Trigger Tumor Cell Death Research Articles

Aloperine Suppresses Cancer Progression by Interacting with VPS4A to Inhibit Autophagosome-lysosome Fusion in NSCLC.

Aloperine (ALO), a quinolizidine-type alkaloid isolated from a natural Chinese herb, has shown promising antitumor effects. Nevertheless, its common mechanism of action and specific target remain elusive. Here, it is demonstrated that ALO inhibits the proliferation and migration of non-small cell lung cancer cell lines in vitro and the tumor development in several mouse tumor models in vivo. Mechanistically, ALO inhibits the fusion of autophagosomes with lysosomes and the autophagic flux, leading to the accumulation of sequestosome-1 (SQSTM1) and production of reactive oxygen species (ROS), thereby inducing tumor cell apoptosis and preventing tumor growth. Knockdown of SQSTM1 in cells inhibits ROS production and reverses ALO-induced cell apoptosis. Furthermore, VPS4A is identified as a direct target of ALO, and the amino acids F153 and D263 of VPS4A are confirmed as the binding sites for ALO. Knockout of VPS4A in H1299 cells demonstrates a similar biological effect as ALO treatment. Additionally, ALO enhances the efficacy of the anti-PD-L1/TGF-β bispecific antibody in inhibiting LLC-derived subcutaneous tumor models. Thus, ALO is first identified as a novel late-stage autophagy inhibitor that triggers tumor cell death by targeting VPS4A.

LC3-associated phagocytosis of neutrophils triggers tumor ferroptotic cell death in glioblastoma

Necrosis in solid tumors is commonly associated with poor prognostic but how these lesions expand remains unclear. Studies have found that neutrophils associate with and contribute to necrosis development in glioblastoma by inducing tumor cell ferroptosis through transferring myeloperoxidase-containing granules. However, the mechanism of neutrophilic granule transfer remains elusive. We performed an unbiased small molecule screen and found that statins inhibit neutrophil-induced tumor cell death by blocking the neutrophilic granule transfer. Further, we identified a novel process wherein neutrophils are engulfed by tumor cells before releasing myeloperoxidase-containing contents into tumor cells. This neutrophil engulfment is initiated by integrin-mediated adhesion, and further mediated by LC3-associated phagocytosis (LAP), which can be blocked by inhibiting the Vps34-UVRAG-RUBCN-containing PI3K complex. Myeloperoxidase inhibition or Vps34 depletion resulted in reduced necrosis formation and prolonged mouse survival in an orthotopic glioblastoma mouse model. Thus, our study unveils a critical role for LAP-mediated neutrophil internalization in facilitating the transfer of neutrophilic granules, which in turn triggers tumor cell death and necrosis expansion. Targeting this process holds promise for improving glioblastoma prognosis.

Therapeutic targeting of TP53 nonsense mutations in cancer.

Mutations in the TP53 tumor suppressor gene occur with high prevalence in a wide range of human tumors. A significant fraction of these mutations (around 10%) are nonsense mutations, creating a premature termination codon (PTC) that leads to the expression of truncated inactive p53 protein. Induction of translational readthrough across a PTC in nonsense mutant TP53 allows the production of full-length protein and potentially restoration of normal p53 function. Aminoglycoside antibiotics and a number of novel compounds have been shown to induce full-length p53 in tumor cells carrying various TP53 nonsense mutations. Full-length p53 protein generated by translational readthrough retains the capacity to transactivate p53 target genes and trigger tumor cell death. These findings raise hopes for efficient therapy of TP53 nonsense mutant tumors in the future.

Abstract 595: Preclinical development of the CDK4 selective inhibitor PF-07220060: Increased CDK4 versus CDK6 inhibition leads to improved anti-tumor efficacy at therapeutic concentrations

