cell proliferation and growth. Compared to normal differentiated cells, MM cells use the glycolytic pathway even when adequate oxygen is present triggering "Glutamine addiction". To investigate the single and combined effects of epigallocatechin-3-gallate (EGCG) and telaglenastat, a glutaminase inhibitor, on the proliferation and apoptosis of the multiple myeloma cell line KM3/BTZ. KM3/BTZ cells were treated with different concentrations of telaglenastat and EGCG alone or in combination to investigate their effect on proliferation and apoptosis using the CCK8 assay, flow cytometry, and western blotting. The Chou-Talalay combination index analysis was used to explore the effect of telaglenastat combined with EGCG, while the Combination Index (CI) was calculated to analyze whether the combination of the two drugs had a synergistic effect. Telaglenastat and EGCG alone as well as in combination (5 μmol/L telaglenastat + 120 μmol/L EGCG) significantly inhibited the proliferation of KM3/BTZ cells compared to the inhibition effect of the control. Additionally, the combined treatment increased the proportion of KM3/BTZ cells in the G2 phase and decreased the proportion of cells in the G1 phase. The apoptosis rate of EGCG alone and the combined treatment was significantly higher than that of the control group. Bax protein expression was highest in the combined treatment group, whereas Bcl-2 expression was lowest, with the combined treatment group having the highest ratio of Bax/Bcl-2. Telaglenastat and EGCG act synergistically to inhibit cell proliferation and promote apoptosis in KM3/BTZ cells, possibly by targeting glutamine metabolism and glycolysis.

Validation of a Novel Rac1 Inhibitor As Potent Secdrug Against Activating Ras Mutant and Drug-Resistant Multiple Myeloma

Multiple myeloma is the second-most common hematopoietic malignancy in the United States. Although several drugs have so far been approved by the FDA, myeloma still remains an incurable disease with dose-limiting toxicities and resistance to primary drugs like proteasome inhibitors (PIs) and Immunomodulatory drugs (IMiDs). Furthermore, activating Ras gene (NRAS, KRAS, and HRAS) mutations are known to occur in >50% of myeloma patients, which are also associated with greater tumor burden, cancer aggressiveness, high recurrence rates, and poor survival. We have recently established a novel drug development/re-purposing pipeline called secDrug that incorporates a pharmacogenomics data-driven computational algorithm to introduce several new drug candidates (secDrugs), including EHT1864 (a Rac1 (Ras superfamily GTPase) inhibitor), CCT018159 (Hsp90 inhibitor), and CP466722 (a reversible ATM inhibitor) as synergistic partners of primary drugs against drug-resistant myeloma. The overall objective of this study is to validate EHT1864 using in vitro and ex vivo models of relapsed and refractory myeloma (RRMM) patients. First, we used single-cell RNA sequencing (scRNAseq) as a novel screening tool to demonstrate that EHT1864 is potentially effective against myeloma subclones based on the enrichment of target genes (Figure 1). For in vitro validation, we used a human myeloma cell line (HMCL) panel representing drug-sensitive (FLAM76), innate/refractory resistance (LP1), and acquired/relapsed resistance (parental and clonally derived PI-resistant and IMiD-resistant HMCL pairs U266 P/VR, RPMI P/VR, MM1S P/VR/LenR). FLAM76 KRas (FLAM76-K12) and FLAM76 Nras (FLAM76-N12) cell lines were generated using Adeno-associated viral (AAV) vector-mediated delivery of CRISPR-Cas9 for genome editing in humans. Ras mutations were confirmed using Sanger DNA sequencing method. Cell viability measured using CellTiter-Glo luminescent cell viability assay showed the potency of EHT1864 as a single-agent (IC50 range 15-40µM) as well as in combination with PIs (represented by Ixazomib) and IMiDs (represented by Lenalidomide). Combination index (CI) values calculated using Chou-Talalay's CI theorem were consistently less than 0.9, while all the four synergy scores (HSA, Bliss, Loewe, and ZIP) were higher than 5, demonstrating high synergy. Cell death due to apoptosis was confirmed using flow cytometry (Annexin-V-Propidium Iodide staining), Caspase 3/7 activity assay, and immunoblotting. Interestingly, EHT1864 was most potent against drug-resistant and Ras-mutant HMCLs. Further, we showed that EHT1864 treatment also affects ROS generation and mitochondrial membrane potential, indicating that EHT1864 induces apoptosis via mitochondria-mediated pathway. Finally, we performed pre-vs-post-treatment genome-wide transcriptome analysis (tumor mRNAseq) and single-cell multi-ome analysis (single-cell RNA sequencing/scRNAseq + single-cell Assay of Transposase Accessible Chromatin sequencing/scATACseq) analysis to derive molecular signatures and pathways representing the basis of (on-target and off-target) efficacy of EHT1864 and drug synergy at the bulk tumor and subclonal levels. Finally, we will perform single-cell proteomics (high-dimensional immunophenotyping using mass cytometry/CyTOF/Cytometry time of flight) in CD138+ bone marrow cells derived from newly-diagnosed and relapsed myeloma patients (ex vivo) to demonstrate efficacy in RRMM. Thus, our work lays a framework to establish EHT1864 as a clinical trial-ready therapeutic option for the management of drug-resistant myeloma. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Simultaneous crosslinking of CD20 and CD38 receptors by drug-free macromolecular therapeutics enhances B cell apoptosis in vitro and in vivo.

