Synergistic Inhibition Of Tumor Growth Research Articles

Polyvalent Aptamer Nanodrug Conjugates Enable Efficient Tumor Cuproptosis Therapy Through Copper Overload and Glutathione Depletion.

Cuproptosis, a recently identified form of copper-dependent cell death, shows promising tumor suppressive effects with minimal drug resistance. However, its therapeutic efficacy is hampered by its dependence on copper ions and the glutathione (GSH)-rich microenvironment in tumors. Here, we have developed polyvalent aptamer nanodrug conjugates (termed CuPEs@PApt) with a nucleosome-like structure to improve tumor cuproptosis therapy by exploiting mitochondrial copper overload and GSH depletion. Polyvalent aptamer (PApt), comprising polyvalent epithelial cell adhesion molecule aptamers for tumor targeting and repetitive PolyT sequences for copper chelation, facilitates efficient loading and targeted delivery of copper peroxide-Elesclomol nanodots (CuPEs). Upon internalization by tumor cells, Elesclomol released from CuPEs@PApt accumulates copper ions in mitochondria to initiate cuproptosis, while lysosomal degradation of CuP nanodots generates exogenous Cu2+ and H2O2, triggering a Fenton-like reaction for GSH depletion to enhance cuproptosis. In vitro and in vivo experiments confirm the efficacy of this strategy in inducing tumor cell cuproptosis and immunogenic cell death, the latter contributing to the activation of the antitumor immune response for synergistic tumor growth inhibition.

Novel gene therapy for drug-resistant melanoma: Synergistic combination of PTEN plasmid and BRD4 PROTAC-loaded lipid nanocarriers

Patients suffering from BRAF mutant melanoma have tumor recurrence within merely 7 months of treatment with a potent BRAF inhibitor (BRAFi) like vemurafenib (VEM). It has been proven that diverse molecular pathways driving BRAFi resistance converge to activation of c-Myc in melanoma. Therefore, we identified a novel combinatorial therapeutic strategy by targeting loss of PTEN tumor suppressor gene and upregulated BRD4 oncoprotein as Myc-dependent vulnerabilities of drug-resistant melanoma. Being promising therapeutic targets, we decided to concomitantly deliver PTEN plasmid and BRD4 targeted PROteolysis-TArgeting Chimera (ARV) to drug the “undruggable” c-Myc in BRAFi-resistant melanoma. Since PTEN plasmid and ARV are distinct in their physicochemical properties, we fabricated PTEN-plasmid loaded LNPs (PL-NANO) and ARV-825 loaded nanoliposomes (AL-NANO) to yield mean particle size of <100 nm and >99% encapsulation efficiency for each therapeutic payload. Combination of PL-NANO and AL-NANO displayed synergistic tumor growth inhibition and substantial apoptosis in in vitro 2D and 3D models. Importantly, simultaneous delivery of PL-NANO and AL-NANO achieved significant upregulation of PTEN expression levels and degradation of BRD4 protein to ultimately downregulate c-Myc levels in BRAFi-resistant melanoma cells. Altogether, lipid nanocarriers delivering this novel lethal cocktail stands as “one-of-a-kind” gene therapy to target “undruggable” c-Myc oncogene in BRAFi-resistant melanoma.

The Combination of Auranofin and Celecoxib Suppresses Local Synovial Sarcoma Progression In Vivo.

Synovial sarcoma (SS) is a rare malignant tumor with a poor survival rate. We previously reported that a combination of auranofin (AUR), a thioredoxin reductase inhibitor, and celecoxib (CE), an anti-inflammatory drug, significantly impedes the local progression of osteosarcoma (OS). However, the role of redox regulation in SS remains to be elucidated. This study aimed to investigate the efficacy of combined treatment of AUR and CE on the local progression of SS in vivo. Nu/nu mice were implanted with the human SS cell line, Aska-SS, and treated with vehicle control, AUR, or a combination of AUR and CE (AUR-CE). Primary tumor size and weight were evaluated for the study duration and upon resection, respectively. Hematoxylin and eosin (H&E) and Ki-67 staining were performed to assess the local progression of SS. A statistically significant reduction in tumor size and weight was observed in the AUR- and AUR-CE-treated groups upon excision compared to that in the vehicle-treated group. The AUR-CE-treated group showed synergistic inhibition of local tumor growth. H&E staining of local SS tumors revealed decreased cell density and nuclear deformation in the AUR- and AUR-CE-treated groups compared to those in the vehicle-treated group. Immunohistochemical staining revealed a statistically significant decrease in Ki-67-positive cells in the AUR-CE-treated group compared to the vehicle-treated group. The combination of AUR and CE showed significant potential for delaying the local progression of SS. These findings support the repurposing of AUR and CE as early treatment options for SS.