Abstract PF-07220060 is a selective inhibitor of CDK4, displaying ~20-fold and ~4-fold increased selectivity for CDK4 versus CDK6 when compared to palbociclib and abemaciclib/ribociclib, respectively. This translates to less PF-07220060 associated neutropenia in beagle dogs and humans when compared to dual CDK4/6 inhibitors. Because neutropenia is the primary culprit necessitating limited and often interrupted clinical dosing of dual CDK4/6 inhibitors, the advantage of PF-07220060 lies in the flexibility to escalate the drug’s exposure in patients and thus realize near-complete target coverage of the CDK4 oncogene in tumors that are dependent on this kinase. Here we select tumor types/indications that show dependency on CDK4 but not CDK6 and includes cells derived from luminal breast cancer and androgen-receptor positive metastatic prostate cancer. Additionally, we investigate certain drugs with which PF-07220060 may be combined to maximize its efficacy in these tumor indications. PF-07220060 sensitizes HR+ HER2- breast cancer to the estrogen inhibitor, fulvestrant and the degrader, ARV-471 (vepdegestrant). PF-07220060 + vepdegestrant showed significantly longer tumor regrowth delay vs monotherapy groups and vs Palbo + vepdegestrant and vs PF-07220060 + fulvestrant*. Similarly, PF-07220060 sensitizes prostate cancer to the androgen receptor antagonist, enzalutamide. In HR+ HER2- breast cancer cells, the senescent cell fraction was markedly increased by co-treatment with PF-07220060 and fulvestrant versus either drug alone, albeit the combination did not trigger tumor cell death. Further addition of the PI3K inhibitor alpelisib was sufficient to enforce tumor shrinkage in vivo. Alternatively, inhibition of CDK4 plus CDK2 also led to tumor shrinkage in xenograft models of HR+, HER2- breast cancer. Finally, our preclinical data indicate that the propensity of CDK6 to compensate for CDK4 inhibition in these tumor types is limited. Compensation by CDK6 was seen in only a fraction of the evaluated in vitro and in vivo models. Even so, in these instances, PF-07220060’s efficacy remained comparable to palbociclib’s when both drugs were used at their therapeutic doses. We conclude that PF-07220060’s anti-tumor efficacy is broadly superior to currently approved dual CDK4/6 inhibitors. Citation Format: Lars Anders, Bernadette Pascual, Britton Boras, Julie Cianfrogna, Scott Garza, Na Li, Jing Tang Yuan, Mark Moen, Nanni Huser, Gary Gallego, Mehran Jalaie, Sacha Ninkovic, Sujin Cho-Schultz, Hong Shen, John Kath, Klaus Dress, Wade Diehl, Sajiv Nair, Rhys Jones, Jennifer Lafontaine, Anwar Murtaza, Aida Sacaan, Sudhakar Chintharlapalli, Todd VanArsdale. Preclinical development of the CDK4 selective inhibitor PF-07220060: Increased CDK4 versus CDK6 inhibition leads to improved anti-tumor efficacy at therapeutic concentrations [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 595.

Erlotinib combination with a mitochondria-targeted ubiquinone effectively suppresses pancreatic cancer cell survival.

Pancreatic cancer is a leading cause of cancer-related deaths. Increased activity of the epidermal growth factor receptor (EGFR) is often observed in pancreatic cancer, and the small molecule EGFR inhibitor erlotinib has been approved for pancreatic cancer therapy by the food and drug administration. Nevertheless, erlotinib alone is ineffective and should be combined with other drugs to improve therapeutic outcomes. We previously showed that certain receptor tyrosine kinase inhibitors can increase mitochondrial membrane potential (Δψm), facilitate tumor cell uptake of Δψm-sensitive agents, disrupt mitochondrial homeostasis, and subsequently trigger tumor cell death. Erlotinib has not been tested for this effect. To determine whether erlotinib can elevate Δψm and increase tumor cell uptake of Δψm-sensitive agents, subsequently triggering tumor cell death. Δψm-sensitive fluorescent dye was used to determine how erlotinib affects Δψm in pancreatic adenocarcinoma (PDAC) cell lines. The viability of conventional and patient-derived primary PDAC cell lines in 2D- and 3D cultures was measured after treating cells sequentially with erlotinib and mitochondria-targeted ubiquinone (MitoQ), a Δψm-sensitive MitoQ. The synergy between erlotinib and MitoQ was then analyzed using SynergyFinder 2.0. The preclinical efficacy of the two-drug combination was determined using immune-compromised nude mice bearing PDAC cell line xenografts. Erlotinib elevated Δψm in PDAC cells, facilitating tumor cell uptake and mitochondrial enrichment of Δψm-sensitive agents. MitoQ triggered caspase-dependent apoptosis in PDAC cells in culture if used at high doses, while erlotinib pretreatment potentiated low doses of MitoQ. SynergyFinder suggested that these drugs synergistically induced tumor cell lethality. Consistent with in vitro data, erlotinib and MitoQ combination suppressed human PDAC cell line xenografts in mice more effectively than single treatments of each agent. Our findings suggest that a combination of erlotinib and MitoQ has the potential to suppress pancreatic tumor cell viability effectively.

Design and Synthesis of New bis-oxindole and Spiro(triazole-oxindole) as CDK4 Inhibitors with Potent Anti-breast Cancer Activity.

