Synergistic Killing Research Articles

Therapeutic Effect of Bortezomib Combined with Dag Regimen in Hyperleukocytic Acute Myeloid Leukemia

AbstractIntroduction: Hyperleukocytic acute myeloid leukemia(HAML) is a high-risk type of acute myeloid leukemia (AML), with white blood cell counts exceeding 100 x 10 ^ 9/L in peripheral blood, accounting for approximately 5%-20% of AML.Bortezomib is the first proteasome inhibitor approved by the US FDA for clinical use.The efficacy of either single agent or in combination with other agents for acute myeloid leukemia is inconclusive. DAG is commonly used as a classical chemotherapy regimen for acute myeloid leukemia.We have found that bortezomib has a therapeutic effect on acute myeloid leukemia in clinical empirical medication, so we investigated the therapeutic effect of bortezomib combined with DAG regimen on HAML. Methods:Primary hyperleukocytic leukemia cells were isolated from bone marrow of hyperleukocytic acute myeloid leukemia patients using ficoll liquid, then the primary hyperleukocytic leukemia cells were treated with bortezomib (0 nm, 5 nm, 10 nm, 20 nm, 50 nm), DAG, and bortezomib combined with DAG regimens at different concentration gradients.Cell viability was detected by flow cytometry. The HAML patient-derived xenograft (PDX) model were used to evaluate the in vivo effect of bortezomib combined with DAG regimen using the same dosing regimen as the clinical medication regimen: bortezomib 1.3 mg/m2, subcutaneous injection, 4 times a course, d1, 4, 8, and 11; doxorubicin liposome injection 5 mg/m2, intravenous drip, every other day, 5 times in total; cytarabine 10 mg/m2, subcutaneous injection, d1-14 every 12 h; and G-CSF 200 μg/m2 per day, subcutaneous injection, d1-14 days. Results: With the increase of concentration gradient, the apoptosis of HAML leukemia cells induced by bortezomib alone and DAG alone was gradually enhanced, while the viable cell count of bortezomib combined with DAG was much lower than that of bortezomib alone or DAG alone. In animal experiments, flow cytometry showed that the ratio of human leukaemia cells in bone marrow blood decreased revealed the most significant decrease in the bortezomib combined with DAG group, absolute counts of CD45 cells in the bone marrow cells of mice in each group also revealed the most significant decrease in the bortezomib combined with DAG group. Morphological examination of myelocyte smears revealed significantly fewer myeloblasts in the bortezomib combined with DAG group than in the other groups. The survival rate in the bortezomib combined with DAG group was better than in the bortezomib alone or DAG alonefor the HAML PDX model. Conclusion:Bortezomib combined with DAG regimen has synergistic killing effect on HAML and may serve as a certain clinical prospect in the treatment of hyperleukocytic acute myeloid leukemia. Disclosures No relevant conflicts of interest to declare.

TMIC-28. COMBINING INDUCED NEURAL STEM CELL THERAPY AND IMMUNOMODULATION IN GLIOBLASTOMA

Abstract Glioblastoma (GBM) is an aggressive malignant adult brain tumor and is characterized by poor prognosis and survival outcome. Insufficient drug accumulation to the tumor site and development of monotherapy drug resistance are critical challenges to overcome in combating GBM. Human induced neural stem cells (hiNSC) therapy is promising due to the stem cells’ innate capability to migrate to the tumors and deliver therapeutics. We previously have developed spheroidal iNSCs and engineered them to constitutively secrete pro-apoptotic protein TRAIL—hiNeuroS-TRAIL. However, investigating a combination therapy along with hiNeuroS-TRAIL therapy is needed to potentially avoid monotherapy resistance and enhance overall treatment durability. One of the GBM hallmarks is its immunosuppressed tumor microenvironment (TME). GBM-associated macrophages and microglia (GAMs) display pro-tumoral phenotypes in TME—inhibiting their ability to target the tumor cells or recruit other immune cells. In humans, higher glioma grade is associated with higher expression of Mer Tyrosine Kinase Receptor (MerTK) in both GBM cells and GAMs. Activation of MerTK in GAMs and GBM cells exacerbates immunosuppression and tumor progression respectively. Inhibiting MerTK activation via a small molecule drug, MRX-2843 and combining with hiNeuroS-TRAIL demonstrated robust synergistic tumor killing effects in vitro (CDI=0.22) and ex vivo (CDI=0.18). Additionally, the preliminary data for in vivo pilot study revealed that the median survival for the combination therapy treated group of mice extended over 24 days whereas the median survival for control, MRX-2843, and hiNeuroS-TRAIL treated groups of mice were 16, 18, and 21 days respectively. We think that combining these two therapies could significantly extend the survival in our murine model of GBM. Therefore, we will be optimizing dose concentration and cell counts for single agent therapies, and we will then repeat in vivo y synergy study with optimized therapies and a larger sample size to fully uncover the potential of this combination therapy.

