Efficient Combination Therapy Research Articles

Assembly of Glycopeptides in Living Cells Resembling Viral Infection for Cargo Delivery

AbstractSelf‐assembly in living cells represents one versatile strategy for drug delivery; however, it suffers from the limited precision and efficiency. Inspired by viral traits, we here report a cascade targeting‐hydrolysis‐transformation (THT) assembly of glycosylated peptides in living cells holistically resembling viral infection for efficient cargo delivery and combined tumor therapy. We design a glycosylated peptide via incorporating a β‐galactose‐serine residue into bola‐amphiphilic sequences. Co‐assembling of the glycosylated peptide with two counterparts containing irinotecan (IRI) or ligand TSFAEYWNLLSP (PMI) results in formation of the glycosylated co‐assemblies SgVEIP, which target cancer cells via β‐galactose‐galectin‐1 association and undergo galactosidase‐induced morphological transformation. While GSH‐reduction causes release of IRI from the co‐assemblies, the PMI moieties release p53 and facilitate cell death via binding with protein MDM2. Cellular experiments show membrane targeting, endo‐/lysosome‐mediated internalization and in situ formation of nanofibers in cytoplasm by SgVEIP. This cascade THT process enables efficient delivery of IRI and PMI into cancer cells secreting Gal‐1 and overexpressing β‐galactosidase. In vivo studies illustrate enhanced tumor accumulation and retention of the glycosylated co‐assemblies, thereby suppressing tumor growth. Our findings demonstrate an in situ assembly strategy mimicking viral infection, thus providing a new route for drug delivery and cancer therapy in the future.

Assembly of Glycopeptides in Living Cells Resembling Viral Infection for Cargo Delivery.

Self-assembly in living cells represents one versatile strategy for drug delivery; however, it suffers from the limited precision and efficiency. Inspired by viral traits, we here report a cascade targeting-hydrolysis-transformation (THT) assembly of glycosylated peptides in living cells holistically resembling viral infection for efficient cargo delivery and combined tumor therapy. We design a glycosylated peptide via incorporating a β-galactose-serine residue into bola-amphiphilic sequences. Co-assembling of the glycosylated peptide with two counterparts containing irinotecan (IRI) or ligand TSFAEYWNLLSP (PMI) results in formation of the glycosylated co-assemblies SgVEIP, which target cancer cells via β-galactose-galectin-1 association and undergo galactosidase-induced morphological transformation. While GSH-reduction causes release of IRI from the co-assemblies, the PMI moieties release p53 and facilitate cell death via binding with protein MDM2. Cellular experiments show membrane targeting, endo-/lysosome-mediated internalization and in situ formation of nanofibers in cytoplasm by SgVEIP. This cascade THT process enables efficient delivery of IRI and PMI into cancer cells secreting Gal-1 and overexpressing β-galactosidase. In vivo studies illustrate enhanced tumor accumulation and retention of the glycosylated co-assemblies, thereby suppressing tumor growth. Our findings demonstrate an in situ assembly strategy mimicking viral infection, thus providing a new route for drug delivery and cancer therapy in the future.

Dual regulation of the orderly aggregation of covalent organic frameworks at the molecular level and nanoscale to achieve efficient phototherapy and gene therapy

Numerous studies have established the promise of combining phototherapy and gene therapy. However, there are persistent obstacles hindering the full optimization of this combined therapeutic approach, such as the maximum development of photosensitizers and the construction of high-performance gene carriers. The LEGO stacking characteristics of COFs have brought infinite imagination to the construction of delivery systems, allowing for the design of responsive nano castles based on different application scenarios. Here, we have constructed a novel cationic multifunctional nano COF nucleic acid delivery system based on photosensitizer and cationic monomers. The long-range ordered structure of COF effectively regulates the spatial distribution of photosensitizer, thereby preserving their phototherapy efficacy. Simultaneously, the inherent cationic porous nanostructure equips COF with exceptional capabilities for delivering nucleic acids. This integrated multifunctional nano delivery system significantly enhances the combined efficacy of phototherapy and gene therapy. The phototherapy capacity of COF enables precise tumor elimination in a spatiotemporal manner, inducing robust ICD and activating immune cells at the tumor site. Furthermore, loaded siPD-L1 significantly enhances immune cell recognition and killing of tumor cells, effectively shielding mice from re-invasion and tumor metastasis. The development of multifunctional nanomedicine based on COF provides new ideas for efficient tumor combination therapy.