Since CDKs have been demonstrated to be overexpressed in a wide spectrum of human malignancies, their inhibition has been cited as an effective technique for anticancer drug development. In this context, new bis-oxindole/spiro-triazole-oxindole anti-breast cancer drugs with potential CDK4 inhibitory effects were produced in this work. The novel series of bis-oxindole/spirotriazole- oxindole were synthesized from the reaction of bis-oxindole with the aniline derivatives then followed by 1,3-dipolar cycloaddition of hydrazonoyl chloride. The structure of these bis-oxindole/spiro-triazole-oxindole series was proven based on their spectral analyses. Most bis-oxindole and bis-spiro-triazole-oxindole compounds effectively inhibited the growth of MCF-7 (IC50 = 2.81-17.61 μM) and MDA-MB-231 (IC50 = 3.23-7.98 μM) breast cancer cell lines with low inhibitory activity against normal WI-38 cells. While the reference doxorubicin showed IC50 values of 7.43 μM against MCF-7 and 5.71 μM against the MDA-MB-231 cell line. Additionally, compounds 3b, 3c, 6b, and 6d revealed significant anti-CDK4 activity (IC50 = 0.157- 0.618 μM) compared to palbociclib (IC50 = 0.071 μM). Subsequent mechanistic investigations demonstrated that 3c was able to trigger tumor cell death through the induction of apoptosis. Moreover, it stimulated cancer cell cycle arrest in the G1 phase. Furthermore, western blotting disclosed that the 3c-induced cell cycle arrest may be mediated through p21 upregulation. According to all of the findings, bis-oxindole 3c shows promise as a cancer treatment targeting CDK4.

Translational readthrough of nonsense mutant TP53 by mRNA incorporation of 5-Fluorouridine

TP53 nonsense mutations in cancer produce truncated inactive p53 protein. We show that 5-FU metabolite 5-Fluorouridine (FUr) induces full-length p53 in human tumor cells carrying R213X nonsense mutant TP53. Ribosome profiling visualized translational readthrough at the R213X premature stop codon and demonstrated that FUr-induced readthrough is less permissive for canonical stop codon readthrough compared to aminoglycoside G418. FUr is incorporated into mRNA and can potentially base-pair with guanine, allowing insertion of Arg tRNA at the TP53 R213X UGA premature stop codon and translation of full-length wild-type p53. We confirmed that full-length p53 rescued by FUr triggers tumor cell death by apoptosis. FUr also restored full-length p53 in TP53 R213X mutant human tumor xenografts in vivo. Thus, we demonstrate a novel strategy for therapeutic rescue of nonsense mutant TP53 and suggest that FUr should be explored for treatment of patients with TP53 nonsense mutant tumors.

Upconversion Optogenetic Engineered Bacteria System for Time-Resolved Imaging Diagnosis and Light-Controlled Lymphoma Therapy

Purpose Engineering bacteria can achieve targeted and controllable cancer therapy by utilizing synthetic biology technology and the characteristics of tumor microenvironment. Besides, the accurate tumor diagnosis and visualization of treatment process are also vital for bacterial therapy. In this study, lymphoma was used as a model. A series of experiments in vitro and in vivo were used to test the tumor targeting and accurate drug release ability of the drug system, to evaluate its visual diagnosis and antitumor effect, and to evaluate the safety of the live biotherapeutics and carrier system. Results As shown in Figure 1, light control engineered bacteria system based on upconversion nanoparticles (UCNP) mediated time-resolved imaging (TRI) was constructed for lymphoma theranostic and therapy. The engineered bacteria were named as EcN-pDawn-φx174E/TRAIL, which lysed by blue light irradiation and then released tumor apoptosis-related inducing ligand (TRAIL). When the tumor was irradiated by blue light, TRAIL was released from bacteria to trigger tumor cell death. And then, UCNP with short luminous lifetime (UCNP(s)) were connected with anaerobic probiotics EcN-pDawn-φx174E/TRAIL to prepare EcN@UCNP(s), which targets tumor through the hypoxia targeting ability of EcN. UCNP with long luminous lifetime were modified with folic acid (FA) to prepare FA@UCNP, which targets tumor through the active targeting ability of FA. EcN@UCNP(s) and FA@UCNP were injected through tail vein, and both of them were co-located at tumor site. They can be distinguished with each other and used to precisely indicate the presence of tumor by TRI. In vitro results showed that the released TRAIL from EcN could induce tumor cell death. In addition, the EcN-pDawn-φx174E/TRAIL+blue group showed the strongest therapeutic effect, which laid foundations for in vivo light control tumor treatment. Subcutaneous Raji, U937 and Jurkat tumor model was constructed to evaluate the blue light control treatment effects. As indicated in bio-distribution studies, there were no bacteria accumulation in heart, spleen and lung tissues, the most bacteria were accumulated in tumor and they expanded over time. To explore whether the bacteria at the tumor site can function normally, the EcN-GFP were used for intuitive observation. The research demonstrated that GFP was expressed in tumor tissues, indicating that EcN-GFP@UCNP(s) targeted tumor, survived and expressed proteins. Besides, the TRAIL expression in tumor tissue was studied by ELISA assay, which showed that TRAIL could be normally expressed and released in therapy group. The mice weight and tumor volume changes were measured every two days. There were no obvious changes in body weight of all mice. In addition, the blood routine and blood biochemical analysis were carried out after the experiment completed, and all indicators were within the normal range. And then HE staining of main organs in all groups explained the biosafety of these therapeutic materials The tumor volume changes in each group were illustrated. In the blue light control tumor inhibition experiment, the therapy group displayed the smallest tumor volume (402 mm3), while the tumor volume in dark group was 1999 mm3, which was similar to that in control (2120 mm3) and FA@UCNP(l) group (2033 mm3). The therapy group revealed the highest tumor growth inhibition compared with other groups. The changes of cytokine concentration in tumor tissues were also measured by ELISA assays. Obviously increased TNF-α, IFN-γ and IL-6 level were observed in therapy group. Besides, more tumor apoptosis was observed in therapy group. All these findings indicated that EcN-pDawn-φX174E/TRAIL could inhibit tumor growth in a blue light-dependent manner. Conclusion In this paper, upconversion optogenetic engineered bacteria system was developed for time-resolved imaging diagnosis and light-controlled cancer therapy. The system had the characteristics of accurate diagnosis and controllable treatment. Both EcN@UCNP(s) and FA@UCNP target tumor tissue and the colocation accurately indicated the existence and location of tumor by TRI. In addition, EcN-pDawn-φX174E/TRAIL accumulated at tumor site were induced by blue light to lyse and release TRAIL, thus inducing tumor cell death. We expect that this engineered bacteria system provides a new technology for intelligent bacterial therapy and the construction of lymphoma theranostics. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Revealing prognostic and tumor microenvironment characteristics of cuproptosis in bladder cancer by genomic analysis.