Nanofibrillar peptide hydrogels for self-delivery of lonidamine and synergistic photodynamic therapy

The use of lonidamine (LND) in photodynamic therapy (PDT) provides a viable approach to develop low-dose PDT modules with high efficacy, for LND potentiates cytotoxicity of photosensitizers through dysregulation of mitochondrial function. Yet, the efficacy of LND is restricted by its low accumulation in cancer cells, especially in the mitochondrial compartments. To address the problem, we design an LND-derived self-assembling peptide molecule (LND-K) that dually targets integrin receptors and mitochondria of cancer cells. The targeted cellular delivery of LND-K gives higher efficacy in ablation of mitochondrial function in melanoma cells A375, as compared to free LND or the control molecule that lacks mitochondria-targeting moieties. To integrate LND-K in a typical PDT module, we develop a nanofibrillar hydrogel system through co-assembly of LND-K and TPPS4, an anionic photosensitizer that forms tight electrostatic interactions with cationic residues of LND-K. Notably, hydrogel formulation of LND-K/TPPS4 facilitates slow release of TPPS4 over 14 days in vitro, and displays a longer retention time than aqueous solution of TPPS4in vivo. By integrating a mitochondria-targeted molecule (LND-K) in a typical PDT module, we achieve synergistic killing of A375 cells with dual drugs, where LND-K not only serves as a chemotherapeutic drug, but also potentiates the cytotoxicities of TPPS4 toward A375 cells in vitro and in vivo. The peptide-based drug self-delivery system promises the development of efficacious combination treatments against cancer, that integrate cell sensitization with existing anticancer modules (e.g., chemotherapy and PDT) for enhanced therapeutic efficacy. Statement of significanceThis study reports the design and synthesis of a lonidamine (LND)-derived self-assembling peptide (LND-K) that dually targets integrin receptors and mitochondria of cancer cells. Under the precision guidance of a mitochondria-targeting sequence, LND-K-containing nanofibers target mitochondria and ablate mitochondrial functions. On one hand, the targeted delivery of LND-K reduces cell viabilities through a chemotherapy route; on the other hand, LND-K sensitizes cancer cells for subsequent PDT treatment with enhanced efficacy, which is mediated by induction of ROS, loss of mitochondrial membrane potential, and decrease of cellular ATP level. We believe that the design of mitochondria-targeted drug delivery systems with a self-assembling molecule provides a new approach to potentiate cytotoxicity of photosensitizers in a low-dose PDT module.

Integrated metabolomic and transcriptomic analyses of the synergistic effect of polymyxin–rifampicin combination against Pseudomonas aeruginosa

BackgroundUnderstanding the mechanism of antimicrobial action is critical for improving antibiotic therapy. For the first time, we integrated correlative metabolomics and transcriptomics of Pseudomonas aeruginosa to elucidate the mechanism of synergistic killing of polymyxin–rifampicin combination.MethodsLiquid chromatography-mass spectrometry and RNA-seq analyses were conducted to identify the significant changes in the metabolome and transcriptome of P. aeruginosa PAO1 after exposure to polymyxin B (1 mg/L) and rifampicin (2 mg/L) alone, or in combination over 24 h. A genome-scale metabolic network was employed for integrative analysis.ResultsIn the first 4-h treatment, polymyxin B monotherapy induced significant lipid perturbations, predominantly to fatty acids and glycerophospholipids, indicating a substantial disorganization of the bacterial outer membrane. Expression of ParRS, a two-component regulatory system involved in polymyxin resistance, was increased by polymyxin B alone. Rifampicin alone caused marginal metabolic perturbations but significantly affected gene expression at 24 h. The combination decreased the gene expression of quorum sensing regulated virulence factors at 1 h (e.g. key genes involved in phenazine biosynthesis, secretion system and biofilm formation); and increased the expression of peptidoglycan biosynthesis genes at 4 h. Notably, the combination caused substantial accumulation of nucleotides and amino acids that last at least 4 h, indicating that bacterial cells were in a state of metabolic arrest.ConclusionThis study underscores the substantial potential of integrative systems pharmacology to determine mechanisms of synergistic bacterial killing by antibiotic combinations, which will help optimize their use in patients.Graphical

Berbamine Hydrochloride inhibits lysosomal acidification by activating Nox2 to potentiate chemotherapy-induced apoptosis via the ROS-MAPK pathway in human lung carcinoma cells.

Autophagy is typically activated in cancer cells as a rescue strategy in response to cellular stress (e.g., chemotherapy). Herein, we found that Berbamine Hydrochloride (Ber) can act as an effective inhibitor of the late stage of autophagic flux, thereby potentiating the killing effect of chemotherapy agents. Lung carcinoma cells exposed to Ber exhibited increased autophagosomes, marked by LC3-II upregulation. The increased level of p62 after Ber treatment indicated that the autophagic flux was blocked at the late stage. The lysosome staining assay and cathepsin maturation detection indicated impaired lysosomal acidification. We found that Nox2 exhibited intensified co-localization with lysosomes in Ber-treated cells. Nox2 is a key enzyme for superoxide anion production capable of transferring electrons into the lysosomal lumen, thereby neutralizing the inner protons; this might explain the aberrant acidification. This hypothesis is further supported by the observed reversal of lysosomal cathepsin maturation by Nox2 inhibitors. Finally, Ber combined with cisplatin exhibited a synergistic killing effect on lung carcinoma cells. Further data suggested that lung carcinoma cells co-treated with Ber and cisplatin accumulated excessive reactive oxygen species (ROS), which typically activated MAPK-mediated mitochondria-dependent apoptosis. The enhanced anti-cancer effect of Ber combined with cisplatin was also confirmed in an in vivo xenograft mouse model. These findings indicate that Ber might be a promising adjuvant for enhancing the cancer cell killing effect of chemotherapy via the inhibition of autophagy. In this process, Nox2 might be a significant mediator of Ber-induced aberrant lysosomal acidification.