Abstract 6487: The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models

Abstract KRASG12C is a potent oncogenic driver which results in downstream hyperactivation of MAPK signaling and unchecked oncogenic growth, while simultaneously increasing replication stress (RS) and accumulation of DNA damage. KRASG12C-specific inhibitors are approved and/or recommended in metastatic non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and while responses are robust, some patients are inherently resistant, and most responders acquire resistance. Resistance mechanisms include reactivation of MAPK signaling, and cancer cells which escape MAPKi are reported to have high levels of genomic instability. WEE1 is a kinase involved in cell cycle progression, allowing cells to repair damaged DNA before entering the next cell cycle phase. Consequently, inhibition of WEE1 leads to premature cell cycle entry and RS, thereby driving cancer cells through the cell cycle with unchecked accumulation of DNA damage, ultimately resulting in mitotic catastrophe and cell death. We hypothesized that high levels of genomic instability and RS in KRASG12C mutant and KRASG12C inhibitor-resistant tumors present a vulnerability to WEE1 inhibition and provide rationale for combining azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor, with KRASG12C inhibitors. Here, we report azenosertib in combination with KRASG12C inhibitors as an effective therapeutic strategy for KRASG12C tumors. In vitro combination of azenosertib with multiple KRASG12C inhibitors demonstrated synergistic cell growth inhibition across a panel of KRASG12C cell lines in both 2D and 3D assays. In vivo treatment with azenosertib in KRASG12C inhibitor-sensitive cell line-derived xenograft (CDX) models of NSCLC (NCI-H358, NCI-H2122, NCI-H1792), CRC (SW837, SW1463), and pancreatic cancer (MIA PaCa-2) demonstrated significant monotherapy activity as well as synergistic tumor growth inhibition (TGI) when combined with KRASG12C inhibitors, including tumor regression in some models. Importantly, the KRASG12C inhibitor-resistant SW1573 NSCLC CDX model demonstrated synergistic TGI in the combo arm (84%) when compared against monotherapy azenosertib (31%) or adagrasib (58%). Similarly, treatment of a KRASG12C inhibitor-resistant patient-derived xenograft (PDX) model of CRC resulted in synergistic TGI in the combination arm (61%) relative to monotherapy azenosertib (17%) or adagrasib (2%). Finally, analysis of biomarkers from both in vitro and in vivo tumor samples demonstrated synergistic increases in protein markers of RS, DNA damage, and apoptosis with combination therapy. Taken together, our results suggest that the combination of azenosertib with KRASG12C inhibitors enhances tumor growth inhibition over single agent therapy and may be an effective treatment option for patients with KRASG12C tumors. Citation Format: Nathan M. Jameson, Gabriel Kim, Catherine Lee, Blake Skrable, Alexandra Shea, Monah Abed, Olivier Harismendy, Jianhui Ma, Doris Kim, Mark R. Lackner. The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6487.

Intratumoral injection of interferon gamma promotes the efficacy of anti-PD1 treatment in colorectal cancer

Immune checkpoint inhibitors (ICIs) offer new options for the treatment of patients with solid cancers worldwide. The majority of colorectal cancers (CRC) are proficient in mismatch-repair (pMMR) genes, harboring fewer tumor antigens and are insensitive to ICIs. These tumors are often found to be immune-deserted. We hypothesized that forcing immune cell infiltration into the tumor microenvironment followed by immune ignition by PD1 blockade may initiate a positive immune cycle that can boost antitumor immunity. Bioinformatics using a public database suggested that IFNγ was a key indicator of immune status and prognosis in CRC. Intratumoral administration of IFNγ increased immune cells infiltration into the tumor, but induced PD-L1 expression. A combined treatment strategy using IFNγ and anti-PD-1 antibody significantly increased T cell killing of tumor cells in vitro and showed synergistic inhibition of tumor growth in a mouse model of CRC. CyTOF found drastic changes in the immune microenvironment upon combined immunotherapy. Treatment with IFNγ and anti-PD1 antibody in CT26 tumors significantly increased infiltration of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs). IFNγ had a more pronounced effect in decreasing intratumoral M2-like macrophages, while PD1 blockade increased the population of CD8+Ly6C + T cells in the tumor microenvironment, creating a more pro-inflammatory microenvironment. Additionally, PD1 induced increased expression of lymphocyte activating 3 (LAG3) in a significant fraction of CD8+ T cells and Treg cells, indicating potential drug resistance and feedback mechanisms. In conclusion, our work provides preclinical data for the Combined immunotherapy of CRC using intratumoral delivery of IFNγ and systemic anti-PD1 monoclonoal antibody.