Drug-Free Macromolecular Therapeutics (DFMT) is a new paradigm in macromolecular therapeutics that induces apoptosis in target cells by crosslinking receptors without the need of low molecular weight drugs. Programmed cell death is initiated via a biomimetic receptor crosslinking strategy using a two-step approach: i) recognition of cell surface antigen by a morpholino oligonucleotide-modified antibody Fab' fragment (Fab'-MORF1), ii) followed by crosslinking with a multivalent effector motif - human serum albumin (HSA) grafted with multiple complementary morpholino oligonucleotides (HSA-(MORF2)x). This approach is effective in vitro, in vivo, and ex vivo on cells from patients diagnosed with various B cell malignancies. We have previously demonstrated DFMT can be applied to crosslink CD20 and CD38 receptors to successfully initiate apoptosis. Herein, we show simultaneous engagement, and subsequent crosslinking of both targets ("heteroreceptor crosslinking"), can further enhance the apoptosis induction capacity of this system. To accomplish this, we incubated Raji (CD20+; CD38+) cells simultaneously with anti-CD20 and anti-CD38 Fab'-MORF1 conjugates, followed by addition of the macromolecular crosslinker, HSA-(MORF2)x to co-cluster the bound receptors. Fab' fragments from Rituximab and Obinutuzumab were employed in the synthesis of anti-CD20 bispecific engagers (Fab'RTX-MORF1 and Fab'OBN-MORF1), whereas Fab' fragments from Daratumumab and Isatuximab (Fab'DARA-MORF1 and Fab'ISA-MORF1) targeted CD38. All heteroreceptor crosslinking DFMT combinations demonstrated potent apoptosis induction and exhibited synergistic effects as determined by Chou-Talalay combination index studies (CI<1). In vitro fluorescence resonance energy transfer (FRET) experiments confirmed the co-clustering of the two receptors on the cell surface in response to the combination treatment. The source of this synergistic therapeutic effect was further explored by evaluating the effect of combination DFMT on key apoptosis signaling events such as mitochondrial depolarization, caspase activation, lysosomal enlargement, and homotypic cell adhesion. Finally, a xenograft mouse model of CD20+/CD38+ Non Hodgkin lymphoma was employed to demonstrate in vivo the enhanced efficacy of the heteroreceptor-crosslinking DFMT design versus single-target systems.

Abstract P073: Pharmacological inhibition of IRAK4 with CA-4948 is beneficial in marginal zone lymphoma models with secondary resistance to PI3K and BTK inhibitors