Injectable “cocktail” hydrogel with dual‐stimuli‐responsive drug release, photothermal ablation, and drug‐antibody synergistic effect

AbstractThe combination of the first‐line standard chemotherapeutic drug doxorubicin hydrochloride (DOX) and the molecular‐targeted drug Herceptin (HCT) has emerged as a promising strategy for human epidermal growth receptor 2 (HER‐2) overexpressing breast cancer treatment. However, insufficient drug accumulation and severe cardiotoxicity are two major challenges that limit its clinical application. Herein, an in situ forming gold nanorods (AuNRs)‐sodium alginate (ALG) hybrid hydrogel encapsulating DOX and HCT was engineered for tumor synergistic therapy involving injectable, dual‐stimuli‐responsive drug release, photothermal ablation, and drug‐antibody synergistic therapy. The photothermal agent AuNRs, anticancer drug DOX, and anticancer antibody HCT were mixed in ALG solution, and after injection, the soluble ALG was quickly transformed into a hydrogel in the presence of Ca2+ in the body. Significantly, the hybrid hydrogel exhibits an extremely high photothermal conversion efficiency of 70% under 808 nm laser irradiation. The thermal effect can also provide photothermal stimulation to trigger the drug release from the gel matrix. In addition, the drug release rate and the releasing degree are also sensitive to the pH. In vitro studies demonstrated that the PEI‐AuNR/DOX/HCT/ALG hydrogel has facilitated the therapeutic efficiency of each payload and demonstrated a strong synergistic killing effect on SK‐BR‐3 cells. In vivo imaging results showed that the local drug delivery system can effectively reduce the nonspecific distribution in normal tissues and increase drug concentration at tumor sites. The proposed hydrogel system shows significant clinical implications by easily introducing a sustainable photothermal therapy and a potential universal carrier for the local delivery of multiple drugs to overcome the challenges faced in HER‐2 overexpressing cancer therapy.

Mesoporous Polydopamine-Based Nanovehicles as a Versatile Drug Loading Platform to Enable Tumor-Sufficient Synergistic Therapy.

The combination of photothermal therapy and chemotherapy are developing as a promising clinical strategy but it urgently needs the high exploration of intelligent multifunctional drug delivery nanovectors. In this paper, we used a versatile method to construct mesoporous polydopamine nanovehicles (MPDA) with the dendritic mesopores loaded with a clinical chemotherapeutic drug, Doxorubicin (MPDA@DOX). The monodisperse nanoagents are spherical with a size of ∼160 nm and pore size of approximately 10 nm. MPDA could efficiently delivery DOX with π-π stacking interaction and acts as the potent photothermal agents. Importantly, MPDA@DOX are preferentially internalized by cancerous cells, then bursting drug release and local hyperthermia generation were observed in conditions representative of the cytoplasm in tumor cells that highly synergistic cell killing effect were found under 808 nm laser irradiation. The fluorescent imaging results of human breast tumor bearing murine model evidenced that MPDA delivery platform have excellent tumor precise targeting effect and in vivo tumor ablation experiment further revealed that MPDA@DOX showed markedly eradicated tumor growth capability under laser exposure. Therefore, this work provided a fascinating strategy based on biocompatible MPDA based drug delivery system for malignant tumors eradication via synergistic therapy.

Targeting Melanin in Melanoma with Radionuclide Therapy.

Nearly 100,000 individuals are expected to be diagnosed with melanoma in the United States in 2022. Treatment options for late-stage metastatic disease up until the 2010s were few and offered only slight improvement to the overall survival. The introduction of B-RAF inhibitors and anti-CTLA4 and anti-PD-1/PD-L1 immunotherapies into standard of care brought measurable increases in the overall survival across all stages of melanoma. Despite the improvement in the survival statistics, patients treated with targeted therapies and immunotherapies are subject to very serious side effects, the development of drug resistance, and the high costs of treatment. This leaves room for the development of novel approaches as well as for the exploration of novel combination therapies for the treatment of metastatic melanoma. One such approach is targeting melanin pigment with radionuclide therapy. Advances in melanin-targeting radionuclide therapy of melanoma can be viewed from two spheres: (1) radioimmunotherapy (RIT) and (2) radiolabeled small molecules. The investigation of mechanisms of the action and efficacy of targeting melanin in melanoma treatment by RIT points to the involvement of the immune system such as complement dependent cytotoxicity. The combination of RIT with immunotherapy presents synergistic killing in mouse melanoma models. The field of radiolabeled small molecules is focused on radioiodinated compounds that have the ability to cross the cellular membranes to access intracellular melanin and can be applied in both therapy and imaging as theranostics. Clinical applications of targeting melanin with radionuclide therapies have produced encouraging results and clinical work is on-going. Continued work on targeting melanin with radionuclide therapy as a monotherapy, or possibly in combination with standard of care agents, has the potential to strengthen the current treatment options for melanoma patients.