Novel two-tiered screening approach identifies synergistic combinations of natural compounds for prostate cancer prevention and treatment.

Prostate cancer (PCa) is the second most common cancer type among American men and it is estimated that in 2023, 34,700 men will die from PCa. Since it can take a considerable amount of time for the disease to progress to clinically evident cancer, there is ample opportunity for effective chemopreventive strategies to be applied for the successful management of PCa progression. In the current study, we have developed a two-tiered metabolomics-based screen to identify synergistic combinations of phytochemicals for PCa chemoprevention. This involves an initial screen for ATP depletion in PCa cells followed by a targeted screen for blocking glutamine uptake in the same cells. One of the phytochemical combinations (enoxolone [ENO] + silibinin [SIL]), identified via this screen, was examined for effects on PCa cell survival, oncogenic signaling and tumor growth in vivo. This combination was found to synergistically reduce cell survival, colony formation and cell cycle progression of PCa cell lines to a greater extent than either agent alone. The combination of ENO and SIL also synergistically reduced tumor growth when administered ad libitum through the diet in a HMVP2 allograft PCa tumor model. Treatment with the combination also significantly reduced STAT3 and mTORC1 signaling pathways in mouse and human PCa cells while significantly reducing levels of critical cell cycle regulatory proteins, contributing to the synergistic inhibition of tumor growth observed. Collectively, the current results demonstrate a novel approach to identifying synergistic combinations of phytochemicals for chemoprevention of PCa and possibly other cancers.

3D-printed NIR-responsive bullets as multifunctional nanodrug platforms for image-guided local chemo-photothermal therapy

Multimodal cancer treatment that combines anticancer drug delivery with other therapeutic strategies, such as photothermal therapy, has shown enormous potential for biomedical applications. However, traditional nanoscale photothermal conversion agents require complicated synthesis processes and lack biosafety, detectability, and mechanical reinforcement ability in the hyperthermia platform. Here, we engineered near-infrared laser (NIR)-responsive bullets as multifunctional nanodrug platforms via a three-dimensional (3D) printing process as an image-guided versatile chemo-photothermal cancer treatment platform. This customizable platform not only serves as a container for anticancer nanodrugs but also possesses excellent radiopacity and high photothermal conversion efficiency, merging on-demand anticancer nanodrug release and hyperthermia effect. Benefiting from the combined chemo-photothermal therapy, the 3D-printed platform showed enhanced cytotoxicity to cancer cells in vitro. Furthermore, in vivo results confirmed effective on-demand nanodrug release and synergistic inhibition of tumor growth upon NIR laser irradiation. 3D-printed NIR-responsive bullets can synergistically enhance anticancer efficiency while evading potential clinical application risks, offering great potential for multimodal cancer therapies.

Leveraging efferocytosis blockade for improved cancer chemo-immunotherapy through synchronized release of doxorubicin and BMS777607 confined within tailored mesoporous silica nanoparticles

Effective antitumor activity of chemotherapy can be counterbalanced by efferocytosis by generating immunosuppressive tumor microenvironment. Targeted blocking efferocytosis at an early stage of cell apoptosis exerts promising benefits on achieving improved chemotherapy, but it appears to have considerably limited outcome due to the uncontrolled release of chemotherapeutic and efferocytosis inhibitors. Herein, a mesoporous silica nanoparticle (MSN)-based nanoconfined assembly strategy is created to co-deliver chemotherapeutic drug doxorubicin (DOX) and efferocytosis inhibitor BMS777607 (BMS) for efficient cancer chemo-immunotherapy. DOX and BMS self-assembly forms nanoparticulate crystal confined within the mesopore of tailor-made MSN, and nanocrystal-encapsulated MSN (denoted as SC-MSN@D/B) enables synchronized drug release behaviors, which is beneficial for efferocytosis blockade at the early stage of apoptosis. Consequently, intravenous injection of SC-MSN@D/B leads to a synergistic inhibition of tumor growth in colon and breast cancer models through inducing a stimulatory immune response, from the secretion of pro-inflammatory cytokines to the inhibition of tumor associated macrophages and regulatory T cells infiltration. The combination of SC-MSN@D/B with checkpoint blockade inhibitors further amplifies the therapeutic effect of on tumor growth and pulmonary metastasis of orthotopic 4T1 breast tumors, and establishes long-term immune memory, offering tremendous potential for a combined efferocytosis-blocking strategy in cancer chemo-immunotherapy.