Small Warriors of Nature: Novel Red Emissive Chlorophyllin Carbon Dots Harnessing Fenton-Fueled Ferroptosis for In Vitro and In Vivo Cancer Treatment.

The appeal of carbon dots (CDs) has grown recently, due to their established biocompatibility, adjustable photoluminescence properties, and excellent water solubility. For the first time in the literature, copper chlorophyllin-based carbon dots (Chl-D CDs) are successfully synthesized. Chl-D CDs exhibit unique spectroscopic traits and are found to induce a Fenton-like reaction, augmenting photodynamic therapy (PDT) efficacies via ferroptotic and apoptotic pathways. To bolster the therapeutic impact of Chl-D CDs, a widely used cancer drug, temozolomide, is linked to their surface, yielding a synergistic effect with PDT and chemotherapy. Chl-D CDs' biocompatibility in immune cells and in vivo models showed great clinical potential.Proteomic analysis was conducted to understand Chl-D CDs' underlying cancer treatment mechanism. The study underscores the role of reactive oxygen species formation and pointed toward various oxidative stress modulators like aldolase A (ALDOA), aldolase C (ALDOC), aldehyde dehydrogenase 1B1 (ALDH1B1), transaldolase 1 (TALDO1), and transketolase (TKT), offering a deeper understanding of the Chl-D CDs' anticancer activity. Notably, the Chl-D CDs' capacity to trigger a Fenton-like reaction leads to enhanced PDT efficiencies through ferroptotic and apoptotic pathways. Hence, it is firmly believed that the inherent attributes of Chl-CDs can lead to a secure and efficient combined cancer therapy.

A graphene oxide-based sequential nanocatalyst for efficient tumor combination therapy

We construct a simple, biocompatible, sequential catalytic nanocatalyst PMO@GO-GOD to realize enhanced chemodynamic therapy and photothermal therapy for combating breast cancers by taking advantage of the peroxidase-like activity of GO.

Gallic Acid Attenuates Cardiovascular and Hematological Complications in Vincristine Treatment via Hydroxyl Radical Scavenging and Endogenous Antioxidant Stimulation in Male Wistar Rats

The menace of cancer and the nightmare of complications of cancer chemotherapy have driven researchers to explore simple but efficient combination therapy that includes antioxidants, in cancer therapy. The ability of gallic acid to correct the toxic complication of Vincristine was investigated. Twenty adult male rats of the Wistar strain were grouped into four, randomly, consisting of five rats each. The untreated control (group A) was given only distilled water, groups B and C 0.025 mg/kg Vincristine sulfate intraperitoneally once a week for two weeks. Group C rats were thereafter administered 100 mg/kg gallic acid daily by gastric gavage for 14 days. At 14 days, blood pressure and ECG were measured in the rats, then blood samples were obtained via the retrorbital venous plexus for determination of haematological parameters and plasma biochemistry. They were then euthanized through cervical dislocation, under ether anaesthesia, and liver, kidneys, heart, and brain samples were collected, weighed, and stored for determination of marker of oxidative stress in the post mitochondrial fractions of each organ. Results of the study showed that rats in group B had hypertension as evidenced by elevated diastolic and systolic as well as mean arterial pressure while QT interval and corrected QT were slightly elongated. They also had lowered RBC, WBC, and granulocyte counts. Markers of oxidative stress, GSH, and SOD were also depleted while H2O2 generation increased in this group, whereas all the observed anomalies were corrected in the group C rats that were administered both Vincristine and gallic acid. This study further showed that Vincristine, at normal recommended therapeutic dosage is toxic, causing anaemia, panleucopenia, and cardiovascular anomalies via oxidative stress and generation of hydroxyl radicals. These were however corrected by concurrent administration of gallic acid