Polymyxin B in Combination with Glycerol Monolaurate Exerts Synergistic Killing against Gram-Negative Pathogens.

The rapid emergence and spread of multidrug-resistant (MDR) bacterial pathogens pose a serious danger to worldwide human health, and resistance to last-resort drugs, such as polymyxins, is being increasingly detected in MDR Gram-negative pathogens. There is an urgent need to find and optimize combination therapies as an alternative therapeutic strategy, with a dry pipeline in novel antibiotic research and development. We found a monoester formed from the combination of lauric acid and glycerol, glycerol monolaurate (GML), possessing prominent antibacterial and anti-inflammatory activity. However, it is still unclear whether GML in combination could increase antimicrobial activity. Here, we reported that polymyxin B (PMNB) combined with GML exhibited a synergistic antimicrobial impact on Gram-negative strains in vitro, including clinical MDR isolates. This synergistic antimicrobial activity correlated with the destruction of bacterial cell structures, eradication of preformed biofilms, and increased reactive oxygen species (ROS) accumulation. We also showed that PMNB synergized with GML effectively eliminated pathogens from bacterial pneumonia caused by Klebsiella pneumoniae to rescue mice. Our research demonstrated that the PMNB and GML combination induced synergistic antimicrobial activity for Gram-negative pathogens in vitro and in vivo. These findings are of great importance for treating bacterial infections and managing the spread of infectious diseases.

Facile synthesis of ε-poly-L-lysine-conjugated ZnO@PDA as photothermal antibacterial agents for synergistic bacteria killing and biofilm eradication

Nanomaterial-based antimicrobial strategies have emerged as powerful tools to combat microbial resistance, avoiding side effects of misusing antibiotics. In this work, the zinc oxide-based nanohybrid (designated as ZnO@PDA-ε-PL) as broad-spectrum antibacterial agents were fabricated in a facile two-step process. To be specific, ZnO was coated with near-infrared (NIR)-photoresponsive polydopamine (PDA), and then coupled with the antimicrobial peptide (AMP) (ε-poly-L-lysine, ε-PL). The as-prepared ZnO@PDA-ε-PL can effectively contact and capture bacteria, and then exhibit an excellent photothermal effect, i.e., the temperature rises rapidly to 53.2 °C (100 μg/ml) under 808 nm NIR light irradiation for 10 min. The synergistic effect of ε-PL, ZnO and photothermal agent PDA contributes to the excellent antibacterial efficiency of ZnO@PDA-ε-PL (<100 μg/ml) with 808 nm irradiation, that is, 99.70% and 100.00% against Gram-negative bacteria (Escherichia coli, E. coli) and Gram-positive bacteria (Staphyloccocus aureus, S. aureus), respectively. Further experiments indicated the ZnO@PDA-ε-PL ultimately lead to bacterial death by disrupting cell integrity, leaking of intracellular components (e.g., nucleic acids). Moreover, ZnO@PDA-ε-PL can effectively remove 70.84% of S. aureus biofilms. Overall, NIR-responsive ZnO@PDA-ε-PL has great potential in the field of infection treatment.

Proteomics Study of the Synergistic Killing of Tigecycline in Combination With Aminoglycosides Against Carbapenem-Resistant Klebsiella pneumoniae.

Co-administration of antibiotics with synergistic effects is one method to combat carbapenem-resistant organisms. Although the synergistic effects of tigecycline combined with aminoglycosides against carbapenem-resistant Klebsiella pneumoniae (CRKP) have been demonstrated in vitro and in animal models, the underlying mechanism remains elusive. Here we used proteomics analysis to assess the short-term bacterial responses to tigecycline and aminoglycosides alone or in combination. Emergence of tigecycline resistance during treatment and the susceptibility of tigecycline-resistant strains to aminoglycosides was further evaluated. The proteomic responses to tigecycline and aminoglycosides were divergent in monotherapy, with proteomic alterations to combination therapy dominated by tigecycline. Adaptive responses to tigecycline were associated with the upregulation of oxidative phosphorylation and translation-related proteins. These responses might confer CRKP hypersensitivity towards aminoglycosides by increasing the drug uptake and binding targets. Meanwhile, tigecycline might perturb adaptive responses to aminoglycosides through inhibition of heat shock response. Tigecycline-resistant strains could be isolated within 24 h exposure even in strains without heteroresistance, and the sensitivity to aminoglycosides significantly increased in resistant strains. Overall, these findings demonstrated that adaption to tigecycline in CRKP was a double-edged sword associated with the synergistic killing in tigecycline–aminoglycoside combination. Evolutionary hypersensitivity can provide novel insight into the mechanisms of antibiotic synergistic effects.