Fatty acid binding protein 5 regulates docetaxel sensitivity in taxane-resistant prostate cancer cells.

Prostate cancer is a leading cause of cancer-related deaths in men in the United States. Although treatable when detected early, prostate cancer commonly transitions to an aggressive castration-resistant metastatic state. While taxane chemotherapeutics such as docetaxel are mainstay treatment options for prostate cancer, taxane resistance often develops. Fatty acid binding protein 5 (FABP5) is an intracellular lipid chaperone that is upregulated in advanced prostate cancer and is implicated as a key driver of its progression. The recent demonstration that FABP5 inhibitors produce synergistic inhibition of tumor growth when combined with taxane chemotherapeutics highlights the possibility that FABP5 may regulate other features of taxane function, including resistance. Employing taxane-resistant DU145-TXR cells and a combination of cytotoxicity, apoptosis, and cell cycle assays, our findings demonstrate that FABP5 knockdown sensitizes the cells to docetaxel. In contrast, docetaxel potency was unaffected by FABP5 knockdown in taxane-sensitive DU145 cells. Taxane-resistance in DU145-TXR cells stems from upregulation of the P-glycoprotein ATP binding cassette subfamily B member 1 (ABCB1). Expression analyses and functional assays confirmed that FABP5 knockdown in DU145-TXR cells markedly reduced ABCB1 expression and activity, respectively. Our study demonstrates a potential new function for FABP5 in regulating taxane sensitivity and the expression of a major P-glycoprotein efflux pump in prostate cancer cells.

Biomimetic Upconversion Nanoplatform Synergizes Photodynamic Therapy and Enhanced Radiotherapy against Tumor Metastasis.

The easy recurrence and high metastasis of fatal tumors require the development of a combination therapy, which is able to overcome the drawbacks of monomodal strategies such as surgery, photodynamic therapy (PDT), and radiotherapy (RT). Taking the complementary advantages of PDT and RT, we present herein the integration of lanthanide-doped upconversion nanoparticles (UCNPs) with chlorin e6 (Ce6)-imbedded RBC membrane vesicles as a near-infrared-induced PDT agent for achieving synchronous depth PDT and RT with reduced radiation exposure. In such a nanoagent, gadolinium-doped UCNPs with strong X-ray attenuation ability act not only as a light transductor to activate the loaded photosensitizer Ce6 to allow PDT but also as a radiosensitizer to enhance RT. PDT with enhanced low-dose RT can achieve synergistic inhibition of tumor growth by producing reactive oxygen species to destroy local tumor cells and inducing strong T-cell-dependent immunogenic cell death to arrest systemic cancer metastasis. This combination of PDT and RT might be a potential appealing strategy for tumor eradication.

Abstract 4246: Synergistic inhibition of prostate tumor growth and progression by the combination of curcumin and ursolic acid in HiMyc mice

Abstract Prostate cancer (PCa) is the second leading cause of cancer death among men in the United States. According to the World Health Organization, approximately one-third of all cancer deaths could be prevented through an increased consumption of natural compounds. Through the use of a two-tiered screening method in PCa cells (depletion of cellular ATP level followed by glutamine uptake) of a phytochemical library, curcumin and ursolic acid were selected for further investigation. Twenty-four hour treatment of LNCaP, 22Rv1 and HMVP2 cells with the combination resulted in significant decreases in expression of members of the miR-17-92 and miR-106b-25 clusters, which are upregulated in PCa and have been shown to be involved in tumorigenesis, cell survival, and metastasis. In the HiMyc mouse model, where mice spontaneously develop primary PCa, supplementing diets with the combination of curcumin (1%) and ursolic acid (0.2%) resulted in significant inhibition of tumor growth and progression compared to diets containing either compound alone. There were no differences in body weight or food consumption in any of the diet groups during the tumor experiment. The current data suggests that the combination of curcumin and ursolic acid synergistically inhibits PCa growth and progression in HiMyc mice, which may be in part due to their effects on microRNA expression. Ongoing studies are further examining the role of specific miRNAs as well as oncogenic signaling pathways potentially involved in the mechanism of synergistic chemopreventive action of this combination. The current data suggest that this novel combination could be a valuable therapeutic option for prevention and/or treatment of PCa. Funding: This work was supported by NIH Grant CA228404. Citation Format: Chelsea A. Friedman, Achinto Saha, John DiGiovanni. Synergistic inhibition of prostate tumor growth and progression by the combination of curcumin and ursolic acid in HiMyc mice. [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 4246.