Enhancement of cancer immunotherapy using CRT valgus tumor cell membranes coated bacterial whole peptidoglycan combined with radiotherapy

Immunotherapy has achieved some success in preclinical and clinical studies, but the immunosuppressive tumor microenvironment (TME) leads to a low response rate of this therapy. In this paper, we describe a calreticulin (CRT) valgus CT-26 tumor cell membranes-coated bacterial whole peptidoglycan (WPG) from P. aeruginosa (CPW/SR) with a high rate of the STING agonist loading. In the construct, WPG from P. aeruginosa (P.WPG) was used as a carrier with the immunoadjuvant function while synergistically promoting the maturation of dendritic cells (DCs) through the delivery of the STING agonist SR-717. CRT valgus tumor cell membranes were identified and internalized by DCs via CRT on the surface. In addition, this construct was able to reverse the immunosuppressive TME in vivo and achieve synergies with radiotherapy by creating a personalized tumor vaccine, therefore achieving more resultful antitumor efficacy. In conclusion, CPW/SR constructed in this paper provides a new approach for achieving efficient cancer immunotherapy and combination therapy.

Camouflaging multifunctional nanoparticles with bacterial outer membrane for augmented chemodynamic/photothermal/starvation/chemo multimodal synergistic therapy of orthotopic glioblastoma

Innovative therapeutic strategy and effective drug delivery are significant challenges in current glioblastoma multiforme (GBM) therapy. The development of efficient combination therapy based on chemodynamic therapy (CDT) has attracted widespread attention in recent years. Herein, a lipopolysaccharide-free bacterial outer membrane vesicles (dOMV) camouflaged biomimetic nanoreactor was constructed for quadruple synergistic therapy of orthotopic GBM. Benefiting from the immune escaping property and blood–brain barrier (BBB) penetration capacity of dOMV, nanoreactors can target cancer cells and accumulate in the tumor site. Firstly, for quadruple synergistic therapy, Glucose oxidase (GOx) can rapidly consume intratumoral glucose to generate H2O2 and gluconic acid for starvation therapy. Secondly, the abundant H2O2 produced by the enzyme-catalyzed reaction can continuously supply Cu9S8-mediated Fenton-like reaction to enhance CDT. Meanwhile, tumor hypoxic microenvironment exacerbated by GOx-induced oxygen depletion activated prodrug AQ4N for chemotherapy. Finally, the second near-infrared (NIR-II) absorbed Cu9S8 increased the temperature in local tumor site upon NIR-II laser irradiation, which can achieve photothermal-enhanced CDT, elevate GOx activity, and accelerate drug release. In vitro and in vivo results demonstrated that the camouflaging multifunctional nanoparticles with bacterial outer membrane (AG@Cu9S8@dOMV) was a promising multifunctional therapeutic nanomedicine platform for highly efficient treatment of GBM, which may be extended to as a treatment option for clinical cancer theranostics.

Combination Therapies with Kinase Inhibitors for Acute Myeloid Leukemia Treatment.

Targeting kinase activity is considered to be an attractive therapeutic strategy to overcome acute myeloid leukemia (AML) since aberrant activation of the kinase pathway plays a pivotal role in leukemogenesis through abnormal cell proliferation and differentiation block. Although clinical trials for kinase modulators as single agents remain scarce, combination therapies are an area of therapeutic interest. In this review, the author summarizes attractive kinase pathways for therapeutic targets and the combination strategies for these pathways. Specifically, the review focuses on combination therapies targeting the FLT3 pathways, as well as PI3K/AKT/mTOR, CDK and CHK1 pathways. From a literature review, combination therapies with the kinase inhibitors appear more promising than monotherapies with individual agents. Therefore, the development of efficient combination therapies with kinase inhibitors may result in effective therapeutic strategies for AML.