Abstract 4012: EGFR inhibition in combination with PI3K/AKT/mTOR and/or Bcl-2 inhibition is a promising novel therapeutic approach for inverted sinonasal papillomas and associated sinonasal carcinomas

Abstract Introduction: Activating EGFR mutations are characteristic of inverted sinonasal papillomas (ISP) and ISP-associated sinonasal carcinomas (SNC). Using standard 2D culture models, our group has shown that targeting EGFR activity in ISP-associated SNC effectively inhibits cellular proliferation in vitro. Targeting EGFR in pre-clinical models that are more representative of in vivo patient responses is a critical next step. Methods: High-throughput drug screening was conducted using a 3D in vitro culture model for two ISP-associated SNC cell lines (SCCNC4 and UM-SCC-112) using a library of &amp;gt;800 curated small molecule inhibitors including inhibitors of EGFR, PI3K/AKT/mTOR (P/A/M), and Bcl-2. Viability was assessed and drug sensitivity scores were calculated. Dual combinations (inhibition of EGFR and P/A/M or Bcl-2) and triple combinations (inhibition of EGFR, P/A/M, and Bcl-2) for select drugs were tested for synergistic killing using the Chou-Talalay and Bliss methods. Treatment-associated transcriptomic changes were monitored using whole-transcriptome RNAseq. Finally, patient-derived organoids (PDOs) from primary sinonasal papilloma or carcinoma tissue were utilized ex vivo for short-term drug screening with EGFR, P/A/M, and/or Bcl-2 inhibitors at maximum human plasma concentration (Cmax) format. Results: Drug screening demonstrated that ISP-associated SNC cell lines are highly resistant to a diversity of standard and targeted therapeutic agents. Indeed, these cell lines demonstrated poorer responses than other highly resistant tumor types previously analyzed by our group, including triple-negative breast cancer and bladder cancer. Our data demonstrated that EGFR inhibition alone does not effectively kill ISP-associated SNC cells in 3D culture; however, dual and triple combinations of EGFR and P/A/M and/or Bcl-2 inhibitors showed synergistic killing. Finally, sinonasal papilloma and carcinoma PDOs showed partial responses to selected EGFR inhibitors in 3D ex vivo culture. Conclusions: While EGFR inhibitors effectively inhibit cellular proliferation in ISP-associated SNC cell lines in 2D culture, drug screening in 3D culture and using PDO material - more representative models of in vivo patient responses - show that EGFR inhibition alone is insufficient to completely eliminate these tumor cells, suggesting that EGFR inhibitors alone are unlikely to generate significant clinical responses for patients with ISP or associated SNCs. However, combining targeting of the P/A/M and/or Bcl-2 pathways revealed synergistic killing of ISP-associated SNC cell lines. Taken together, these data demonstrate that compensatory oncogenic and/or anti-apoptotic pathways must be targeted to potentiate EGFR inhibition for successful treatment of ISP and associated SNCs. Citation Format: Athena M. Apfel, Zhaoping Qin, Albert Liu, Matthew B. Soellner, Sofia D. Merajver, Aaron M. Udager, Nathan M. Merrill. EGFR inhibition in combination with PI3K/AKT/mTOR and/or Bcl-2 inhibition is a promising novel therapeutic approach for inverted sinonasal papillomas and associated sinonasal carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4012.

Abstract 5339: BCL2A1 is expressed in myelofibrosis specimens and JAK2-mutated UKE-1 cells, yet does not inhibit synergistic cell killing by BCL-XL inhibitor navitoclax plus JAK1/2 inhibitors, including ruxolitinib

Abstract Myelofibrosis (MF) is driven by mutually exclusive mutations in JAK2, CALR, and MPL in hematopoietic stem cells, leading to constitutively active JAK-STAT signaling and consequently, increased proliferation and cell survival. The JAK1/2 inhibitor ruxolitinib (Rux) is standard of care in front line patients; however, JAK inhibition alone does not induce long-term remission or disease-modifying effects. The BCL-2 family pro-survival proteins BCL-XL and MCL-1 have been implicated in resistance to Rux, and combined activity between the BCL-XL/BCL-2 inhibitor navitoclax (Nav) and JAK2 inhibition has been described in preclinical models. The combination of Nav + Rux resulted in clinically meaningful responses (e.g., improvements in spleen volume, constitutional symptoms, anemia, and bone marrow fibrosis) in patients with suboptimal response to, or progression while on, Rux (Harrison et al., EHA 2021, EP1087). Nav does not have strong affinity for MCL-1 or BCL2A1. BCL2A1 has garnered recent attention given its potential role in tumor survival and drug resistance; however, its role in MF is unknown. Given the potential of Nav + Rux in MF, we evaluated the expression of BCL-2 pro-survival proteins, including BCL2A1, in MF patient specimens and assessed the ability of BCL2A1 to serve as a resistance factor to Nav + Rux in vitro. DNA/RNA was extracted from PBMCs from healthy donors (n=15), JAK1/2-inhibitor naïve patients with MF (n=18), and suboptimal responders or progressors (R/R MF) currently on stable dose of Rux (n=72). When compared to healthy donors, expression of BCL2L1, MCL1 and BCL2A1, but not BCL2, was higher in naïve and R/R MF patients, with greatest increase observed in BCL2L1. Increase in median expression in naïve patients vs healthy donors for BCL2L1, MCL1, and BCL2A1 was ~4-, 1.5-, and 2-fold, respectively. A similar trend was observed with R/R MF patients vs healthy donors. We next evaluated BCL-2 family expression in in vitro models of MPN. Notably, MCL1 expression was high in UKE-1, SET-2 and HEL cell lines, while high expression of BCL2A1 was only observed in UKE-1. The combination of Nav plus either Rux, fedratinib or momelitinib induced synergistic cell killing in all lines. These data suggest that although MCL1 and BCL2A1 are expressed in JAK2-mutated cells and other malignancies characterized by constitutive JAK/STAT signaling, they do not confer resistance to a Nav + JAK-inhibitor combination. In summary, our data demonstrate dysregulated pro-survival BCL-2 family member expression, including MCL1 and BCL2A1, in MF patient samples and JAK2-mutated cell lines, and further implicate BCL-XL as a major survival factor in both naïve and Rux-treated R/R MF patients. Together these data support a Nav/JAK1/2-inhibitor combination for both JAK1/2-inhibitor naïve patients, as well as those no longer benefiting from Rux therapy. Citation Format: Marion Refici, Ziping Yang, Jacob Riehm, Darren Phillips, Andrew Souers, Jason Harb. BCL2A1 is expressed in myelofibrosis specimens and JAK2-mutated UKE-1 cells, yet does not inhibit synergistic cell killing by BCL-XL inhibitor navitoclax plus JAK1/2 inhibitors, including ruxolitinib [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5339.