Abstract 577: ENPP1 inhibitor ZX-8177 in combination with chemotherapy or radiation exhibits synergistic anti-tumor effects via STING-mediated response

Abstract 2’3’-cGAMP serves as a second messenger binding to the stimulator of interferon genes (STING) to induce the STING-dependent interferon response, triggering downstream antiviral and anti-tumor mechanism. Ecto-nucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1) hydrolyzes the extracellular 2’3’-cGAMP and attenuates the immune stimulatory tumor microenvironment, and thereby facilitates tumor progression. Inhibition of ENPP1 activity in tumors that produce a significant amount of 2’3’-cGAMP, potentially activates the anti-tumor immune response. We previously presented a selective ENPP1 inhibitor ZX-8177 with high potency in biochemical and MDA-MB-231 cell-based enzymatic assays (IC50 values are 9.5 nM and 11 nM, respectively). In multiple syngeneic mouse models, ZX-8177 alone (2mg/kg, i.p., BID) exhibited 37-60% tumor growth inhibition (TGI) and significantly increased NK cells, T cells (Granzyme B+CD8+), and M1/M2 macrophages ratio in the tumor microenvironment. Radiation and many chemotherapy regimens can result in DNA-damaging and activation of innate immunity. In this report, we further assessed the synergistic effect on tumor inhibition in combination of ZX-8177 with chemotherapies or radiation in multiple in vivo mouse models. Synergistic anti-tumor efficacy was observed in CT-26 model with the combination of Mitomycin C (0.5 mg/kg, BIW) and ZX-8177 (25 mg/kg, oral, BID), and in 4T1 and Pan02 models with the combination of Cisplatin (5 mg/kg, BIW) and ZX-8177 (25 mg/kg, oral, BID). Flow cytometry analysis demonstrated the increase of tumor infiltrating NK cells in the ZX-8177 treated tumor tissues. Meanwhile, tumor tissues showed a higher level of 2’3’-cGAMP and the increased gene expression of type I interferon (IFN) related genes, i.e. CCL5 and CXCL10. In Pan02 syngeneic mouse model, ZX-8177 (25 mg/kg, oral, BID) plus radiation at 20 Gy displayed an enhanced anti-tumor efficacy compared with radiation only treatment (100% TGI vs. 78.5% TGI, p &amp;lt; 0.005). Two of five mice achieved tumor free in the combination group at the end of treatment. Increased level of tumor p-STING, 2’3’-cGAMP, IFN-related genes (i.e., IFN-r, IFN-β, and CXCL10), and cytotoxic T cells-related markers (i.e., Perforin, Granzyme A and B) was observed in the combination group, correlated with the synergistic effect on tumor growth. These in vivo studies demonstrated the synergistic inhibition of tumor growth in combination of ZX-8177 with radiation or chemotherapies through activation of STING-mediated innate immunity. Currently, ZX-8177 is under IND-enabling pre-clinical evaluation. First-in-human study of ZX-8177 is in planning as a single agent and combination therapies. Citation Format: Xiaoli Qin, Ying-ying Li, Shuaijun Sun, Xiaomin Li, Zhiyuan Peng, Deming Kong, Siyu Shui, Jinfu Yang, Xiaolin Alan Hao. ENPP1 inhibitor ZX-8177 in combination with chemotherapy or radiation exhibits synergistic anti-tumor effects via STING-mediated response [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 577.

Abstract 1377: DCC-3116, a first-in-class selective inhibitor of ULK1/2 kinases and autophagy, synergizes with encorafenib and cetuximab in BRAF V600E mutant colorectal cancer models