Insights from a 30-year journey: function, regulation and therapeutic modulation of PD1.

PD1 was originally discovered in 1992 as a molecule associated with activation-induced cell death in T cells. Over the past 30 years, it was found that PD1 has a critical role in avoiding overactivation-induced cell death and autoimmunity, whereas its inhibition unleashes anticancer immunity. Here, we outline the journey from the discovery of PD1 to its role as a breakthrough target in cancer immunotherapy. We describe its regulation and function and examine how a mechanistic understanding of PD1 signalling suggests a central function in setting the T cell activation threshold, thereby controlling T cell proliferation, differentiation, exhaustion and metabolic status. This threshold theory, in combination with new insights into T cell metabolism and a better understanding of immune cell modulation by the microbiota, can provide guidance for the development of efficient combination therapies. Moreover, we discuss the mechanisms underlying immune-related adverse events after PD1-targeted therapy and their possible treatment.

Trojan nanobacteria hybridized with prodrug nanocapsules for efficient combined tumor therapy

Trojan nanobacteria hybridized with prodrug nanocapsules for efficient combined tumor therapy

Molecular Engineering of NIR-II/IIb Emitting AIEgen for Multimodal Imaging-Guided Photo-Immunotherapy.

In view of the great challenges related to the complexity and heterogeneity of tumors, efficient combination therapy is an ideal strategy for eliminating primary tumors and inhibiting distant tumors. A novel aggregation-induced emission (AIE) phototherapeutic agent called T-TBBTD is developed, which features a donor-acceptor-donor (D-A-D) structure, enhanced twisted molecule conformation, and prolonged second near-infrared window (NIR-II) emission. The multimodal imaging function of the molecule has significance for its treatment time window and excellent photothermal/photodynamic performance for multimode therapy. The precise molecular structure and versatility provide prospects for molecular therapy for anti-tumor applications. Fluorescence imaging in the NIR-II window offers advantages with enhanced spatial resolution, temporal resolution, and penetration depth. The prepared AIE@R837 NPs also have controllable performance for antitumor photo-immunotherapy. Following local photo-irradiation, AIE@R837 NPs generate abundant heat, and 1 O2 directly kills tumor cells, induces immunogenic cell death (ICD) as a photo-therapeutic effect, and releases R837, which enhances the synergistic effect of antigen presentation and contributes to the long-lasting protective antitumor immunity. A bilateral 4T1 tumor model revealed that this photo-immunotherapy can eliminate primary tumors. More importantly, it has a significant inhibitory effect on distant tumor growth. Therefore, this method can provide a new strategy for tumor therapy.

Virtual screening reveals aprepitant to be a potent inhibitor of neutral sphingomyelinase 2: implications in blockade of exosome release in cancer therapy.

Exosomes are membrane-derived nano-vesicles upregulated in pathological conditions like cancer. Therefore, inhibiting their release is a potential strategy for the development of more efficient combination therapies. Neutral sphingomyelinase 2 (nSMase2) is a key component in exosome release; however, a clinically safe yet efficient nSMase2 inhibitor remains to be used discovered. Accordingly, we made an effort to identify potential nSMase2 inhibitor(s) among the approved drugs. Virtual screening was performed and aprepitant was selected for further investigation. To evaluate the reliability of the complex, molecular dynamics were performed. Finally, using the CCK-8 assay in HCT116 cells, the highest non-toxic concentrations of aprepitant were identified and the nSMase2 activity assay was performed to measure the inhibitory activity of aprepitant, in vitro. To validate the screening results, molecular docking was performed, and the retrieved scores were in line with the screening results. The root-mean-square deviation (RMSD) plot of aprepitant-nSMase2 showed proper convergence. Following treatment with different concentrations of aprepitant in both cell-free and cell-dependent assays, nSMase2 activity was remarkably decreased. Aprepitant, at a concentration as low as 15µM, was able to inhibit nSmase2 activity in HCT116 cells without any significant effects on their viability. Aprepitant is therefore suggested to be a potentially safe exosome release inhibitor.