Abstract Background. Marginal zone lymphoma (MZL) is a heterogeneous B-cell malignancy for which no standard treatment exists. B-cell receptor (BCR) signaling dysregulation is a hallmark of MZL and can mimic antigen dependent BCR activation. However, single treatment with BCR signaling inhibitors shows limited complete response rate and relapsing patients. IRAK4 is a protein kinase downstream the Toll-like receptor signaling. CA-4948 is the first-in-class inhibitor of IRAK4 and it is in phase 1/2 studies for patients with relapsed/refractory clinical studies with favorable activity in lymphomas and myeloid neoplasms. Here, we assessed IRAK4 inhibition with CA-4948 against a panel of MZL cell lines in which we have developed secondary resistance to inhibitors of PI3K (idelalisib, copanlisib) or BTK (ibrutinib). Methods. Models with secondary resistance were developed by continuous exposing cell lines derived from splenic MZL (Karpas 1718 and VL51) to high doses of drugs for long period of times. Resistant and parental lines were exposed to increasing doses of CA-4948, alone or in combination with compounds they are resistant to. Cell viability was tested by MTT assay (72h treatment). Synergy of combinations was evaluated according to the Chou-Talalay combination index (CI), as well as potency and efficacy, estimated according to the MuSyC algorithm. Results. The parental Karpas1718, bearing a MYD88 L265P mutation, had an IC50 of 3.29 µM for CA-4948 as single agent, while the parental VL51 and all resistant models presented IC50 values in the range of 19-35 µM. IRAK4 inhibition with CA-4948 had strong benefit when combined to downstream BCR inhibitors. In resistant cells, CA-4948 was strongly synergistic with idelalisib, ibrutinib and, at lesser extent, with copanlisib. CA-4948 in combination with ibrutinib was strongly synergistic especially in the VL51 ibrutinib resistant model compared to the parental one. CA-4948 (from 1 to 5 µM) recovered sensitivity to ibrutinib at IC50 values close to the parental condition. Similarly, CA-4948 in combination with idelalisib was also synergistic and increased sensitivity to idelalisib in idelalisib resistant VL51. Less benefit was seen adding CA-4948 to copanlisib: the combination was synergistic in VL51 parental cell, but with only weak efficacy. In the Karpas1718, CA-4948 was synergistic either in idelalisib resistant or in parental, with improved benefit in the latter, in which synergistic effect was observed when combined with both idelalisib and ibrutinib. The synergistic activity of CA-4948 was mainly due to improved efficacy rather than potency of the combinatorial partners. Conclusions. Our results in MZL cell lines show that the IRAK4 inhibitor CA-4948 is synergistic with small molecules targeting BCR signaling, especially with idelalisib and ibrutinib. Data also suggest that concentrations of CA-4948 comparable to those achieved in patients might overcome or reduce secondary resistance to the tested tyrosine kinase inhibitors. Citation Format: Francesca Guidetti, Alberto J. Arribas, Filippo Spriano, Laura Barnabei, Reinhard von Roemeling, Elizabeth Martinez, Emanuele Zucca, Francesco Bertoni. Pharmacological inhibition of IRAK4 with CA-4948 is beneficial in marginal zone lymphoma models with secondary resistance to PI3K and BTK inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P073.

Abstract 1355: A novel andrographolide analogue (3A.1) synergizes with Taxane derivatives in aggressive metastatic prostate cancers through upregulation of heatshock proteins and downregulation of MAT2A-mediated cell migration and invasion

