Induction Of Death Research Articles

Tumor-treating fields in cancer therapy: advances of cellular and molecular mechanisms.

Tumor-Treating Fields (TTFields) use intermediate-frequency and low-intensity electric fields to inhibit tumor cells. However, their mechanisms are still not well understood. This article reviews their key antitumor mechanisms at the cellular and molecular levels, including inhibition of proliferation, induction of death, disturbance of migration, and activation of the immune system. The multifaceted biological effects in combination with other cancer treatments are also summarized. The deep insight into their mechanism will help develop more potential antitumor treatments.

Apoptotic cells may drive cell death in hair follicles during their regression cycle.

Intravital microscopy in live mice has shown that the elimination of epithelial cells during hair follicle regression involves supra-basal cell differentiation and basal cell apoptosis through synergistic action of TGF-β (transforming growth factor) and mesenchymal-epithelial interactions. In this process the basal epithelial cells are not internally committed to death and the mesenchymal dermal papilla (DP) plays essential role in death induction. Because the DP cells are not necessary for completion of the cycle but only for its initiation it is still an open question what is the mechanism leading to the propagation of apoptosis towards the regenerative stem cell population. Here, we use a quantitative analysis of the length of hair follicles during their regression cycle. The data are consistent with a propagation mechanism driven by apoptotic cells inducing apoptosis in their neighboring cells. The observation that the apoptosis slows down as the apoptotic front approaches the stem cells at the end of the follicle is consistent with a gradient of a pro-survival signal sent by these stem cells. An experiment that can falsify this mechanism is proposed.

Premature Death of Sleep Fragmented Males is Associated With An Accumulation of Reactive Oxygen Species In The Gut

Abstract 70 million Americans report insufficient sleep due to work or lifestyle choices. Decreased duration and poor sleep quality are associated with high rates of CVD and is strongly associated with increased risk of all-cause mortality. The effects of gender have been studied in CVD, but the impact of sexual dimorphism in Sleep Fragmented (SF)-associated atherogenesis is not known. Recently, we found that high fat diet (HFD) fed male Apoe −/−mice on SF experienced premature death. 80% of males did not survive past week 2 of SF, while 80% of females survived past week 8 under same conditions. We sought to identify a cause of premature death in HFD fed male Apoe −/−mice following sleep fragmentation. Interestingly, HFD fed male Ldlr −/−mouse did not experience premature death when SF, suggesting that ApoE absence, not hyperlipidemia, plays an essential function in the induction of death. Furthermore, oral 17β-Estradiol was able to rescue HFD fed male Apoe −/−mice from SF induced premature death. Compared to controls, SF, HFD fed male Apoe −/−mice had decreased body weight and increased circulating LPS, ROS and cell-free dsDNA at death. Finally, the small intestines SF, HFD fed male Apoe −/−mice had increased ROS and decreased tight junction protein (Tjp1) expression, suggesting compromised intestinal barrier function. Our data suggests that a combination of known risk factors for CVD (male sex, ApoE genotype, and HFD) results in premature death when combined with inadequate sleep. Furthermore, our data suggests that this specific combination of co-factors might result in premature death due to a systemic increase in oxidative stress, possibly caused by leakage of LPS from the gut, which may be alleviated through the administration of 17β-Estradiol. Supported by project–initiation funds from the Center for Integrative Neuroscience and Inflammatory Diseases and AHA Predoctoral (20PRE35180156)

Cytotoxic Activity of Atmospheric Cold Plasma Jet Towards 3D Human Breast Cancer Cell Model

The treatment of solid tumors with a cold atmospheric plasma jet (CAP) is an innovative approach, which began to be actively developed only in the last decade. As a consequence, the studies aimed at revealing the conditions of selectivity of such effects on tumor cells, including in 3D tumor models, are important. It is known that the main cytotoxic effects of CAP are caused by reactive oxygen and nitrogen species, which are formed in the plasma flow and the availability of which for the cells in the classical 2D and 3D cultivation models may be different. We used multicellular spheroids of MCF7-EGFR cells with hyperexpression of epidermal growth factor receptor (EGFR), the parental MCF7 breast adenocarcinoma cell line, and MCF10A non-transformed human breast cells. Irradiation of MCF7-EGFR spheroids led to destruction of multicellular 3D structures into individual cells with activation of death processes. It was shown that cells of CAP-irradiated spheroids underwent phagocytosis by activated macrophages. When comparing direct exposure to CAP and cultivation of MCF7-EGFR spheroids in CAP-irradiated medium (CAP-IM), a higher content of reactive oxygen and nitrogen species in spheroid cells was found when cultured in CAP-IM, which further leads to a greater cytotoxic effect than in direct irradiation. The cytotoxicity of CAP-IM has been shown to be valid longer when such medium is stored at 4 than at −20°С. Thus, it was shown that the treatment of spheroids with CAP-IM was more effective in death induction than direct CAP irradiation.