Stimulus-responsive curcumin-based polydopamine nanoparticles for targeting Parkinson’s disease by modulating α-synuclein aggregation and reactive oxygen species

Parkinson’s disease (PD) is the second most common neurodegenerative disease, posing a significant threat to an individual’s health, particularly among the elderly. An incomplete understanding of the pathogenesis underlying PD renders the single therapeutic model ineffective. Oxidative stress and abnormal alpha-synuclein (α-Syn) aggregation are two crucial factors accounting for dopaminergic neuron loss. Therefore, combined PD therapy that inhibits and reduces α-Syn aggregation and regulates the balance of reactive oxygen species (ROS) holds promise. In this study a novel nanodrug, polydopamine-based curcumin-loaded nanoparticles (PC NPs), based on polydopamine (PDA)-assembled curcumin (Cur) nanoparticles was designed, which was then modified with a peptide obtained from rabies virus glycoprotein (RVG) 29 (obtained RPC NPs) to target the brain. Cur and PDA of ROS-responsive nanoparticles (RPC NPs) significantly scavenged a broad spectrum of ROS. Cur inhibited and removed α-Syn aggregation, thus protecting PC12 cells from rotenone-induced cell death. RPC NPs were also shown to prolong the lifespan of Caenorhabditis elegans and restored dopamine-dependent behaviors, including area-restricted searching, ethanol avoidance, and the basal slowing response, by using 6-hydroxydopamine (6-OHDA)-pretreated C. elegans wild-type Bristol (N2) and the nematode transgenic hus111 strain as models. In vivo animal studies showed that RPC NPs significantly lowered dopaminergic neuron damage and improved the neurobehavioral abnormalities in PD mice, while having no noticeable systemic side effects. RPC NPs also decreased α-Syn accumulation and reduced oxidative stress in the striatum and substantia nigra. This multifunctional synergetic strategy may provide a new candidate for highly efficient combination therapy in patients with PD.

Programmed-stimuli responsive carrier-free multidrug delivery system for highly efficient trimodal combination therapy

Programmed response, carrier-free, and multimodal therapy drug delivery systems (DDS) are promising solutions to multidirectional cytotoxic effects, inefficient antitumor, and severe side effects for cancer therapy. Here, three widely used clinical drugs, interferon α1b (IFNα1b), indocyanine green (ICG), and doxorubicin (DOX), were prepared into carrier-free DDS IFNα1b-ICG-DOX (IID) by a simple one-step method without additional any reagents. IID can achieve smart and programmed DDS by combining low pH and near-infrared (NIR) light stimuli-responsive controlled release. In pH = 7.4 environments, our IID is about 380 nm in size with negative charge rounded particles; while they enter into the acid environment (pH < 7), hydrogen ions (H+) trigger DOX release, their size becomes larger and the surface charge turns positive. These larger particles are rapidly disintegrated after exposure to NIR light and then the remaining DOX, IFNα1b, and ICG are released. In vivo, the IID with larger size and positive charge resulting from low pH is is easy to accumulate in tumor tissue. Tumors can be exposed to NIR light when needed to control the release of these three drugs. Hence, DOX, ICG, and IFNα1b can be enriched in the tumor to the high efficiency of combined chemotherapy, photothermal therapy, and immunotherapy.