Abstract BACKGROUND: Prostate cancer (PCa) is the 2nd leading cause of non-cutaneous cancer deaths among men in the USA with many progressing to aggressive metastatic castration resistant PCa (mCRPC; PCa unresponsive to androgen deprivation). Conventional treatment with taxanes (TX; docetaxel (DTX) or cabazitaxel (CBZ)) increases survival rates only slightly. The andrographolide analog, 19-tert-butyldiphenylsilyl-8,7-epoxy andrographolide (3A.1) has shown anticancer activity against various cancers. In this study, we investigated the effect of 3A.1 alone and in combination with DTX and CBZ against models of aggressive PCa. METHODS: Androgen receptor negative (AR-ve) mCRPC cell lines were treated with CBZ, DTX and 3A.1 as single-agent or combination over a broad concentration range, and in vitro cytotoxicity was determined. Chou-Talalay's combination index (CI) theorem was used to determine synergism and predicted dose reduction of taxanes. Post-treatment effect of single-agent and combination regimens on gene expression profile (GEP) was assessed using mRNA sequencing. Differentially expressed genes (DEGs) and molecular pathways involved in 3A.1 mechanism of action and drug synergy were identified using DESeq2, edgeR and Ingenuity pathway analysis (IPA). Protein expression of top DE genes was confirmed by immunoblotting. Cell cycle analysis, scratch/wound healing, and COMET assays were used to functionally validate the top treatment-associated genes. RESULTS: Exposure to 3A.1 alone exhibited a dose- and time-dependent antitumor activity in mCRPC. CI values of all 3A.1+ TX combinations were less than 0.5, indicating synergism. Co-treatment of 3A.1 with TX reduced the required dose of DTX by 9.5 to 18-folds (p&amp;lt;0.05), and CBZ by 3 to 14-folds (p&amp;lt;0.05). Our caspase 3/7 assay (apoptosis) results agreed with in vitro cytotoxicity data. RNAseq followed by IPA analysis identified that the top treatment-induced DEGs belong to cell migration, growth, apoptosis (MMP1, HSPB1, PSRC1), protein ubiquitination (HNRNPH2, HSPA1A/HSPA1B), and DNA repair (H2BC4, 11) pathways, along with cancer progression. Most importantly, our top downregulated DEG was MAT2A which has earlier been shown to be involved in cell migration and invasion. Our analysis using Immunoblotting, COMET, and Wound healing/Scratch (cell migration) assays corroborated with these findings. Finally, using in silico analysis on the TCGA (The Cancer Genome Atlas) database, we found that MAT2A and highly co-expressed (r&amp;gt;0.7) genes, TRA2B and SF1, were associated with worser Gleason score and nodal metastasis status in prostate adenocarcinoma patients (PRAD-TCGA). CONCLUSION: These results suggest that 3A.1 may be useful in increasing the anticancer efficacy of taxanes to treat aggressive prostate cancer. Citation Format: Taraswi Mitra Ghosh, Teeratas Kansom, Suman Mazumder, Joshua Davis, Ahmed Alnaim, Aedan Bird, P Opanasopit, Amit K. Mitra, Robert Arnold. A novel andrographolide analogue (3A.1) synergizes with Taxane derivatives in aggressive metastatic prostate cancers through upregulation of heatshock proteins and downregulation of MAT2A-mediated cell migration and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1355.

The ETS Inhibitors Yk-4-279 and TK-216 Interfere with Spib and Synergize with Lenalidomide in Diffuse Large B Cell Lymphoma of the Activated B Cell-like Type (ABC DLBCL)