Qilian Formula Inhibits Tumor Cell Growth in a Bone Metastasis Model of Lung Cancer.

Bone metastasis is frequently common in advanced lung cancer with the major issue of a pathological fracture. Previous studies suggested that Astragalus membranaceus (Qi) and Ampelopsis japonica (Lian), which are used as folk medicine in China, have potential effects on inhibiting tumor growth and protecting bones, respectively. In this study, an experiment on the inhibitory effect of the Qilian formula (AAF) in vivo was designed to examine tumor growth in bone and osteoclast formation. The bone metastasis xenograft models were established by implanting NCI-H460-luc2 lung cancer cells into the right tibiae bones of mice. After confirming the model's viability through optical imaging 7 days post-implantation, 2 groups, namely the AAF group and the control group, were administered 0.3 mL of AAF extract (9 g/kg/day) or normal saline via intragastric delivery for a duration of 4 weeks. Throughout the study, we longitudinally assessed tumor burden, bone destruction, and weight-bearing capacity in vivo using reporter gene bioluminescence imaging (BLI), micro-CT, and dynamic weight-bearing (DWB) tests. Mechanistic insights were gained through Hematoxylin-eosin (H&E) staining, immunohistochemical (IHC) analysis, western blotting, and flow cytometry. Qilian formula produced significant inhibition to the progress of bone destruction and tumor burden in the right tibiae bone in the treatment group. It was further evidenced by molecular imaging in vivo via small animal micro-CT and BLI with parametric quantification, characterizing significantly lower uptake of BLI signal in the treated tumor lesions and improving the pathological changes in the microstructure of bone. Furthermore, DWB tests revealed that Qilian formula treatment significantly maintained the weight-bearing capacity. According to immunohistochemical analysis, the effect of the Qilian formula appeared to involve the suppression of osteoclast formation by lower expression of the tartrate-resistant acid phosphatase. Cell apoptosis and death induction were evidenced by a higher percentage of Bal2、BAX and caspase 3 expressions of Qilian formula-treated tumor tissues. Our study demonstrated a significant inhibitory effect of the Qilian formula on the progression of osteolytic invasion in vivo by suppressing osteoclastogenesis and promoting apoptotic cell death.

Abstract 5380: Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death

Abstract Background and goals: ONC201 an experimental anticancer drug, targeting mitochondrial caseinolytic peptidase, ClpP is currently under investigation. Current data on the effect of ONC201 along with observations that ONC201 causes cytotoxic effect, there are data demonstrating that ONC201 is rather cytostatic and does not induce cell death. Recently, we demonstrated ONC201-induced arrest of proliferation and a decrease in the number of mitochondrial nucleoids. Here we extended our study to describe the effect of short-and long-term exposure of BT474 cells to ONC201. Observations: Proliferation and cell cycle arrest. ONC201 in a dose-dependent manner (0-50 µM) inhibited the proliferation of BT474 cells without induction of apoptotic cell death. Cells exposed to 10 µM ONC201 accumulated in G0/G1 phase (49.4 ± 11.5% vs 78.7 ± 5.1%) and declined in the S-phase of the cell cycle (39.2 ± 6.6% vs 9.0 ± 2.1%). In parallel, ONC201 exposure induced a decline in Cyclin E and Cdk2 protein levels. Nucleoids and mtDNA. Exposure of BT474 cells, to 10 µM ONC 201, decreased the number of mitochondrial nucleoids from 249 ± 52 to 155 ± 38 per cell after 24 h. Longer 72 h exposure further reduced the number of nucleoids to 84 ± 36 per cell. ONC201 exposure also induced reduction of mitochondrial size from 2.8 ± 0.5 µm2 to 1.5 ± 0.2 µm2 and depletion of mtDNA. Short-term and long-term consequences of cell exposure to ONC201. Short-term treatment (24 h exposure) demonstrated the reversible effect of ONC201 on the expression of stress proteins (ATF4, CHOP, and GDF-15), cell cycle regulatory proteins (cyclin E, Cdk2), and partial restoration of mitochondrial nucleoids (to 214 ± 46 nucleoids per cell). On the contrary, long-term treatment (72 h) resulted in the irreversible and persistent arrest of proliferation, decline of cell cycle regulatory proteins, and activation of stress proteins. Respiration. ONC201 exposure suppressed State 3, State 4, and uncoupled respiration in digitonin-permeabilized BT474 cells. Complex I activity was decreased from 3.3 ± 0.4 ng-atoms O/min/106 cells to 0.5 ± 0.1 and 0.4 ± 0.3 ng-atoms O/min/106 cells, in cells exposed to ONC201 for 24h and 72h, respectively. Similarly, ONC201 decreased the activity of Complex II from 3.5 ± 0.5 ng-atoms O/min/106 cells to 1.5 ± 0.6 and 0.3 ± 0.1 ng-atoms O/min/106. Conclusions: The consequences of ONC201 treatment were dependent on the duration of exposure to the drug. Short-term (24 h) exposure of BT474 cells resulted in reversible induction of stress proteins (ATF4, CHOP, and GDF-15), which were reversed following 120 h incubation in drug-free media. On the contrary, long-term (72 h) exposure of cells to ONC201 induced irreversible and persistent arrest of proliferation and elevation of expression of stress proteins. Thus, ONC201 can induce prolonged and irreversible elevation of stress proteins and persistent G1/S cell cycle arrest. Citation Format: Artem A. Mishukov, Irina V. Odinokova, Serazhutdin A. Abdullaev, Vitaly K. Zhalimov, Emily M. Fennel, Lucas J. Aponte-Collazo, Paul R. Graves, Lee M. Graves, Ekhson L. Holmuhamedov. Anticancer compound ONC201 induces prolonged stress and arrest of proliferation in BT474 human breast cancer cells without induction of cell death [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 5380.