Cancer-Responsive Multifunctional Nanoplatform Based on Peptide Self-Assembly for Highly Efficient Combined Cancer Therapy by Alleviating Hypoxia and Improving the Immunosuppressive Microenvironment

Hypoxia, as a main feature of the tumor microenvironment, has greatly limited the efficacy of photodynamic therapy (PDT), as well as its clinical application. Here, a multifunctional composite nanoplatform, the peptide/Ce6/MnO2 nanocomposite (RKCM), has been constructed to alleviate tumor hypoxia and increase the efficacy of PDT using rationally designed peptide fibrils to encapsulate chlorin e6 (Ce6) inside and to mineralize MnO2 nanoparticles on the surface. As a result, RKCM significantly improved the PDT efficacy by increasing reactive oxygen species (ROS) generation, decreasing tumor cell viability, and inhibiting tumor growth and metastasis. Besides, decreased HIF-1α expression and increased immune-activated cell infiltration were also observed in RKCM/laser treatment xenograft. Mechanically, (1) Ce6 can induce singlet oxygen (1O2) generation under laser irradiation to give photodynamic therapy (PDT); (2) MnO2 can react with H2O2 in situ to supply additional O2 to alleviate tumor hypoxia; and (3) the released Mn2+ ions can induce a Fenton-like reaction to generate •OH for chemical dynamic therapy (CDT). Moreover, RKCM/laser treatment also presented with an abscopal effect to block the occurrence of lung metastasis by remolding the pre-metastasis immune microenvironment. With these several aspects working together, the peptide/Ce6/MnO2 nanoplatform can achieve highly efficient tumor therapy. Such a strategy based on peptide self-assembly provides a promising way to rationally design a cancer-responsive multifunctional nanoplatform for highly efficient combined cancer therapy by alleviating hypoxia and improving the immune microenvironment.

Metal-rich cascade nanosystem for dual-pathway ferroptosis resistance regulation and photothermal effect for efficient tumor combination therapy.

Despite the therapeutic response of ferroptosis in various tumors, ferroptosis resistance has been found in numerous studies, significantly hindering the progress of ferroptosis anti-tumor therapy. Herein, we propose a metal-rich cascade nanosystem (Simvastatin-HMPB-Mn@GOx) combined with the dual-pathway regulation of ferroptosis resistance and photothermal therapy for efficient tumor combination therapy. The manganese-bonded hollow mesoporous Prussian blue (HMPB-Mn) serves as the photothermal agent and metal donor, and dissociates multivalent metal ions Mn2+, Fe3+ and Fe2+ to consume glutathione and amplify the Fenton reaction. Glucose oxidase (GOx) absorbed serves as the converter to provide hydrogen peroxide (H2O2) for the cascade Fenton reaction, causing a high burst of hydroxyl radicals (˙OH) and lipid peroxidation. Simvastatin innovatively acts as a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) inhibitor to decrease the expression of coenzyme Q10 (CoQ10) and glutathione peroxidase 4 (GPX4), eventually defeating ferroptosis resistance. The nanosystem acted in both classical and non-classical ferroptosis pathways and showed significant ferroptosis- and hyperthermia-induced anti-tumor efficacy both in vitro and in vivo. Thus, this study offers a promising way for ferroptosis and phototherapy to achieve complete tumor regression.

NIR-II Light Leveraged Dual Drug Synthesis for Orthotopic Combination Therapy.

Pd-catalyzed bioorthogonal bond cleavage reactions are widely used and frequently reported. It is circumscribed by low reaction efficiency, which may encumber the therapeutic outcome when applied to physiological environments. Herein, an NIR-II light promoted integrated catalyst (CuS@PDA/Pd) (PDA - polydopamine) is designed to accelerate the reaction efficiency and achieve a dual bioorthogonal reaction for combination therapy. As NIR-II light can penetrate deeply into tissue, the Pd-mediated cleavage reaction can be promoted both in vitro and in vivo by the photothermal properties of CuS, beneficial to orthotopic 4T1 tumor treatment. In addition, CuS also catalyzes the synthesis of active resveratrol analogs by the CuAAC reaction. These simultaneously produced anticancer agents result in enhanced antitumor cytotoxicity in comparison to the single treatments. This is a fascinating study to devise an integrated catalyst boosted by NIR-II light for dual bioorthogonal catalysis, which may provide the impetus for efficient bioorthogonal combination therapy in vivo.