Background. The two main DLBCL subtypes are the germinal center B cell (GCB) and the ABC types, characterized by individual biologic and clinical features. Since up to 40% DLBCL patients are still not cured with the standard treatments there is the need of novel therapies. YK-4-279 is a small molecule that inhibits binding of the EWS1-FLI1 fusion protein to RHA resulting in growth arrest and apoptosis in Ewing sarcoma cells. Its derivative TK-216 is the first in class inhibitor of the ETS-family of transcription factors in phase I (NCT02657005 for relapsed or refractory Ewing sarcoma). Both compounds have shown promising preclinical anti-lymphoma activity. Here, we present novel data on their mechanism of action in DLBCL.Methods. Cell lines were exposed to YK-4-279 or TK-216 alone or in combination with other compounds for 72h using Tecan D300e Digital Dispenser and 96 well plates; cell proliferation was measured with MTT; synergy with Chou-Talalay combination index. Gene expression profiling (GEP) was performed with the Illumina HumanHT 12 Expression BeadChips. In vivo studies were done in NOD-SCID mice and treatments started with 60mm3 tumor volumes sc.Results. While TK-216 has shown strong in vitro and in vivo anti-lymphoma activity, only in vitro data are available for YK-4-279 in lymphomas. Here, we first confirmed the anti-tumor activity also of the latter compound in the same ABC-DLBCL model (TMD8 xenograft). Compared with control group (n=10), mice treated with YK-4-279 (100 mg/Kg, BID; n=9) clearly presented a reduction in tumor growth at D8 and D11 (P<0.01) and D13 (P not available since control group had to be stopped due to tumor volume).In accordance with our previous combination data showing a specific synergism of TK-216 when combined with the immunomodulatory drug (IMID) lenalidomide in ABC DLBCL, when YK-4-279 was combined with the BTK-inhibitor ibrutinib, the PI3K-delta inhibitor idelalisib, the BET inhibitor OTX-015 and lenalidomide in four DLBCL cell lines (2 ABC, 2 GCB), the biggest benefit was achieved with the combination of YK-4-279 plus lenalidomide with synergism in both ABC DLBCL. Finally, since lenalidomide is active in mantle cell lymphoma (MCL), the synergism of the combination of TK-216 and lenalidomide was confirmed also in two MCL cell lines.With the aim to understand the mechanism of action of the two small molecules, we correlated the baseline RNA expression levels of the different ETS factors with sensitivity to the drugs. SPIB was the gene presenting the most significant negative correlation with both YK-4-279 and TK-216, especially among the ABC DLBCL cell lines (P<0.05). Interestingly, SPIB is a known oncogene for ABC DLBCL (Lenz et al, PNAS 2008) and is involved in the response to lenalidomide in ABC DLBCL (Yang et al, Cancer Cell 2012). YK-4-279 inhibits the binding of EWS1-FLI1 to the helicases RHA and DDX5 (Selvanathan et al, PNAS 2012). Thus, we assessed whether YK-4-279 and TK-216 can have a similar effect on SPIB and whether they induce cellular effects similar to lenalidomide. Protein modelling demonstrated that the 3D structure of FLI1 and SPIB are highly overlapping. Co-IP experiments showed the binding of SPIB to RHA and DDX5 in two ABC DLBCL cell lines. The binding to RHA and, at lesser extent, to DDX5 was decreased exposing the cells to TK216 or YK-4-279 (500 nM, 4-10h). Similarly to lenalidomide (Yang et al, Cancer Cell 2012), TK-216 decreased IRF4 and upregulated IRF7 protein in cells.Finally, GEP of two ABC DLBCL cell lines exposed to the active (S)- or to the inactive (R)-enantiomer (500 nM, 4-8h) showed that (S)-YK-4-279 caused an important upregulation of multiple snoRNAs, an effect compatible with an interference of the compound on helicases and RNA editing.Conclusions. In ABC DLBCL, the ETS inhibitor YK-4-279 and its clinical derivative TK-216 interfere with SPIB and helicases involved in RNA editing. Moreover, both compounds act similarly to lenalidomide inhibiting IRF4 and upregulating IRF7 and synergize with the IMID in both ABC DLBCL and MCL. EC and FS equally contributed. DisclosuresZucca:Jannsen: Consultancy, Honoraria, Other: Advisory role; Jannsen: Consultancy, Honoraria, Other: Advisory role; Roche: Honoraria, Research Funding; Celltrion Healthcare: Consultancy, Other: Advisory Role; Takeda: Consultancy, Other: Advisory role; Bayer: Consultancy, Other: Advisory Role; Celltrion Healthcare: Consultancy, Other: Advisory Role; Takeda: Consultancy, Other: Advisory role; Gilead Science: Consultancy, Other: Advisory role; Gilead Science: Consultancy, Other: Advisory role; Celgene: Honoraria, Research Funding; Mundipharma: Research Funding; Mundipharma: Research Funding; Celgene: Honoraria, Research Funding; Roche: Advisory role, Honoraria, Research Funding; Sandoz: Consultancy, Other: Advisory role; Sandoz: Consultancy, Other: Advisory role; Bayer: Consultancy, Other: Advisory Role. Stathis:Roche: Consultancy, Other: Advisory board; Merck: Research Funding; Celgene: Research Funding; Pfizer: Research Funding; Amgen: Honoraria. Jessen:Oncternal Therapeutics: Employment. Lannutti:Oncternal Therapeutics: Employment. Toretsky:Oncternal Therapeutics: Membership on an entity's Board of Directors or advisory committees. Bertoni:Menarini: Research Funding; Cellestia: Research Funding; Bayer: Research Funding; Acerta Pharma: Research Funding; Piqur: Research Funding; Immunogen: Research Funding.

Modulation of Cholesterol Metabolism Improves Response to Enzalutamide Treatment in Prostate Cancer