Abstract 1281: SGN-B7H4V shows immunomodulatory activity through induction of immunogenic cell death

Abstract SGN-B7H4V is a novel investigational antibody-drug conjugate composed of a B7-H4-directed human monoclonal antibody conjugated to the validated vedotin drug linker, which incorporates the microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linkage. This vedotin drug linker system has been clinically validated by multiple ADC programs, including brentuximab vedotin, enfortumab vedotin, tisotumab vedotin, and polatuzumab vedotin. B7-H4 is an immune checkpoint ligand with elevated expression on a variety of solid tumor types, including breast, ovarian, and endometrial tumors. SGN-B7H4V is designed to bind and internalize the B7-H4/ADC complex from the surface of malignant cells and release the cytotoxic payload MMAE. The antitumor activity of SGN-B7H4V may be multimodal as SGN-B7H4V can induce tumor cell death through several mechanisms, including MMAE-mediated direct cytotoxicity and bystander killing as well as antibody-mediated functions including antibody-dependent cellular cytotoxicity (ADCC) and phagocytosis (ADCP). Previously, vedotin ADCs have been described to elicit antitumor immune responses in part through induction of immunogenic cell death (ICD) mediated by the MMAE payload. These immunomodulatory effects potentially position vedotin ADCs to uniquely synergize with checkpoint inhibitors, supported by recent clinical activity observed when vedotin ADCs are paired with anti-PD1 agents. Here, we characterize SGN-B7H4V-mediated ICD and subsequent immunomodulatory activity. We also evaluate the contribution of SGN-B7H4V-induced immune activation to antitumor activity in combination with an anti-PD1 agent in an immunocompetent mouse model. In vitro, tumor cells treated with SGN-B7H4V showed several hallmarks of immunogenic cell death, including calreticulin exposure and release of ATP. In vivo, treatment of B7-H4-expressing tumors with SGN-B7H4V led to immune changes in the tumor microenvironment, including recruitment of macrophages and T cells. Finally, SGN-B7H4V drove robust, curative activity in an immunocompetent tumor model as a monotherapy and paired well with an anti-PD1 agent. Altogether, these data support the evaluation of SGN-B7H4V as a monotherapy in a first-in-human phase 1 clinical study and potential future clinical combinations with immunotherapies. Citation Format: Elizabeth E. Gray, Michelle Ulrich, Angela Epp, Patrick Younan, Kelly Hensley, Sean Allred, Julie Hahn, Kristen Gahnberg, Piper M. Treuting, John J. Gosink, Robert Thurman, Alyson J. Smith, Jason Schrum, Natalya Nazarenko, Shyra J. Gardai. SGN-B7H4V shows immunomodulatory activity through induction of immunogenic cell death [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 1281.

Abstract 6111: OBI-3424, an AKR1C3-activated prodrug, exhibits in vivo synergistic anti-tumor effect in combination with pembrolizumab by induction of immunogenic cell death