Abstract Background: Cancer cells activate autophagy through ULK1/2 kinases as an adaptive stress response (ASR) mechanism to therapies targeting the RTK/RAS/MAPK/PI3K pathways, limiting antitumor response. BRAF signals through the MAPK pathway while EGFR signals upstream through both the MAPK and PI3K pathways, suppressing ULK1/2 kinases and autophagy. Inhibition of mutant BRAF and EGFR reverses this suppression, activating the autophagy ASR1-4. The BRAF V600E mutation occurs in ~10% of colorectal cancer (CRC) patients. Approved treatments for these patients include the BRAF V600E inhibitor encorafenib in combination with the EGFR antibody, cetuximab. Treatment with encorafenib + cetuximab (E+C) is initially successful, but drug resistance develops either through RTK/MAPK resistant mutations or ASR pathways including autophagy. DCC-3116 is a selective, investigational, potent, first-in-class inhibitor of the ULK1/2 kinases in clinical development in combination with targeted therapies that activate the autophagic ASR pathway. Herein we demonstrate ULK1/2 kinases and autophagy are activated upon treatment with E+C. A combination of E+C with DCC-3116 leads to inhibition of ULK1/2-mediated ATG13 phosphorylation (pATG13) and autophagy in vitro. In a PK/PD model, DCC-3116 inhibits E+C induced pATG13 and leads to synergistic tumor growth inhibition (TGI) in a preclinical model of BRAF V600E mutated CRC. Methods: ULK1/2 activity was measured in cells using an ELISA for the ULK substrate phospho-ATG13 (pATG13). Autophagic flux was measured by monitoring mCherry/GFP tagged LC3 in HT-29 and Colo-205 cells. Xenograft models were performed at a CRO. Results: E+C treatment led to the activation of ULK1/2 2-3-fold in BRAF V600E mutated CRC cell lines. DCC-3116 inhibited both E+C -induced and basal pATG13 with IC50 values of 80 nM and 234 nM in HT-29 and Colo-205 cells, respectively. E+C induced autophagic flux ~3-fold which was potently inhibited by DCC-3116 with IC50 values of 65 nM in HT-29 and 40 nM in Colo-205 cells. In a PK/PD model, DCC-3116 inhibited ULK1/2-mediated pATG13 induced by E+C. The combination of DCC-3116 with E+C resulted in greater TGI compared to single agent or the combination of E+C in BRAF V600E models. Conclusions: These preclinical data demonstrate that BRAF inhibitors in combination with EGFR blockade such as E+C activate ULK-mediated autophagy as an ASR resistance mechanism which can be inhibited by DCC-3116, providing the rationale to study the combination of DCC-3116 with E+C in BRAF V600E mutated CRC patients. DCC-3116 is currently in a Phase 1 clinical trial in patients with advanced solid tumors. (NCT04892017).

Abstract P2-24-05: TAVO412 - A Novel Trispecific cMet x EGFR x VEGF Antibody for Triple Negative Breast Cancer

Abstract Novel Multispecific Antibody with Superior Activity in TNBC INTRO: TNBC (Triple Negative Breast Cancer) has a diverse etiology and high unmet need. We created a novel multispecific antibody with unique 2:1:1 ratio of EGFR:cMet:VEGF binding arms with excellent activity against TNBC. METHOD: Multiple in vitro EGFR &amp; cMet signaling pathway inhibition, proliferation inhibition, and ADCC, ADCP, and CDC were conducted. Various in vivo studies using the TNBC cell lines (MDA-MB-468, MDA-MB-231, HCC70, BT20) with diverse levels of EGFR and cMet receptor density levels were tested. RESULTS: Strong tumor growth inhibition was found in multiple TNBC animal models (56% tumor growth inhibition (TGI) in BT20, 26% TGI in HCC70, 62% TGI in MDA-MB-231). Tavo412 demonstrated better activity than comparator molecules in several in vivo models using lung, gastric, and pancreatic cancer cell lines (data not shown). A one month GLP toxicity study in cynomolgus monkeys has been completed with well-expected tolerability and pharmacokinetic profiles. Besides shutting down three important cancer pathways, TAVO412 showed high potency because it also has strong EGFR and VEGF binding at pH 6 in the tumor microenvironment. Moreover, Tavo412 has exemplary Fc effector function engaging the innate immune responses that include ADCC, ADCP, and CDC. There is also a synergistic tumor growth inhibition in the presence of a standard of care molecule such as doxorubicin (61% in HCC70 with TAVO412 + doxorubicin, 66% TGI in BT20 with TAVO412 + doxorubicin, 71% TGI in MDA-MB-231 with TAVO412 + doxorubicin). In addition, gastric, pancreatic, and lung cancer in vivo models showed excellent TGI profiles. CONCLUSIONS: Tavo412 has the potential activity to be a superior multispecific antibody to treat patients with triple negative breast cancer. Phase 1 clinical testing in patients is anticipated in early 2023. Citation Format: Mark Chiu. TAVO412 - A Novel Trispecific cMet x EGFR x VEGF Antibody for Triple Negative Breast Cancer [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-24-05.