Abstract Objectives Prostate cancer (PCa) growth is mediated by androgens via activation of androgen receptor (AR). Accordingly, androgen deprivation therapy (ADT) is the gold standard for the treatment of advanced PCa, but progression to castration-resistant PCa (CRPC) follows. Enzalutamide (ENZ) is an AR antagonist used for the management of CRPC. However, patients acquire resistance to the drug in a short period. As cholesterol metabolism is dysregulated in PCa and lipogenesis is upregulated by AR signaling, we hypothesized that inhibition of cholesteryl ester formation and suppression of lipogenesis by blockage of sterol-O-acyltransferase 1 (SOAT1) could enhance the response to ENZ. Methods The mRNA expression of SOAT1 in PCa patients was analyzed using Oncomine and TCGA database. Survival analysis of TCGA PCa patients data was executed using cBioPortal. 22RV1 cells inherently resistant to ENZ, were challenged to avasimibe (AVA), a pharmacological inhibitor of SOAT1, with or without ENZ. Results We found SOAT1 mRNA is significantly overexpressed in prostate carcinoma compared to the normal tissue in separate datasets (Taylor 3; Grasso; Liu). Analysis of the TCGA PCa dataset among patients who had undergone ADT showed longer disease-free status (P = 0.039) and a trend toward better overall survival status (P = 0.066) in low SOAT1 mRNA expressing patients. Moreover, SOAT1 mRNA expression positively correlated with AR mRNA expression with Pearson coefficient of 0.5 (P = 7.475e-6). We also found that combination of 20 μM ENZ and 2 μM AVA reduced 52% of cell counts after 5 days and 53% of colony formation after 2 weeks, whereas AVA resulted in 20% and 28% reduction, respectively, with no effect from ENZ. Synergism of these two drugs was observed based on MTT assay with Chou-Talalay's combination index method. 4 μM AVA treatment for 24h downregulated AR downstream nkx3.1 mRNA level with no difference in AR or ARV7 mRNA levels. AVA treatment also downregulated lipogenic SCD1 mRNA level, inhibition of which was previously reported to enhance response to ENZ. Conclusions SOAT1 is a potential biomarker predictive of the success of ENZ treatment in PCa according to TCGA data. Our in vitro study further supports that SOAT1-regulated cholesterol metabolism is an important factor for the ENZ response. Funding Sources Purdue Research Foundation, Ralph W. and Grace M. Showalter Research Trust.

Synergistic potential of dual andrographolide and melatonin targeting of metastatic colon cancer cells: Using the Chou-Talalay combination index method

Colorectal cancer (CRC) mortality has diminished for decades due to new and improved treatment profiles. However, CRC still ranks as the third most diagnosed cancer in the US. Therefore, a new therapeutic approach is needed to overcome colospheroids inhibition and drug resistance. It is well documented that andrographolide (AGP) and melatonin (MLT) have anti-carcinogenic properties. Our goal was to evaluate their synergistic effects on metastatic colon cancer cells (mCRC) and colospheroids. HT-29 and HCT-15 mCRC cells were simultaneously treated with serial dilutions of AGP and MLT for 24, 48 and 72 h. Cell viability was monitored using the MTT assay. The Chou-Talalay method for drug combination is based on the median effect equation, providing a theoretical basis for the combination index and the isobologram equation. This allows quantitative determination of drug interactions using the CompuSyn software, where CI < 1, = 1, and >1 indicates synergistic, additive, and antagonistic effects respectively. Our results demonstrate that AGP and MLT in combination show synergism with CI values of 0.35293 and 0.34152 for HT-29 and HCT-15 respectively and a fractional inhibition of Fa = 0.50–0.90, as shown by the Fa-CI plot and isobologram. The synergism value was validated in colospheroids (HT-29-s and HCT-15-s) based on morphology, viability, and colony formation and in 5-FU drug resistant cell (HT-29R and HCT-116R) viability. The mechanism(s) of decreased cell viability are due to the induction of ER stress proteins and angiogenic inhibition. Our results provide rationale for using AGP in combination with MLT on mCRC.

Flavokawain B and Doxorubicin Work Synergistically to Impede the Propagation of Gastric Cancer Cells via ROS-Mediated Apoptosis and Autophagy Pathways.