Objectives: Bladder cancer (BLCA) is the most common malignant tumor in the urinary system, while the prognosis of muscle-invasive bladder cancer (MIBC) is poor. Cuproptosis might be a promising therapeutic approach to trigger tumor cell death. This study aimed to figure out the role of cuproptosis in BLCA and constructed a new cuproptosis scoring system to guide clinical diagnosis and individualize treatments. Methods: Consensus clustering was used to classify 490 patients with BLCA from TCGA and GEO cohorts. Survival outcomes and functional enrichment analyses were performed between the different subtypes. The cuproptosis scoring system was constructed by LASSO-Cox analysis. ESTIMATE, CIBERSORT, and ssGSEA were used to investigate the tumor microenvironment (TME). Drug sensitivity was evaluated with pRRophetic. An immunotherapy cohort was used to investigate the treatment response. The cuproptosis scoring system was verified in our own cohort with quantitative real-time PCR. Results: An overview of 12 cuproptosis genes (CuGs) in the TCGA database was depicted. Based on the mRNA expression profiles of CuGs, patients were classified into two cuproptosis molecular patterns. Based on the differential genes between the two cuproptosis patterns, the patients were classified into two cuproptosis gene clusters. There were distinct survival outcomes, signaling pathways, and TME between the two subtypes. A 7-gene cuproptosis scoring system was constructed. Patients with high cuproptosis scores showed worse OS and more immunosuppressing TME than those with low cuproptosis scores. The two cuproptosis score groups had distinct mutation profiles. Patients with high cuproptosis scores tended to be sensitive to chemotherapy drugs, but insensitive to immune checkpoint inhibitors (ICIs) treatment. Conclusion: This study depicted the landscape of cuproptosis in BLCA. We constructed a cuproptosis scoring system to predict the prognosis of BLCA patients. There were significant differences in survival outcomes, TME, mutation profiles, and drug sensitivities in high and low cuproptosis score patients. The cuproptosis scoring system could help oncologists comprehensively understand the tumor characteristic of BLCA and make individualized treatment strategies.

A Mini Review on Molecules Inducing Caspase-Independent Cell Death: A New Route to Cancer Therapy.

Most anticancer treatments trigger tumor cell death through apoptosis, where initiation of proteolytic action of caspase protein is a basic need. But under certain circumstances, apoptosis is prevented by the apoptosis inhibitor proteins, survivin and Hsp70. Several drugs focusing on classical programmed death of the cell have been reported to have low anti-tumorogenic potency due to mutations in proteins involved in the caspase-dependent programmed cell death with intrinsic and extrinsic pathways. This review concentrates on the role of anti-cancer drug molecules targeting alternative pathways of cancer cell death for treatment, by providing a molecular basis for the new strategies of novel anti-cancer treatment. Under these conditions, active agents targeting alternative cell death pathways can be considered as potent chemotherapeutic drugs. Many natural compounds and other small molecules, such as inorganic and synthetic compounds, including several repurposing drugs, are reported to cause caspase-independent cell death in the system. However, few molecules indicated both caspase-dependent as well caspase-free cell death in specific cancer lines. Cancer cells have alternative methods of caspase-independent programmed cell death which are equally promising for being targeted by small molecules. These small molecules may be useful leads for rational therapeutic drug design, and can be of potential interest for future cancer-preventive strategies.