Abstract Immunogenic cell death (ICD) involves the activation of cytotoxic T lymphocyte-driven adaptive immunity with long-term immunological memory. OBI-3424 is a highly selective prodrug that is activated by AKR1C3 to release a potent bis-alkylating agent. This study aims to investigate whether OBI-3424 can induce ICD and create a tumor microenvironment that benefits the combination therapy of OBI-3424 with immune checkpoint inhibitors. OBI-3424 induced ICD was examined in vitro by incubation of the prodrug with AKR1C3 positive cells followed by the detection of damaging-associated molecular patterns (DAMPs). The ICD-related immunity was assessed in vivo using advanced severe immunodeficient mice that were engrafted with human peripheral blood mononuclear cells (PBMCs). Anti-tumor effect of OBI-3424 in combination with pembrolizumab was evaluated in a xenograft model using PBMC-humanized mice. Incubation of OBI-3424 with AKR1C3 positive cells H460 and HepG2 induced the release of DAMPs including calreticulin, HMGB1, and ATP, in dose- and time-dependent manners. The detection of the DAMPs indicated that OBI-3424 induced ICD in vitro. The OBI-3424-induced ICD and its related immunity were also assessed in vivo. PBMC-humanized mice were immunized with OBI-3424- or PBS-treated HepG2 cells and then challenged with live HepG2 cells. No tumor growth was noted in mice that were immunized with OBI-3424-treated cells, indicating that the dying HepG2 cells induced by OBI-3424 elicited an adaptive, tumor-specific immune response. Furthermore, OBI-3424 showed a synergistic anti-tumor effect in combination with pembrolizumab in HepG2 xenograft model using PBMC-humanized mice. OBI-3424 plus pembrolizumab exhibited significantly stronger inhibition on tumor growth (TGI 77.2%) when compared with the treatment of OBI-3424 (TGI 27.8%) or pembrolizumab (TGI -15.3%) alone. Analysis of tumor-infiltrating lymphocytes (TILs) showed that OBI-3424 treatment induced the populations of activated cytotoxic CD8 T-cells (CD45+/CD8+/CD69+ and CD45+/CD8+/Granzyme), activated helper CD4 T-cells (CD45+/CD4+/CD69+), and mature dendritic cells (CD11b+/CD86+). In addition, OBI-3424 treatment also increased PD-1 expression on CD8 and CD4 cells, which in turn potentiated the anti-tumor effect of pembrolizumab. We demonstrated that OBI-3424 was able to induce ICD as shown by the release of DAMPs in vitro and tumor-specific immunity in vivo. OBI-3424 also created a tumor microenvironment that enhances the function of pembrolizumab, supported by the synergistic effect in animals with the cotreatment of the two drugs. The results suggest that a combination therapy of OBI-3424 and anti-PD-1 in human clinical study is warranted. OBI-3424 is currently in Phase 1/2 clinical trials for solid tumor and acute lymphoblastic leukemia (NCT03592264 and NCT04315324). Citation Format: Chun-Chung Wang, Wan-Fen Li, Chih-Chan Lee, Lu-Tzu Chen, Jhih-Jie Yang, Jiann-Shiun Lai, Ming-Tain Lai. OBI-3424, an AKR1C3-activated prodrug, exhibits in vivo synergistic anti-tumor effect in combination with pembrolizumab by induction of immunogenic cell death [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 6111.

Abstract 199: A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments

Abstract Mantle cell lymphomas (MCL) are rare B-cell neoplasms belonging to the non-Hodgkin’s lymphomas and often show an aggressive clinical course. MCL respond relatively briefly to conventional chemotherapy and are characterized by frequent relapses (Dreyling et al., Ann Oncol, 2017). Therapy for MCL remains a challenge also due to limited cultivation time of primary cells. In vitro suspension culture of primary human MCL cells is not easily feasible (Bryant et al., Lab Invest, 2000) and cells undergo spontaneous apoptosis ex vivo (Zhang et al., Blood, 2012; Medina et al., Heamatologica, 2012). Useful in vitro MCL models that allow long-term culture of primary MCL cells could be helpful in the biological and therapeutic study of MCL. For this purpose, we established a 3D hydrogel culture model for cultivation of primary MCL cells that were first tested with cell lines. After 3 days of culture, the cells cultured in the hydrogel can successfully be analysed for cell viability (live-dead confocal laser scanning microscope-based assay, flow cytometry), metabolism (pH, glucose, lactate), and biomarker expression by immunohistochemistry. The 3D hydrogel cultivation model allows a variety of analytical methods, also because it is reversible and the cells can easily be separated as single cell suspension from hydrogel for further analytical purposes, such as flow cytometric analyses. Next, we cultured primary MCL cells in the alginate hydrogel 3D culture system. MCL cells were isolated from blood of different patients and separated by Ficoll centrifugation. The isolated cell population was identified as MCL cells by double-staining of anti-CD20 and anti-CD5 followed by flow cytometric analysis. Cells were further cultured in medium as conventional suspension culture or in alginate hydrogel as 3D culture. Cell death was assessed with AnnexinV by staining phospholipids of apoptotic cells, allowing flow cytometric quantification. All samples had less than 6% AnnexinV positive (+) cells at day 0 before the experiment started. After 2 days cultivation, primary MCL cells showed a strong induction of cell death in suspension culture with 65.5% (±4.2%) AnnexinV+ cells versus 21.9% (±0.6%) AnnexinV+ cells in 3D hydrogel culture. Subsequently after 3 days cultivation, 74.2% (±12.2%) AnnexinV+ cells in suspension versus 33.5% (±1.3%) AnnexinV+ cells in the hydrogel 3D culture were determined. Therefore, we conclude that our novel 3D culture system might be a more suitable cultivation method for primary MCL cells than conventional suspension culture in cell culture medium. We have developed a 3D cultivation system with alginate hydrogel that allows nearly tripling the in vitro cultivation time of primary MCL. This could pave the way for new long-term treatments in research in the future and serve a better understanding of the cells. Citation Format: Kathrin Böpple, Annette M. Staiger, Heike Horn, German Ott, Walter E. Aulitzky, Meng Dong. A step closer to in-depth analyses: Cultivation of primary mantle cell lymphoma cells in 3D enables in vitro long-term experiments [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 199.