Simple SummaryAmong various kinds of treatment strategies for cancers, combination therapy has attracted significant attention due to its beneficial effects than the individual effects of the same compounds. Based on this idea, this study has investigated the synergistic effects of combination treatment of a natural anti-cancer agent flavokawain B (FKB) and a chemotherapeutic agent Doxorubicin on human gastric cancer cells and the underlying molecular mechanisms were deciphered through in vitro and in vivo approaches. Experimental data obtained in this study provided promising application prospects of FKB + Doxrubicin combination treatment in human gastric cancer cells.Chalcone flavokawain B (FKB) possesses a chemopreventive and anti-cancer activity. Doxorubicin is a chemotherapeutic DNA intercalating agent widely used in malignancy treatment. The present study investigated whether synergistic effects exist between the combination of FKB (1.25–5 µg/mL) and doxorubicin (0.5 µg/mL) on the apoptosis and autophagy in human gastric cancer (AGS) cells, and the possible in vitro and in vivo mechanisms. The MTT assay measured cell viability. Various apoptotic-, autophagy-associated protein expression was determined by the Western blot technique. FKB+doxorubicin synergy was estimated by the Chou-Talalay combination index (CI) method. In vivo studies were performed on BALB/c mice. Results showed that compared to FKB/doxorubicin treatments, low doses of FKB+doxorubicin suppressed AGS cell growth. FKB potentiated doxorubicin-induced DNA fragmentation, apoptotic cell death, and enhanced doxorubicin-mediated mitochondrial, death receptor pathways. FKB+doxorubicin activated increased LC3-II accumulation, p62/SQSTM1 expression, and AVO formation as compared to the FKB/doxorubicin alone treatments indicating autophagy in these cells. The death mechanism in FKB+doxorubicin-treated AGS cells is due to the activation of autophagy. FKB+doxorubicin-mediated dysregulated Bax/Bcl-2, Beclin-1/Bcl-2 ratios suggested apoptosis, autophagy induction in AGS cells. FKB+doxorubicin-induced LC3-II/AVOs downregulation was suppressed due to an apoptotic inhibitor Z-VAD-FMK. Whereas, 3-methyladenine/chloroquine weakened FKB+doxorubicin-induced apoptosis (decreased DNA fragmentation/caspase-3). Activation of ERK/JNK may be involved in FKB+doxorubicin-induced apoptosis and autophagy. FKB+doxorubicin-triggered ROS generation, but NAC attenuated FKB+doxorubicin-induced autophagic (LC3 accumulation) and apoptotic (caspase-3 activation and PARP cleavage) cell death. FKB+doxorubicin blocked gastric cancer cell xenografts in nude mice in vivo as compared to FKB/doxorubicin alone treatments. FKB and doxorubicin wielded synergistic anti-tumor effects in gastric cancer cells and is a promising therapeutic approach.

Abstract A53: Synergistic interaction of HDACi and PLK1i in Group 3 MYC-amplified medulloblastoma

Abstract Medulloblastoma (MB) is one of the common malignant brain tumors in children. Patients with MYC-amplified Group 3 MBs exhibit particularly poor survival rates even after intensive therapy. Surviving patients often suffer from long-term side effects. This calls for new therapeutic strategies, such as targeted therapy options. The sensitivity of MYC-amplified MBs to class I histone deacetylase (HDAC) inhibition was previously shown. In order to elucidate potential combination partners, we have identified PLK1 as one of the top regulated genes following treatment of MYC-amplified Group 3 MB cells with class I HDACi entinostat. Our aim here is to investigate possible combination treatments with entinostat and several PLK1i (volasertib, GSK461364, onvansertib). The cell metabolic activity was evaluated using MYC-amplified and nonamplified MB, high-grade glioma (HGG) and neuroblastoma (NB) cell lines after single and combination treatments. The interaction effect was determined by combination index (CI) (Chou-Talalay CI calculation method). Results were validated assessing cell viability, cell cycle profile, and caspase activity upon treatment with single agents or combination. On-target activity was examined using immunoblotting for pTCTP and H3K27ac. We have confirmed our findings in an inducible MYC cell line. The gene expression profile was analyzed in HD-MB03 cell line after entinostat, volasertib, or combination treatment. We demonstrate that the MYC target gene PLK1 is significantly downregulated upon HDACi treatment. Based on this, we hypothesized that inhibition of both class I HDACs and PLK1 could have synergistic effects. MYC-amplified cell lines were more sensitive than nonamplified cell lines to treatment with all PLK1i investigated, showing 3 to 10 times lower IC50. PLK1i and entinostat interacted synergistically (CI below 0.9) at lower concentrations in MYC-amplified compared to non-amplified MB cell lines. Similar results were obtained for MYC or N-MYC-amplified HGG and NB cell lines. We also observed the loss of viability and loss of fraction of cells in G1 phase in MYC-amplified MB cells after treatment with entinostat and PLK1i. MYC target genes were significantly downregulated in the MYC-amplified MB cell line HD-MB03 after treatment with PLK1i and entinostat. Moreover, we demonstrated reduction of MYC levels and faster MYC degradation upon volasertib treatment. Our data suggest that MYC-amplification might serve as a predictive biomarker for PLK1i treatment in MB and other entities. The combination of entinostat and PLKi could be a candidate therapy for future clinical trials for MYC-amplified Group 3 MB, and possibly other tumors harboring MYC amplification. Citation Format: Gintvile Valinciute, Jonas Ecker, Thomas Hielscher, Christin Schmidt, Marc Remke, Gianluca Sigismondo, Jeroen Krijgsveld, Stefan M. Pfister, Olaf Witt, Till Milde. Synergistic interaction of HDACi and PLK1i in Group 3 MYC-amplified medulloblastoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr A53.