Lipid bilayer composition as a determinant of cancer cell sensitivity to tumoricidal protein-lipid complexes.

Complexes formed by the alpha1 N-terminal peptide of alpha-lactalbumin and oleic acid (alpha1-oleate) interact with lipid bilayers. Plasma membrane perturbations trigger tumor cell death but normal differentiated cells are more resistant, and their plasma membranes are less strongly affected. This study examined membrane lipid composition as a determinant of tumor cell reactivity. Bladder cancer tissue showed a higher abundance of unsaturated lipids enriched in phosphatidylcholine, PC (36:4) and PC (38:4), and sphingomyelin, SM (36:1) than healthy bladder tissue, where saturated lipids predominated and the lipid extracts from bladder cancer tissue inhibited the tumoricidal effect of the complex more effectively than healthy tissue extracts. Furthermore, unsaturated PC in solution inhibited tumor cell death, and the complex interacted with giant unilamellar vesicles formed by PC, confirming the affinity of alpha1-oleate for fluid membranes enriched in PC. Quartz Crystal Microbalance with dissipation monitoring (QCM-D) detected a preference of the complex for the liquid-disordered phase, suggesting that the insertion into PC-based membranes and the resulting membrane perturbations are influenced by membrane lipid saturation. The results suggest that the membrane lipid composition is functionally important and that specific unsaturated membrane lipids may serve as "recognition motifs" for broad-spectrum tumoricidal molecules such as alpha1-oleate.

The T/Tn-Specific Helix pomatia Lectin Induces Cell Death in Lymphoma Cells Negative for T/Tn Antigens.

Simple SummaryChanges in glycosylation, such as incomplete synthesis and higher density of O-glycans on the cell surface, are frequently observed in cancer cells. Several types of truncated O-glycan structures, e.g., T/Tn antigens, are suspected to disrupt molecular interactions between tumor microenvironment and immune cells, for instance, facilitating cancer immune-escape. Therefore, numerous exogenous lectins targeting aberrant O-glycans are interesting tools for cancer diagnosis, prognosis, and therapy. However, the ability of exolectins to detect subtle alterations in the glycome of tumor cells and to interfere in tumor/healthy cell interactions remains largely unknown. The present article reports for the first time that the Helix pomatia (HPA) lectin, a well-known T/Tn-specific lectin, currently used as a tool in cancer diagnostics, kills Tn-positive leukemia cells and Tn-negative lymphoma cells but does not affect healthy lymphocytes. Thus, HPA could be used to discriminate between tumor and healthy cells, and detect subtle alterations in the glycosylation profile.Morniga G is a T/Tn-specific lectin, inducing cell death in Tn-positive leukemias but not in healthy lymphocytes. Helix pomatia lectin (HPA) is another T/Tn-specific lectin, currently used as tool for cancer diagnostics. The HPA-mediated tumor cell death was evaluated on human leukemia and mouse lymphoma cells, and compared to the effect of Morniga G. Both lectins induced an equivalent percentage of cell death in Tn-positive Jurkat human leukemia. In contrast, EL4 mouse lymphoma resisted Morniga G-mediated cytotoxicity but were killed by HPA at concentrations of 2.5 μg/mL (0.032 nM) and higher. In both malignant cells, HPA-mediated cell death showed features compatible with apoptosis (annexin-externalization, caspase-activation, mitochondrial membrane depolarization, and ROS production). Cytometry analysis indicated that EL4 cells are T/Tn-negative. Because previous results showed a high amount of N-acetylgalactosamine (GalNAc, sugar present in Tn antigen) on EL4 cell surface, this GalNAc could be involved in the formation of truncated O-glycans other than the T/Tn residues. When compared to Morniga G, bioinformatic analysis suggested that HPA benefits from an extended carbohydrate-binding site, better adapted than Morniga G to the accommodation of more complex branched and truncated O-glycans (such as core 2). Finally, HPA killed EL4 cells but not healthy lymphocytes in a mixture of lymphoma cells + lymphocytes, suggesting that HPA selectively triggers tumor cell death.

Bufalin engages in RIP1-dependent and ROS-dependent programmed necroptosis in breast cancer cells by targeting the RIP1/RIP3/PGAM5 pathway.