Abstract 5681: Metabolic targeting of NRF2 potentiates the efficacy of TRAP1 inhibitor GTPP in colorectal cancer

Abstract Tumor necrosis factor receptor-associated protein 1 (TRAP1) is a mitochondrial homolog of HSP90 chaperones. It plays an important role in protection against oxidative stress and apoptosis by regulating reactive oxidative species (ROS) and may also exert the cytoprotective activities by regulating mitochondrial permeability transition pore opening. Recent studies have suggested that TRAP1 may regulate metabolism and mitochondrial homeostasis in cancer. To further elucidate the mechanistic role of TRAP1 in regulating tumor cell survival, we used cellular models to investigate cellular effects and signaling pathways regulated by TRAP1 in several colon cancer cell lines. Inhibition of TRAP1 by a small molecule inhibitor, Gamitrinib-triphenylphosphonium (GTPP), in colon cancer cells led to ROS production, ER stress, reduction of cell viability and induction of cell death. Interestingly, we observed wide-range of responses to TRAP1 inhibition among these colon cancer cell lines. In GTPP sensitive cell lines (SW48), GTPP induced upregulation of GRP78, CHOP and cleavage form PARP proteins. In contract, in GTPP resistant cells (DLD1 and RKO), GTPP treatment highly induced activation of antioxidant gene NRF2, leading to better cell survival under oxidative environment. NRF2 target genes HO1 and NQO1 were upregulated in GTPP-treated DLD1 cells, making these cells more resistant to GTPP treatment. Using reporter assay, we further found that transcriptional activation of antioxidative responds element (ARE) was increased in GTPP-treated DLD1 and RKO cells (GTPP resistant), but not in SW48 cells (GTPP sensitive). Combination of NRF2 inhibitor ML385 and GTPP led to excessive ROS production and exacerbated GTPP-induced cell death in GTPP resistant cells. These new findings for the first-time link TRAP1 and NRF2 in a common pathway regulating ROS production in colon cancer. Taken together, dual targeting of TRAP1 and NRF2 may potentially overcome the tumor cell resistance through disruption of mitochondrial redox homeostasis. Citation Format: Hong-Yuan Tsai, Ru Chen, Noah Shroyer, Sheng Pan. Metabolic targeting of NRF2 potentiates the efficacy of TRAP1 inhibitor GTPP in colorectal cancer [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 5681.

Abstract 1754: Amatoxin-based antibody-drug conjugates induce immunogenic cell death and improve the anti-tumor efficacy of immune checkpoint inhibitors in humanized mouse models

Abstract Background: Amatoxin-based antibody-drug conjugates (ATACs) comprise a new class of antibody-drug conjugates (ADCs) using amanitin as a toxic payload. Amanitin binds to the RNA polymerase II and thereby efficiently inhibits the cellular transcription process. In the present study, we show that ATACs belong to the class of immune activating drugs which exhibit synergistic anti-tumor efficacy with immune checkpoint inhibitors (ICIs) in vivo by induction of immunogenic cell death (ICD). ICIs are a new class of cancer therapeutics utilizing patients` immune system to kill cancer cells. ICIs rely on the activity of the immune system to develop their full potential. Therefore, drugs that are heating up cold tumors and make them visible to the patients` immune system and by that enhancing the anti-tumor efficacy of ICIs, e.g., ATACs, are on high demand for the treatment of tumor patients. Material and methods: Cell line: Her2+ cell lines: NCI-N87; CD19+ cell line: Raji Antibody: anti-Her2 and anti-CD19 engineered monoclonal antibody produced at Heidelberg Pharma Toxic warhead: Cysteine reactive amanitin-linker constructs were synthesized at Heidelberg Pharma and conjugated site-specifically to the antibody. Animal models: Raji cells with human peripheral blood mononuclear cells (PBMCs), NCI-N87 cells or HCBx-10/HCBx-11 PDX (Xentech, France) tumor pieces were implanted subcutaneously in mice. Treatment: ATAC: single dose i.v.; immune checkpoint inhibitors: Q3Dx6 or Q3Dx5. Results: In vivo treatment of heterogenous Her2low PDX tumors with an anti-Her2-ATAC led to a significant tumor growth delay and complete tumor remission. Furthermore, increased expression of HMGB1 and surface exposure of calreticulin (CRT), two hallmarks of ICD, was observed in the same PDX tumors as well as in a Her2+ CDX (NCI-N87) tumor after the treatment with an anti-HER2 ATAC. In addition, the combined treatment of ATACs and different ICIs targeting CTLA-4 (Ipilimumab), PD-L1 (Pembrolizumab) or PD-1 (Avelumab) had a synergistic effect in a humanized lymphoma CDX model in the presence of human PBMCs. Combination treatment led to enhanced tumor growth inhibition and more tumor free animals as compared to single treatments with ATAC or ICI. Conclusions: The strong anti-tumor efficacy of ATACs even in heterogenous patient-derived xenograft models in combination with the induction of ICD in vitro and in vivo suggest that the anti-tumor effect of ATACs is accompanied by the activation of the immune system. This hypothesis is strengthened by the finding that ATACs and ICIs show a synergistic effect in vivo. The presented data highlights the general concept of the synergistic effect of ATACs and ICIs which applies to several types of ICIs thus strengthening the scientific rationale for combination treatments in clinical trials. Citation Format: Christian Orlik, Franziska Ebeling, Kristin Decker, Irina Dranova, Anikó Pálfi, Christoph Mueller, Torsten Hechler, Andreas Pahl, Michael Kulke. Amatoxin-based antibody-drug conjugates induce immunogenic cell death and improve the anti-tumor efficacy of immune checkpoint inhibitors in humanized mouse models [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 1754.