Abstract 1268: Early proliferative response via [18]FLT-PET/CT and serum TK1 activity are predictive biomarkers for synergistic effect of sequential cell cycle targeted therapy in sarcoma

Abstract Introduction: Sarcomas are rare, aggressive tumors that make up a higher percentage of overall cancer mortality in children and young adults. Primary management of sarcoma patients includes surgical resection with adjuvant or neoadjuvant radiation/chemotherapy. Even with aggressive multimodal treatment regimens, patients with high-grade sarcomas experience suboptimal prognosis and advanced disease is almost universally fatal. Innovative biomarker driven targeted therapies are in urgent need to improve survival in these patients. Discoveries in molecular and genetic basis of sarcoma have revealed new targets that deserve consideration. Inactivation of retinoblastoma (Rb) pathway, which causes unabated cell proliferation, through overexpression of cyclin D1 and/or CDK4/6 has been detected in the majority of sarcomas even when wild-type Rb protein is expressed. Hence, inhibition of cell cycle progression through reactivation of the tumor suppressor function of Rb by inhibiting CDK4/6 activity poses a potential avenue for new therapeutic options. Methods: A panel of 4 sarcoma cell lines and 4 patient derived xenograft (PDX) models were used: 2 with an intact Rb pathway (Rb+ve, p16-ve) and 2 with an altered Rb pathway (Rb low/-ve, p16+ve). CDK4/6 inhibitor, abemaciclib, was used to reactivate the Rb pathway followed by treatment with gemcitabine or WEE1-kinase-inhibitor, to target the enriched S and G2/M phase cells post-abemaciclib removal. 18F-FLT PET imaging and serum thymidine kinase 1 (TK1) were used to monitor changes in cell proliferation and treatment response in both in vitro and in vivo. Changes in cell cycle biomarkers in response to treatment were also interrogated. Results: Rb+ve sarcoma cell lines were sensitive to the anti-proliferative activity of abemaciclib leading to their reversible G1 arrest, while Rb null cells did not arrest in any phase of cell cycle in response to CDK4/6 inhibition. Following abemciclib removal, Rb+ve models (but not Rb-ve) showed significant increase in 18F-FLT biotracer uptake concomitant with elevated serum TK1 and enrichment of cells in S phase. The enrichment of cells in S phase maximized therapeutic efficacy of S-phase or G2/M targeting agents leading to synergistic cell killing in Rb+ve cell lines and PDX models. Conclusion: Our study presents an innovative treatment strategy inhibiting the Rb pathway followed by S or G2/M pathway sequentially resulting in synergistic cell killing in those cells/tumors with wild-type Rb. We also incorporate real-time PET imaging and serum TK1 as non-invasive indicators of response to the treatment strategy. The study with PDX models also identified biomarkers of cell proliferation to be used in conjunction with PET imaging to provide the pre-clinical rationale to translate our findings into clinical trials for this aggressive disease. Citation Format: Tuyen Duong Nguyen, Marc A. Pina, Yan Wang, Cansu Karakas, Tuyen N. Bui, Osama Mawlawi, Alexander J. Lazar, Davis R. Ingram, Peter J. Hougton, Kelly K. Hunt, Khandan Keyomarsi. Early proliferative response via [18]FLT-PET/CT and serum TK1 activity are predictive biomarkers for synergistic effect of sequential cell cycle targeted therapy in sarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1268.

An open-label, multicenter, randomized phase II study of atezolizumab and bevacizumab with Y90 TARE in patients with unresectable hepatocellular carcinoma (HCC).