Breast cancer causes high mortality among females worldwide. Bufalin has recently been shown to trigger tumor cell death, although the mechanism of cytotoxicity remains unclear. The cytotoxicity of bufalin in breast cancer cells was examined using an MTT assay. The modes of death and intracellular reactive oxygen species production were measured by flow cytometry. We also observed cellular morphologic changes by Hoechst 33342 and propidium iodide staining and transmission electron microscopy. Western blotting was performed to determine the expression levels of related proteins. Our results showed that bufalin reduced cellular viability and promoted reactive oxygen species production, which could be inhibited by Nec-1 and N-acetylcysteine. Necroptosis was detected by Hoechst 33342 and propidium iodide staining and transmission electron microscopy. Western blot analysis showed that bufalin induced necroptosis by upregulating the necroptosis mediator RIP1 and the RIP1/RIP3/PGAM5 pathway. Taken together, these findings indicated that bufalin induces breast cancer cell necroptosis by targeting the RIP1/RIP3/PGAM5 pathway.

A light-induced nitric oxide controllable release nano-platform based on diketopyrrolopyrrole derivatives for pH-responsive photodynamic/photothermal synergistic cancer therapy.

Emerging treatment approaches, such as gas therapy (GT), photodynamic therapy (PDT) and photothermal therapy (PTT), have received widespread attention. The development of an intelligent multifunctional nano-platform responding to tumor microenvironments for multimodal therapy is highly desirable. Herein, a near-infrared (NIR) light-responsive nitric oxide (NO) photodonor (4-nitro-3-trifluoromethylaniline, NF) and a pH-sensitive group (dimethylaminophenyl) have been introduced into a diketopyrrolopyrrole core (denoted as DPP-NF). The DPP-NF nanoparticles (NPs) can be activated under weakly acidic conditions of lysosomes (pH 4.5-5.0) to generate reactive oxygen species (ROS) and enhance photothermal efficiency. The fluorescence detection demonstrated that NO controllable release can be realized by "on-off" switching of the NF unit under NIR light irradiation or dark conditions. The controllable NO release of DPP-NF NPs can not only trigger tumor cell death by DNA damage, but also overcome PDT inefficiencies caused by hypoxia in tumors. Additionally, DPP-NF NPs displayed 45.6% photothermal conversion efficiency, making them superior to other reported DPP derivatives. In vitro studies showed that DPP-NF NPs possessed low dark toxicity and high phototoxicity with a half-maximal inhibitory concentration of about 38 μg mL-1. In vivo phototherapy indicated that DPP-NF NPs exhibited excellent tumor phototherapeutic efficacy with passive targeting of the tumor site via the enhanced permeability and retention (EPR) effect. These results highlight that the nano-platform has promising potential for NO-mediated multimodal synergistic phototherapy in clinical settings.

Abstract 2330: BI5: a novel SMAC mimetic that triggers tumor cell death and potentiates PD-1 mediated cancer therapy

Abstract Background: Inhibitors of apoptosis proteins (IAPs) regulate cellular apoptosis by interfering with the proteolytic activities of caspases. IAP inhibitors (SMAC mimetics) have been developed to restore the defective apoptosis that characterizes many tumour cells. Emerging evidence demonstrates that IAPs are critical components of immune-modulatory pathways that control innate and adaptive immunity. Accordingly, SMAC mimetics hold the promise of both inducing tumour cell killing and stimulating the immune system to recognize and eliminate dying tumour cells. Here we show that BI5 primes immune components and synergises with PD-1 checkpoint inhibitors to promote eradication of syngeneic tumors. Methods: Here we report the efficacy and modulation of the immune response by a potent and selective SMAC mimetic, BI5. We characterised the effect of BI5 on tumor growth inhibition as a single agent and in combination with an anti-PD-1 antibody in syngeneic mouse tumor models. A detailed 17-colour multi-color flow cytometry analysis was used to investigate the mechanisms by which the SMAC mimetic interacts with anti-PD-1 therapy in vivo. Results: Treatment of the syngeneic mouse tumor models MBT-2 and EMT-6 with the SMAC mimetic in combination with an anti-PD-1 antibody results in remarkable tumor regressions in vivo. Importantly, the combined effect of the SMAC mimetic and anti-PD-1 on tumor growth was dependent on the adaptive immune system in vivo. Mechanistic studies show that degradation of IAP triggers tumor cell death, which leads to a potent activation of dendritic cells in the draining lymph nodes and a subsequent influx of T and NK cells into the tumor microenvironment. Interestingly, in the presence of the SMAC mimetic alone, an induction of PD-1 expression on tumor-infiltrating CD8+ T cells was observed, which in turn resulted in the exhaustion of these cells and tumor outgrowth. In the presence of the anti-PD-1 antibody, T cells are reactivated leading to potent and long term tumor eradication. Conclusion: We show that our SMAC mimetic leads to a potent induction of immunogenic cell death and sets up a “virtuous cycle” by potentiating dendritic cell and T cell mediated immune responses that further promote induction of cell death. These effects are potentiated by checkpoint inhibitors, leading to long term tumor control. Tumours with minimal T-cell infiltration are poorly responsive to PD-1 monotherapy. These studies indicate that SMAC mimetics, such as BI5, represent promising and tolerated combination partners for checkpoint inhibitors in patients that lack a strong immune inflammatory signature. Citation Format: Markus Reschke, Maria Antonietta Impagnatiello, Ulrich Reiser, Dirk Scharn, Walter Spevak, Alexander Savchenko, Andreas Wernitznig, Martina Sykora, Rebecca Langlois, Elisabeth Zier, Daniel Zach, Sabine Kallenda, Pilar Garin-Chesa, Jens Quant, Mark Pearson, Darryl McConnell, Norbert Kraut, Juergen Moll. BI5: a novel SMAC mimetic that triggers tumor cell death and potentiates PD-1 mediated cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2330. doi:10.1158/1538-7445.AM2017-2330