Abstract 3011: Development of mitochondrial fission inhibitors in inducing non-apoptotic cell death in triple-negative breast cancer

Abstract Triple-negative breast cancer (TNBC) is the most aggressive subtype of metastatic breast cancer with poor clinical prognosis. Presently used neoadjuvant chemotherapy against TNBC, such as taxanes, and/or anthracyclines show an initial treatment response but follows with drug resistance, tumor relapse, side effects and high proclivity to develop metastases. Recent studies point out that targeting dysregulated mitochondrial dynamics, that is now linked to the tumor initiation, progression, and metastasis in TNBC, as an effective therapeutic strategy. In particular, mitochondrial fission factors such as dynamin-related protein 1 (Drp1) is upregulated in TNBC and is known to affect mitochondrial energetics and alter metabolism which favors tumor progression. We have designed and developed small molecule inhibitors to specifically target Drp1 as a viable therapeutic strategy for metastatic TNBC (mTNBC). We recently identified a new class of structurally constrained thienopyridine (TPH) probes, and particularly a new analog TPH104c that showed higher binding affinity in silico to Drp1 as compared to Mdivi-1, a weak inhibitor of Drp1. Surface plasmon resonance studies suggest that TPH104c binds with Drp1 with high affinity. Similarly, mechanistic studies using Western blotting and immunofluorescence analysis confirmed that TPH104c significantly downregulated the levels of total and phosphorylated form of Drp1 at serine 616 in MDAMB-231 and BT-20 TNBC cells. Interestingly, Drp1 inhibition by TPH104c led to a hitherto unreported form of caspase- and reactive oxygen species (ROS)-independent non-apoptotic form of cell death. Rather than shrinking in size, or formation of apoptotic blebs which are classical features of apoptosis, TPH104c treated TNBC cells were enlarged, rounded, and swollen in size. These cells eventually lost their proliferative ability and were significantly less confluent than control cells. Additionally, in TPH104c-treated cells, neither the mitochondrial membrane potential was lost, nor the caspases were activated as also indicated by a lack of rescue of cell death when incubated with pan-caspase inhibitor. Further studies are in progress to understand the role of Drp1 inhibition in the induction of non-apoptotic cell death which will further help to optimize an effective lead compound for the future pre-clinical development of promising anticancer agents targeting mTNBC. Citation Format: Saloni Malla, Shikha Kumari, David Terrero, Dayanidhi Raman, Amit K. Tiwari. Development of mitochondrial fission inhibitors in inducing non-apoptotic cell death in triple-negative breast cancer [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 3011.

Abstract 5384: Cell growth of sunitinib-resistant renal cell carcinoma was inhibited by Shikonin through necroptosis induction and Akt/mTOR signaling inhibition