Copper carbonate nanoparticles as an effective biomineralized carrier to load macromolecular drugs for multimodal therapy

Macromolecular drugs have attracted great interest as biotherapy to cure previously untreatable diseases. For clinical translation, biomacromolecules encounter several common druggability difficulties, such as in vivo instability and poor penetration to cross physiologic barriers, thus requiring sophisticated systems for drug delivery. Inspired by the natural biomineralization via interaction between inorganic ions and biomacromolecules, herein we rationally screened biocompatible transition metals to biomineralize with carbonate for macromolecules loading. Among the metal ions, Cu2+ was found to be the best candidate, and its superiority over the widely studied Ca2+ minerals was also demonstrated. Capitalized on this finding, copper carbonate nanoparticles were prepared via a simple mixing process to co-load glucose oxidase (GOx) and a HIF-α DNAzyme (DZ), achieving ultra-high loading capacity of 61%. Upon encapsulation into nanoparticles, enzymatic activity of both drugs was passivated to avoid potential side-effects during circulation, while the drugs could be rapidly released within 1 h in response to acidic pH to fully recover their activities. The nanoparticles could accumulate into tumor via intravenous injection, facilitate the cell membrane penetration, and release the payloads of GOx, DZ and Cu2+ inside cells to exert a series of anti-tumor effects. GOx caused tumor starvation by catalytic glucose consumption, and the concomitantly generated H2O2 byproduct boosted the Cu2+-mediated chemodynamic therapy (CDT). Meanwhile, the DZ silenced HIF-α expression to sensitize both starvation therapy and CDT. As a result, a synergistic tumor growth inhibition was achieved. This work provides a simple method to prepare biomineralized nanoparticles, and offers a general approach for macromolecular drugs delivery via Cu2+-based biomineralization.

Bioinspired low-density lipoprotein co-delivery system for targeting and synergistic cancer therapy

Epithelial-mesenchymal transition (EMT) is the culprit of tumor invasion and metastasis. As a critical transcription factor that induces EMT, snail is of great importance in tumor progression, and knocking down its expression by small interfering RNA (siRNA) may inhibit tumor metastasis. Herein, we developed a core-shelled bioinspired low-density lipoprotein (bio-LDL) in which snail siRNA-loaded calcium phosphate nanoparticles were wrapped as the core and doxorubicin was embedded in the outer phospholipids modified with a synthetic peptide of apoB100 targeting LDL receptor-abundant tumor cells. Bio-LDL exhibited pH-responsive release, lysosomal escape ability, enhanced cytotoxicity and apoptotic induction. Bio-LDL could significantly inhibit the expression of snail and regulate EMT-related proteins to reduce tumor migration and invasion in vitro. Bio-LDL also displayed favorable tumor targeting and synergistic inhibition of tumor growth and metastasis in vivo. Therefore, the multifunctional bio-LDL will be a promising co-delivery vector and holds potential value for clinical translation.

FOXM1 inhibitor-loaded nanoliposomes for enhanced immunotherapy against cancer

Immune checkpoint inhibitors (ICIs) have emerged as effective cancer therapeutic agents. However, immune-related adverse effects, high treatment costs, and low therapeutic efficacy against rapidly growing tumors limit their clinical applications. Here, we developed thiostrepton-loaded nanoliposomes (ThioLipos) as novel therapeutic drugs to overcome the limitations of ICIs and for enhancing the efficacy of cancer immunotherapy. Thiostrepton, a forkhead box M1 inhibitor, induced a significant reduction in programmed death ligand-1 (PD-L1) levels and viability of human and murine colon cancer cells. The therapeutic potential of ThioLipos in cancer immunotherapy was evaluated using a MC38 syngeneic tumor model. For mice with rapidly growing colon tumors, ThioLipo treatment alone resulted in therapeutic outcomes similar to those of ICI treatment. Moreover, combined therapy with ThioLipos and ICIs (anti-4-1BB or anti-PD-1 antibodies) resulted in synergistic tumor growth inhibition when compared with ThioLipo or ICI treatment alone, and no side effects were observed in normal tissues. These results suggest that ThioLipos can be used as promising nano-immunotherapeutics for overcoming the limitations of ICIs and enhancing the efficacy of cancer immunotherapy.