A dose-escalation study of imipridone ONC201 administered every one (QW) or three weeks (Q3W) in advanced solid tumors and multiple myeloma.

2592 Background: ONC201 is an orally active, first-in-class small molecule activator of the integrated stress response that selectively upregulates ATF4 to trigger tumor cell death. A phase I study of ONC201 exploring different dosing regimens and drug exposure was conducted to determine the maximum tolerated dose (MTD), and recommended phase II dose (RP2D). Methods: A modified accelerated-titration dose escalation design was employed to enroll patients onto 2 sequential dose-escalation arms: ONC201 Q3W and QW. Dose escalation proceeded with the following order: 125, 250, 500, and 625mg (Q3W) and 250, 375, 500 and 625 (QW). Dose exposure ranged from 125 mg/6 wks to 1875 mg/3 wks. Key eligibility: advance/refractory solid tumor or myeloma, ECOG 0/1, and no active CNS disease. Adverse events, SAEs, laboratory values, physical exam findings, EKGs and bio-samples (for PK/PD) are collected. Pre and post ONC201 dose biopsies are being obtained from the 500mg weekly cohort and above. Results: 17 pts (12F:5M) with treatment-refractory tumors have been enrolled to date. Dose/#pts Q3W: 125/6, 250/1, 500/1, and 625/1; QW: 250/4, 375/4. Two patients have been enrolled at 500mg QW after database lock and not included in this assessment. Median (range) age: 57 yrs (27-72). ECOG 0/1: 2/15. MTD has not been reached. No DLTs observed. Tumor types: 8 CRC, 3 pancreatic, 2 sarcoma and 1 each cervical, endometrial, NSCLC (adeno) and small bowel. Of 17 pts, 10 (59%) had ≥1 tx-related adverse events (TRAEs), possibly related. Most common TRAEs (≥15% pts): fatigue (9,53%), anorexia (5,29%), nausea (4,24%), vomiting (4,24%), abdominal pain (3,18%), and arthralgias (2,12%). Grade ≥ 3 TRAEs were observed in 3 pts (18%). Pharmacokinetic and pharmacodynamic analysis is ongoing. Fresh frozen and paraffin-embedded biopsies (baseline and week 2) are being assessed for tumor markers implicated in the mechanism of action. No objective responses by RECIST have been seen. Final cohort is enrolling. Conclusions: ONC201 was well tolerated throughout the Q3W dosing and weekly dosing has been well tolerated to date without an apparent increase in AEs. Final enrollment summary, AEs and PK/PD data will be presented. Clinical trial information: NCT02609230.

First-in-human trial of ONC201 in patients with refractory solid tumors.

2514Background: ONC201 is an orally active, first-in-class small molecule activator of the integrated stress response that selectively upregulates ATF4 to trigger tumor cell death. Preclinical studies have established the compelling antitumor efficacy of the small molecule in a range of advanced solid tumors as well as its excellent safety profile in animals. A first-in-human phase I study of ONC201 was conducted to determine its recommended phase II dose (RP2D) in patients with advanced solid tumors. Methods: This open-label study treated 10 patients during the single-patient cohort dose escalation portion of the trial. The human starting dose was 125mg, which represents 1/10th of the no-adverse-adverse-effect-level in animal toxicology studies using standard allometric conversion. Results: The RP2D was defined as 625 mg and no Grade > 1 drug-related adverse events occurred on this trial, which enrolled patients at increasing dose levels of 125mg, 250mg, 375mg, 500mg, and finally 625mg. Pharmacokinetic a...

Tumor suppressor Spred2 interaction with LC3 promotes autophagosome maturation and induces autophagy-dependent cell death.