Abstract Despite the establishment of new therapy options during the last decades, advanced renal cell carcinoma (RCC) remains an uncurable disease, due to evolving drug-resistance. New, more efficient treatment strategies are still in urgent demand. Shikonin (SHI) from Traditional Chinese Medicine has exhibited anti-tumor properties in several tumor entities. Thus, in the current investigation we evaluated the impact of SHI on progressive growth and metastatic behavior, not only in therapy-sensitive (parental) but also in therapy-resistant RCC cells. Parental and sunitinib-resistant RCC cells, Caki-1, 786-O, KTCTL-26, and A498, were treated with SHI. Tumor cell growth, proliferation, clonogenic capacity, cell cycle phase distribution, induction of cell death (apoptosis and necroptosis) and the expression and activity of regulating and signaling proteins were evaluated. Moreover, the adhesion and chemotactic activity of the RCC cells after exposure to SHI were investigated. SHI did not affect cell motility but led to significant inhibition of progressive tumor cell growth in both therapy-sensitive and therapy-resistant RCC cells. Thereby, SHI significantly inhibited the growth, proliferation and clone formation in parental and sunitinib-resistant RCC cells by G2/M phase arrest through down-regulation of cell cycle activating proteins, blocking cell division. Furthermore, SHI induced apoptosis and necroptosis by activating necrosome complex proteins. Concomitantly, SHI impaired the Akt/mTOR pathway, pivotal for cell survival and growth. Thus, SHI may hold promise as an additive option in treating patients with advanced and therapy-resistant RCC. Citation Format: Eva Juengel, Sascha D. Markowitsch, Olesya Vakhrusheva, Yasminn Akele, Jovanna Kitanovic, Patricia Schupp, Kimberly S. Slade, Anita Thomas, Igor Tsaur, Rene Mager, Thomas Efferth, Axel Haferkamp. Cell growth of sunitinib-resistant renal cell carcinoma was inhibited by Shikonin through necroptosis induction and Akt/mTOR signaling inhibition [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 5384.

Abstract 703: Novel histone lysine demethylase inhibitors disrupt PAX3-FOXO1-driven transcriptional output in fusion-positive rhabdomyosarcoma

Abstract BACKGROUND: The PAX3/7-FOXO1 (P3F) fusion transcription factor is the oncogenic driver in fusion-positive rhabdomyosarcoma (FP-RMS). P3F drives oncogenesis in FP-RMS through transcriptional modulation of downstream target genes. Thus, P3F represents a unique vulnerability in FP-RMS. A screen for inhibitors of P3F action identified novel histone lysine demethylase (KDM) inhibitors characterized in this study. MATERIALS/METHODS: A specific luciferase assay which monitors P3F activity was utilized to screen 62,643 compounds. The top candidate with unknown mechanism of action, PFI-63, and its analog PFI-90, were characterized. Western blotting was used to detect MYOG, PARP, and PAX3-FOXO1. KDM enzyme inhibition assays were conducted. Ligand-observed NMR analysis was used to determine binding of PFI-90 to KDM3B. KDM knockdown using CRISPRi was carried out. ChIP-seq analysis on H3K4me3, H3K9me2, H3K27me3, H3K27ac, and PAX3-FOXO1 was performed. Mouse xenograft models of FP-RMS were used to determine in vivo efficacy. RESULTS: 64 compounds that inhibited P3F activity without general inhibition of transcription or induction of cell death were further characterized. PFI-63 and a more water-soluble analog, PFI-90, were identified. GSEA of RNA-seq showed activation of apoptosis and myogenesis while P3F targets were repressed. Activation of apoptosis and myogenesis were validated by Western blotting showing PARP cleavage and increased MYOG levels, respectively. RNA-seq suggested that PFI-63 and PFI-90 were KDM inhibitors. In vitro enzymatic inhibition assays confirmed activity against multiple KDMs with most potent inhibition of KDM3B. Western analysis for methylation at H3K4 and H3K9 showed increases after PFI-90 treatment. NMR techniques confirmed biophysical binding of PFI-90 to KDM3B. RNA-seq of KDM knockdowns demonstrated that KDM3B knockdown most closely recapitulated PFI-90’s downregulation of P3F targets. Knockdown of KDM1A recapitulated PFI-90’s upregulation of myogenesis and apoptosis. ChIP-seq analysis showed increased levels of H3K9me2 at P3F sites while H3K4me3 was increased in muscle differentiation and apoptosis. In two different in vivo xenograft models of FP-RMS, PFI-90 treatment delayed tumor progression vs DMSO control. CONCLUSION: We identified novel multi-KDM inhibitors with highest potency for KDM3B. Downregulation of P3F by KDM3B inhibition is associated with increased H3K9me2 at P3F sites. PFI-90 also inhibits KDM1A which increases H3K4me3 at myogenesis and apoptosis genes. Thus, PFI-90 is a novel multi-KDM inhibitor whose biological effect on FP-RMS is by inhibition of KDM3B and KDM1A. Pre-clinical validation via FP-RMS xenograft models showed that PFI-90 delayed tumor progression. PFI-90 thus represents a promising novel compound for the treatment of FP-RMS, and potentially, other transcriptionally driven cancers. Citation Format: Yong Yean Kim, Girma Woldemichael, Berkley Gryder, Silvia Pomella, Ranuka Sinniah, Josh Kowalczyk, Young Song, Mehal Churiwal, Joseph Barchi, John Schneekloth, Xinyu Wen, Hsein-Chao Chou, Barry Okeefe, John Shern, Robert Hawley, Javed Khan. Novel histone lysine demethylase inhibitors disrupt PAX3-FOXO1-driven transcriptional output in fusion-positive rhabdomyosarcoma [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 703.