Aurora-A/FOXO3A/SKP2 axis promotes tumor progression in clear cell renal cell carcinoma and dual-targeting Aurora-A/SKP2 shows synthetic lethality

Renal cell carcinoma (RCC) is a common malignant tumor in the world. Histologically, most of RCC is classified as clear cell renal cell carcinoma (ccRCC), which is the most prevalent subtype. The overall survival of patients with ccRCC is poor, thus it is urgent to further explore its mechanism and target. S-phase kinase-associated protein 2 (SKP2) is overexpressed in a variety of human cancers and is associated with poor prognosis by enhancing tumor progression. However, it is unclear whether or how SKP2 is involved in ccRCC progression. Here, we reported that overexpression of SKP2 enhanced cell proliferation of ccRCC, while SKP2 depletion exhibited the opposite effect. Bioinformatic analyses found that SKP2 was positively correlated with Aurora-A (Aur-A) in ccRCC. The protein and mRNA levels of SKP2 were elevated or reduced by Aur-A overexpression or silencing, respectively. It was further found that Aur-A caused an increase phosphorylation of FOXO3A, which is a negatively transcription factor for SKP2. Interestingly, SKP2 mediated ubiquitylation and degradation of FOXO3A depend on the kinase activity of Aur-A. The combination of Aur-A inhibitor MLN8237 and SKP2 inhibitor SZL P1-41 showed a synergistic tumor growth inhibition in vivo and in vitro of ccRCC models. Thus, our data reveal that Aurora-A/FOXO3A/SKP2 axis promotes tumor progression in ccRCC, and the double inhibition of SKP2 and Aur-A shows significant synergistic effect, which indicates a potential new therapeutic strategy for ccRCC.

Dual inhibition of TGFβ signaling and CSF1/CSF1R reprograms tumor-infiltrating macrophages and improves response to chemotherapy via suppressing PD-L1

TGFβ contributes to chemoresistance in advanced colorectal cancer (CRC) via diverse immune-microenvironment mechanisms. Here, we found that cancer cell autonomous TGFβ directly triggered tumor programmed cell death 1 ligand 1 (PD-L1) upregulation, resulting in resistance to chemotherapy. Inhibition of tumor PD-L1 expression sensitized cancer cells to chemotherapy, reduced lung metastasis and increased the influx of CD8+ T cells. However, chemorefractory cancer cell-derived CSF1 recruited TAMs for TGFβ-mediated PD-L1 upregulation via a vicious cycle. High infiltration of macrophages was clinically correlated with the status of tumor PD-L1 after chemotherapy treatment in CRC patients. We found that depletion of immunosuppressive CSF1R+ TAM infiltration and blockade of the TGFβ receptor resulted in an increased influx of cytotoxic CD8+ T and effector memory CD8+ cells, a reduction in regulatory T cells, and a synergistic inhibition of tumor growth when combined with chemotherapy. These findings show that CSF1R+ TAMs and TGFβ are the dominant components that regulate PD-L1 expression within the immunosuppressive tumor microenvironment, providing a therapeutic strategy for advanced CRC patients.

Abstract 835: Estrogen receptor expression and ER dependent breast tumor growth are dependent on translation initiation factor EIF4A

Abstract The majority of human breast cancers are dependent on Estrogen Receptor Alpha (ER) and sensitive to its inhibition. In advanced, ER+ dependent breast cancers, resistance usually develops and is associated with insensitivity of the estrogen receptor to inhibition. Mutations that activate PI3K signaling occur in over 40% of ER-driven breast cancers. The PI3K pathway regulates cap-dependent protein translation by controlling mTOR complex I (mTORC1). Inhibitors of PI3K/mTOR are effective in this setting when given with anti-estrogens, but induce ER activity and expression. We now show that despite reducing global cap-dependent translation, PI3K/mTOR inhibition does not reduce ER translation or expression. Translation of ER instead depends on the translation initiation factor, EIF4A. Inhibitors of EIF4A significantly reduce the expression of WT and mutant ER, with attendant blockage of breast cancer model growth in vivo, including models driven by estrogen-independent ER fusions that are unaffected by estrogen receptor antagonists. The utility of EIF4A inhibition can be enhanced when combined with Fulvestrant, a degrader of ER. Combining inhibition of ER translation and induction of ER degradation causes synergistic deep and durable inhibition of ER expression and tumor growth. Inhibition of ER translation represents a new potent strategy for treating ER-dependent breast cancers with acquired resistance to current therapies. Citation Format: Jacob A. Boyer, Madeline A. Dorso, Corina Amor, Jason Reiter, Jianing Xu, Elisa de Stanchina, Hans-Guido Wendel, Sarat Chandarlapaty, Neal Rosen. Estrogen receptor expression and ER dependent breast tumor growth are dependent on translation initiation factor EIF4A [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 835.