TPS4177 Background: The anti–PD-L1 antibody atezolizumab (ATEZO) prevents PD-L1 from interacting with PD-1 and B7.1, thus reinvigorating antitumor T cell activity. Anti-VEGF bevacizumab (BEV) increases dendritic cell maturation, enhances T cell infiltration, and reduces myeloid-derived suppressor cells and regulatory T cells in tumors. ATEZO + BEV is FDA approved for first-line treatment of advanced HCC based on the IMbrave 150 study. Locoregional radiotherapy (e.g., Y90 TARE) enhances the diversity of the intratumoral T cell receptor repertoire and increases tumor antigen release. We hypothesize that the Y90 TARE + BEV + ATEZO combination induces synergistic tumor killing and prolongs progression-free survival in patients (pts) receiving Y90 TARE (HR = 0.6 when compared to Y-90 TARE alone). Methods: Eligible pts have HCC that cannot be surgically resected (confirmed by pathology review), is at least BCLC stage B, and is outside Milan criteria. Other requirements include ECOG PS of 0-1 at screening, measurable disease by RECIST 1.1, no prior systemic therapy, and FLR estimated at ≥ 40% post local therapy. Pts must have a pretreatment liver biopsy taken and then be randomized 1:1 to TARE (Arm A) or TARE + ATEZO + BEV (Arm B). Pts will have TARE mapping followed by TARE treatment. In Arm B, pts will begin TARE treatment followed by BEV + ATEZO (4 wks [± 1 wk] later). Pts will have abdominal MRI or CT scans every 12 weeks and CT scans of the chest every 24 wks. The primary study objective is to assess and compare pts' progression-free survival (per mRECIST 1.1) in each study arm. The main secondary objective is to determine the safety and tolerability (CTCAE v5) of pts in Arm B. Exploratory objectives are to define the use of cellular and circulating biomarkers in the prediction of improved clinical outcomes of pts in Arm B. Symptoms experienced by pts in both arms using patient-reported outcomes will be assessed. Disease progression will be captured by both RECIST 1.1 and mRECIST. We plan to assess the safety of Y90 TARE + BEV + ATEZO in the first 10 pts randomized to Arm B for two cycles, and if there are no grade ≥ 3 unexpected toxicities possibly, probably, or definitely related to combined TARE + BEV + ATEZO, continue to accrue 128 pts in total (current enrollment n- = 5). Pts will continue study treatment (Arm B) for a total of 24 months from initiation of TARE or until intolerable toxicity or disease progression occur, whichever is earlier. Enrollment began in September 2020. Clinical trial information: NCT04541173.

Identifying Small Molecule Compounds that Have Synergistic Effects With Cisplatin and Other Platinum Drugs Using High Throughput Matrix Screening

PURPOSE High grade serous ovarian cancer (“HGSOC”) is the seventh most common cancer worldwide among women. The disease has no unique symptoms and often women are diagnosed in advance stages. The World Health Organization estimates that 225,500 women are diagnosed and 140,200 will die of this disease per year. In the United States, the 5-year survival rate is 43%, and 85% of all HGSOC patients experience recurrence despite aggressive treatment. More efficacious drug combination treatments are needed. Recently it was found that platinum drugs are inactivated in dimethyl sulfoxide (DMSO). Thus drug discovery platforms that use it as a solvent are not able to study platinum drugs in combination with other drugs easily. Advances in Echo technology at the National Center for Advancing Translational Sciences (NCATS) enabled acoustic transfer of platinum drugs in aqueous solutions. This allows us to find novel small molecule/platinum drug combinations that can be tested in clinical trials. METHODS We ran single-agent dose-response experiments to correctly estimate the dose-ranges needed for the matrix screens utilizing the OVCAR8 human carcinoma cell line. Then, we conducted our pilot matrix screen with compounds that are commonly combined with cisplatin for HGSOC treatment. Lastly, we spotted cisplatin against the Mechanism Interrogation PlatE drug library to identify novel combinations for clinical use against HGSOC. Compounds that show synergistic cell killing will give insight into novel drug combinations to treat HGSOC. RESULTS Several synergistic and antagonistic effects were found. These results were refined with smaller-scale screening and tested in other HGSOC cell lines. CONCLUSION The novel combinations discovered will allow chemotherapy patients to receive lower dosages of platinum drugs while maintaining or improving efficacy of cytotoxicity of cancer cells. This will help alleviate the side-effects of platinum drugs experienced by HGSOC patients.

IKAROS and MENIN coordinate therapeutically actionable leukemogenic gene expression in MLL-r acute myeloid leukemia.

Acute myeloid leukemia (AML) remains difficult to treat and requires new therapeutic approaches. Potent inhibitors of the chromatin-associated protein MENIN have recently entered human clinical trials, opening new therapeutic opportunities for some genetic subtypes of this disease. Using genome-scale functional genetic screens, we identified IKAROS (encoded by IKZF1) as an essential transcription factor in KMT2A (MLL1)-rearranged (MLL-r) AML that maintains leukemogenic gene expression while also repressing pathways for tumor suppression, immune regulation and cellular differentiation. Furthermore, IKAROS displays an unexpected functional cooperativity and extensive chromatin co-occupancy with mixed lineage leukemia (MLL)1-MENIN and the regulator MEIS1 and an extensive hematopoietic transcriptional complex involving homeobox (HOX)A10, MEIS1 and IKAROS. This dependency could be therapeutically exploited by inducing IKAROS protein degradation with immunomodulatory imide drugs (IMiDs). Finally, we demonstrate that combined IKAROS degradation and MENIN inhibition effectively disrupts leukemogenic transcriptional networks, resulting in synergistic killing of leukemia cells and providing a paradigm for improved drug targeting of transcription and an opportunity for rapid clinical translation.