Engineering biomimetic ATP-responsive Se-containing core-shell cascade nanozyme for efficient tumor combination therapy

The ineffectiveness of cancer chemodynamic treatment (CDT) is intimately connected to intracellular antioxidant defense mechanisms. While great efforts have been made to interfere with the oxidative protection mechanisms within tumor cells, this remains a difficult challenge. Herein, inspired by the glutathione peroxidase (GPx) function for consuming GSH, an artificial selenase was designed to combine with the Fenton reaction to prepare a metal complex nanocarrier (IM-Se-Ph@TA-Fe NPs), in which glucose oxidase (GOx) is loaded to obtain the multienzyme synergic nanozyme GOx@IM-Se-Ph@TA-Fe NPs. In response to the high expression of ATP in the tumor, released GOx could specifically reduce the molecular oxygen to H2O2 by consuming intratumoral glucose and cause tumor starvation. Following that, the generated H2O2 supported the intracellular Fenton reaction and consumed intracellular GSH under the catalysis of selenase to enhance multimodal cancer therapy. The strategy of interfering with the intracellular antioxidant system by utilizing artificial selenase and GOx increases the efficacy of chemodynamic therapy and offers new concepts for cancer combination therapy.

RF07 | PSUN356 Synergistic Combination of HSP90 and PI3K inhibitors in Adrenocortical Cancer

Abstract Adrenocortical cancer (ACC) is a rare and aggressive cancer (0.5-2 cases/ million/ year) with a poor 5-year survival (&amp;lt;40%). Most patients inevitably succumb to the wide-spread metastasis because of the lack of effective systemic treatment options or to complications from uncontrolled hypercortisolism. Therefore, it is critically important to identify new effective treatments that can be readily used in a clinical trial. In this direction, we identified a potent synergistic combination of Heat Shock Protein 90 (HSP90) and PI3K inhibitors via quantitative high-throughput drug screening (qHTS) for ACC, using a pharmaceutical library comprised of ∼5000 drugs (FDA approved or investigational), and evaluation of drug synergy by a computerized drug combination matrix analysis. The synergistic efficacy of HSP90 inhibitor STA9090 and PI3K inhibitor, PIK75 was validated in vitro, in ACC cell lines H295R and SW13, by proliferation assay, which also determined the most efficacious doses of individual and combinatorial drugs. The drug combination significantly induced apoptosis and disintegrated the 3D-ACC spheroids as compared to the individual drugs, with a simultaneous upregulation of apoptotic protein markers (cleaved-Caspase3, cleaved-PARP), inhibition of the phosphorylation of PI3K pathway members (AKT1, mTOR, S6K, 4EB1 and GSK3α/β), and alteration in the G2/M phase of the cell cycle, in ACC cells. The synergetic effect of the drug combination was also effective in reducing the metastatic potential as revealed by invasion/migration assay, which was confirmed by the downregulation of the mesenchymal protein (Vimentin, N-cadherin, SNAIL and Zeb1) markers. Consistent with our data, observations from the gene expression data from The Cancer Genome Analysis (TCGA) &amp; Gene Expression Omnibus (GEO) databases, revealed significant mRNA overexpression of HSP90AA1, HSP90AB1 HSP90B1 and its clients AKT2, CDK1, CDK4, NR3C1 indicating both HSP90 and PI3K/AKT pathways are highly activated in ACC cohorts. Overexpression and a positive correlation of mesenchymal markers TWIST1, ZEB1, VIM with HSP90AB1 and PI3KCA, in GEO and TCGA respectively, was consistent with the invasive potential of the ACC cells. In conclusion, our studies on the combination of HSP90 and PI3K inhibitors demonstrated a promising preliminary synergistic efficacy by inhibiting proliferation and epithelial-to-mesenchymal transition of ACC. Our findings can lead to development of an efficient mechanism-specific novel combination therapy that can be expedited to clinical trial in advanced ACC patients. Presentation: Saturday, June 11, 2022 1:12 p.m. - 1:17 p.m., Sunday, June 12, 2022 12:30 p.m. - 2:30 p.m.