Mass‐Action Law Based Pharmacodynamics General Theory, Equation, Algorithm, and Computer Simulation for Econo‐Green Biomedical R&amp;D and for Quantitative/indexed Drug Evaluation Guidance

The Mass‐Action Law (MAL) based Median‐Effect Equation (MEE) is the Unified General Theory of Pharmacodynamics (PD) ‐‐Biodynamics (BD) – Bioinformatics (BI) (MAL‐PD/BD/BI) [Chou TC, Pharmacol. Rev. 58: 621–681, 2006]. The derived MEE yields the algorithms that provide efficient, quantitative, econo‐green, digital, automated computer simulations for bio‐medical research &amp; development (R&amp;D). Its extension yields Combination Index Equation (CIE), which quantitatively defines drug combination synergy (CI&lt;1), additive effect (CI=1) and antagonism (CI&gt;1) [in: Chou TC and Talalay P. Adv. Enz. Regul. 22: 27–55, 1984], which has been cited 6,275 times in over 1,249 biomedical journals worldwide, encompassing nearly all disciplines of biomedical sciences, as of Oct. 2, 2019. The derived MEE theory reveals the following new basic paradigms: 1. “Dose” and “Effect” are interchangeable. 2. All dose‐effect curves of MAL can be transformed into straight lines.3. The theoretical minimum of only 2‐data points are required to simulate a dose‐effect curve, where the 3rd point is Dose Zero, and the 4th point is the median‐effect dose (Dm), which is the universal reference‐point and the dynamic common‐link. 4. The fact that only 2‐data‐points theory are required to simulate a dose‐effect curve, is of particularly importance, since fewer number of doses are needed in animals studies or in clinical protocol trial designs. 5. It is noted that most clinical trials had used only single dose. However, a single dose (for a single drug or for a drug‐combination dose) generates a Potency “Point”, but a “point” has no “shape”, and PD requires both “Potency” and “Shape”. 6. The PD indicates that Phase I clinical trial protocol‐design shall use 2 to 3 doses for single drug, and only ten doses (3+3+3+1=10) for two‐drug combination synergy quantification, using Chou‐Talalay combination index (CI) method and computer software. Thus, the new PD paradigm is the theoretical basis for simple, efficient, quantitative “econo‐green” bio‐R&amp;D and drug PD parameter evaluations. 7. The explicit digital definition of “Synergism” of drug combination is now established. 8. The PD software, e.g., CompuSyn, allows automated generations of five graphic transformations for diagnostic plots [i.e., the dose‐effect curves, the median‐effect plots (for curve linearization), the CI‐Plot and the Isobologram (for synergy/antagonism determination), as well as the DRI‐Plot (for the dose‐reduction calculation in synergistic combinations). These automatic illustrations by computer simulation is completed within 1 second after data entries. Thus, the MAL‐PD/BD unified general theory is proven to have broad and cost‐effective utilities in biomedical R&amp;D, provide automated quantitative/indexed basic parameters, diagnosis and conclusions for drug evaluations, and streamline the drug‐evaluation regulatory processes.Support or Funding InformationSelf Funding (Retired Professor)The Mass‐Action Law PD/BD Theory, Equations, and New Paradigm Revelations.Figure 1Mass‐Action Law Based Pharmacodynamics, Biodynamics and Bioinformatics Theory: Applications, Citation Metrics and Trend.Figure 2