Photocatalytic Quantum Dot-Armed Bacteriophage for Combating Drug-Resistant Bacterial Infection.

Multidrug‐resistant (MDR) bacterial infection is one of the greatest challenges to public health, a crisis demanding the next generation of highly effective antibacterial agents to specifically target MDR bacteria. Herein, a novel photocatalytic quantum dot (QD)‐armed bacteriophage (QD@Phage) is reported for combating green fluorescent protein‐expressing Pseudomonas aeruginosa (GFP‐P. aeruginosa) infection. The proposed QD@Phage nanosystem not only specifically binds to the host GFP‐P. aeruginosa while preserving the infectivity of the phage itself, but also shows a superior capacity for synergistic bacterial killing by phage and by the photocatalytic localized reactive oxygen species (ROS) generated from anchored QD components. Notably, this highly targeted QD@Phage nanosystem achieves robust in vitro antibacterial elimination for both planktonic (over 99.9%) and biofilm (over 99%) modes of growth. In a mouse wound infection model, this system also shows remarkable activity in eliminating the wound infection and promoting its recovery. These results demonstrate that the novel QD@Phage nanosystem can diversify the existing pool of antibacterial agents and inspire the development of promising therapeutic strategies against MDR bacterial infection.

Interplay between Amoxicillin Resistance and Osmotic Stress in Helicobacter pylori.

Rising antibiotic resistance rates are a growing concern for all pathogens, including Helicobacter pylori. We previously examined the association of specific mutations in PBP1 with amoxicillin resistance and fitness in H. pylori and found that V374L and N562Y mutations were associated with resistance, but also resulted in fitness defects. Furthermore, we found that hyperosmotic stress differentially altered the fitness of strains bearing these mutations; survival of the V374L strain was decreased by hyperosmotic stress, but the N562Y strain showed increased cell survival relative to that of wild-type G27. The finding that amoxicillin-resistant strains show environmentally dictated changes in fitness suggests a previously unexplored interaction between amoxicillin resistance and osmotic stress in H. pylori. Here, we further characterized the interaction between osmotic stress and amoxicillin resistance. Wild-type and isogenic PBP1 mutant strains were exposed to amoxicillin, various osmotic stressors, or combined antibiotic and osmotic stress, and viability was monitored. While subinhibitory concentrations of NaCl did not affect H. pylori viability, the combination of NaCl and amoxicillin resulted in synergistic killing; this was true even for the antibiotic-resistant strains. Moreover, similar synergy was found with other beta-lactams, but not with antibiotics that did not target the cell wall. Similar synergistic killing was also demonstrated when KCl was utilized as the osmotic stressor. Conversely, osmolar equivalent concentrations of sucrose antagonized amoxicillin-mediated killing. Taken together, our results support a previously unrecognized interaction between amoxicillin resistance and osmotic stress in H. pylori. These findings have interesting implications for the effectiveness of antibiotic therapy for this pathogen. IMPORTANCE Rising antibiotic resistance rates in H. pylori are associated with increased rates of treatment failure. Understanding how stressors impact antibiotic resistance may shed light on the development of future treatment strategies. Previous studies found that mutations in PBP1 that conferred resistance to amoxicillin were also associated with a decrease in bacterial fitness. The current study demonstrated that osmotic stress can enhance beta lactam-mediated killing of H. pylori. The source of osmotic stress was found to be important for these interactions. Given that relatively little is known about how H. pylori responds to osmotic stress, these findings fill important knowledge gaps on this topic and provide interesting implications for the effectiveness of antibiotic therapy for this pathogen.

Mismatch repair proteins play a role in ATR activation upon temozolomide treatment in MGMT-methylated glioblastoma

The methylation status of the O6-methylguanine methyltransferase (MGMT) gene promoter has been widely accepted as a prognostic biomarker for treatment with the alkylator, temozolomide (TMZ). In the absence of promoter methylation, the MGMT enzyme removes O6-methylguanine (O6-meG) lesions. In the setting of MGMT-promoter methylation (MGMT-), the O6-meG lesion activates the mismatch repair (MMR) pathway which functions to remove the damage. Our group reported that loss of MGMT expression via MGMT promoter silencing modulates activation of ataxia telangiectasia and RAD3 related protein (ATR) in response to TMZ treatment, which is associated with synergistic tumor-cell killing. Whether or not MMR proteins are involved in ATR activation in MGMT-cells upon alkylation damage remains poorly understood. To investigate the function of MMR in ATR activation, we created isogenic cell lines with knockdowns of the individual human MMR proteins MutS homolog 2 (MSH2), MutS homolog 6 (MSH6), MutS homolog 3 (MSH3), MutL homolog 1 (MLH1), and PMS1 homolog 2 (PMS2). Here, we demonstrate that MSH2, MSH6, MLH1 and PMS2, specifically, are involved in the activation of the ATR axis after TMZ exposure, whereas MSH3 is likely not. This study elucidates a potential mechanistic understanding of how the MMR system is involved in ATR activation by TMZ in glioblastoma cells, which is important for targeting MMR-mutated cancers.