The tumor suppressor Spred2 (Sprouty-related EVH1 domain-2) induces cell death in a variety of cancers. However, the underlying mechanism remains to be elucidated. Here we show that Spred2 induces caspase-independent but autophagy-dependent cell death in human cervical carcinoma HeLa and lung cancer A549 cells. We demonstrate that ectopic Spred2 increased both the conversion of microtubule-associated protein 1 light chain 3 (LC3), GFP-LC3 puncta formation and p62/SQSTM1 degradation in A549 and HeLa cells. Conversely, knockdown of Spred2 in tumor cells inhibited upregulation of autophagosome maturation induced by the autophagy inducer Rapamycin, which could be reversed by the rescue Spred2. These data suggest that Spred2 promotes autophagy in tumor cells. Mechanistically, Spred2 co-localized and interacted with LC3 via the LC3-interacting region (LIR) motifs in its SPR domain. Mutations in the LIR motifs or deletion of the SPR domain impaired Spred2-mediated autophagosome maturation and tumor cell death, indicating that functional LIR is required for Spred2 to trigger tumor cell death. Additionally, Spred2 interacted and co-localized with p62/SQSTM1 through its SPR domain. Furthermore, the co-localization of Spred2, p62 and LAMP2 in HeLa cells indicates that p62 may be involved in Spred2-mediated autophagosome maturation. Inhibition of autophagy using the lysosomal inhibitor chloroquine, reduced Spred2-mediated HeLa cell death. Silencing the expression of autophagy-related genes ATG5, LC3 or p62 in HeLa and A549 cells gave similar results, suggesting that autophagy is required for Spred2-induced tumor cell death. Collectively, these data indicate that Spred2 induces tumor cell death in an autophagy-dependent manner.

Combination Therapy Targeting Ribosome Biogenesis and mRNA Translation Synergistically Extends Survival in MYC-Driven Lymphoma.

Ribosome biogenesis and protein synthesis are dysregulated in many cancers, with those driven by the proto-oncogene c-MYC characterized by elevated Pol I-mediated ribosomal rDNA transcription and mTORC1/eIF4E-driven mRNA translation. Here, we demonstrate that coordinated targeting of rDNA transcription and PI3K-AKT-mTORC1-dependent ribosome biogenesis and protein synthesis provides a remarkable improvement in survival in MYC-driven B lymphoma. Combining an inhibitor of rDNA transcription (CX-5461) with the mTORC1 inhibitor everolimus more than doubled survival of Eμ-Myc lymphoma-bearing mice. The ability of each agent to trigger tumor cell death via independent pathways was central to their synergistic efficacy. CX-5461 induced nucleolar stress and p53 pathway activation, whereas everolimus induced expression of the proapoptotic protein BMF that was independent of p53 and reduced expression of RPL11 and RPL5. Thus, targeting the network controlling the synthesis and function of ribosomes at multiple points provides a potential new strategy to treat MYC-driven malignancies. Treatment options for the high proportion of cancers driven by MYC are limited. We demonstrate that combining pharmacologic targeting of ribosome biogenesis and mTORC1-dependent translation provides a remarkable therapeutic benefit to Eμ-Myc lymphoma-bearing mice. These results establish a rationale for targeting ribosome biogenesis and function to treat MYC-driven cancer.

ME-06 * ENDOGENOUS NEURAL PRECURSOR CELLS INDUCE CELL DEATH OF HIGH-GRADE ASTROCYTOMAS AND HAVE A TROPISM TO TUMORS IN THE HUMAN BRAIN

Data from mouse models suggest that high-grade gliomas (HG-gliomas) origin from somatic mutant neural stem and precursor cells (NPCs). We found that endogenous NPCs also defend the brain against HG-gliomas. We observed that NPCs migrate from the subventricular zone to HG-gliomas in mice and we found that NPCs, which are genetically distinct from the tumor mass, accumulate at the border of high-grade gliomas in humans. The vascular endothelial growth factor pathway controls the tropism of mouse and human NPCs to gliomas. Tumour associated NPCs reduce glioma expansion and prolong survival by releasing a group of fatty acid ethanolamides that have agonistic activity on the vanilloid receptor (transient receptor potential vanilloid subfamily member-1; TRPV1). TRPV1 expression is much higher in HG-gliomas than in tumor-free brain and TRPV1 stimulation triggers tumor cell death. NPC-mediated tumor suppression can be mimicked by systemic administration of synthetic vanilloids and phytovanilloids suggesting that TRPV1 agonists hold potential as new HG-astrocytoma therapeutics. Finally, our data reveal that brain tumour suppression is an intrinsic feature of NPCs, but not other stem and precursor cells. Factors secreted from induced pluripotent stem cells (iPS cells) did not trigger glioma cell death, but when iPS cells were differentiated into NPCs they released anti-tumourigenic vanilloids. Overall, our data reveal that NPCs are attracted to high-grade gliomas in mice and humans, that NPCs have a specific role in suppressing HG-gliomas and that NPC-released anti-tumorigenic factors can be used as pre-clinical HG-glioma therapeutics.