Abstract 1766: SGN-ALPV a novel, investigational vedotin ADC demonstrates highly effective targeting of oncofetal phosphatases ALPP and ALPPL2 in preclinical models

Abstract Antibody-drug conjugates (ADC) leverage the specificity of antibodies to direct the delivery of potent cytotoxic agents to cancer cells. To fully leverage this specificity, targets with significant differential tumor versus normal tissue expression are ideal for ADC development. Placental alkaline phosphatases, ALPP and ALPPL2, are proteins present during fetal development but are found in several tumor types, making them an attractive ADC target. These cell membrane-attached phosphatases form homo- and heterodimers and play key roles in nucleotide recycling. SGN-ALPV is a novel investigational vedotin ADC comprised of a humanized IgG1 monoclonal antibody conjugated to the microtubule disrupting agent monomethyl auristatin E (MMAE) via a protease-cleavable peptide linker that has been clinically validated in multiple ADC programs. Here we characterize the target antigens, ALPP and ALPPL2, and evaluate SGN-ALPV antitumor activity in preclinical models. Immunohistochemistry and genomic data mining showed that ALPP and ALPPL2 have a highly restricted normal tissue expression yet high expression in several solid tumor types including ovarian, endometrial, germ cell, non-small cell lung and gastric carcinomas. Importantly, normal tissue expression is restricted to placenta and reproductive tissue, with low levels in lung tissue. SGN-ALPV utilizes a humanized antibody, h12F3, that is highly specific for both human and cynomolgus monkey ALPP and ALPPL2 proteins, but not other related phosphatases. In vitro, upon binding to SGN APLV, the antigens, ALPP and ALPPL2, are internalized to lysosomal vesicles releasing the MMAE payload, whose cytotoxic features drive mitotic arrest, apoptosis, and the induction of immunogenic cell death. Additionally, SGN-ALPV mediates antibody dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP) in vitro but lacks complement-dependent cytotoxicity. In preclinical studies, SGN-ALPV exhibits robust antitumor activity in cell line- and patient-derived xenograft models of ovarian, lung, pancreatic, and gastric carcinoma including models with both homogenous and heterogeneous expression of ALPP and ALPPL2, consistent with robust bystander activity of vedotin ADCs. Although, the contribution of ADCC and ADCP in vivo is currently unknown, it is expected that the antitumor activity of SGN-ALPV is mediated by MMAE cytotoxicity. SGN-ALPV was well tolerated in non-human primates (NHP) and exhibited linear pharmacokinetic characteristics, with a toxicity profile consistent with other vedotin-based ADCs. In summary, differential expression of ALPP and ALPPL2 in the tumor versus normal tissue, antibody specificity, antitumor activity, and tolerability of SGN-ALPV provide a strong rationale for the initiation of a planned first-in-human Phase 1 clinical study. Citation Format: Sarah Anderson, Nanna Hansen, Robert Lawrence, Aroon T. Chande, David Ortiz, Christopher Carosino, Esther Trueblood, Nicole Stevens, Kerry Klussman, Angela Epp, Bill Arthur, Shyra Gardai, Hector Rincon. SGN-ALPV a novel, investigational vedotin ADC demonstrates highly effective targeting of oncofetal phosphatases ALPP and ALPPL2 in preclinical models [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 1766.

Abstract 411: Reversibility of tumor adaptations to PI3K inhibition decreases as a function of time

Abstract The PI3K pathway is a key regulator of metabolism, cell proliferation and migration and some of its components (e.g. PIK3CA, HER2 and PTEN) are frequently altered in cancer by genetic events that deregulate its output. However, inhibitors of components of the pathway have only modest antitumor effects and, in some instances, have resulted in “tumor flares” in patients upon cessation of therapy. Low efficacy has been attributed to toxicity and to adaptive resistance. Inhibition of the PI3K pathway has been associated with relief of feedback inhibition of receptor signaling (RTKs) by a variety of mechanisms including increased receptor expression and decreased expression of the PTEN phosphatase, a major negative regulator of the pathway. Now we show that changes in expressions of RTKs, PTEN, anti-apoptosis proteins like Mcl1 and transcription factors known to drive oncogenic and survival programs in tumors like c-Myc, c-Jun, FOXOs are induced upon continuous long-term PI3K inhibition (2 weeks and longer). These changes increase as a function of duration of inhibition and become less reversible with time in drug. Unbiased clustering of RNA seq of cells treated with PI3K inhibitors revealed clusters of gene expression changes based upon short or long-term inhibition. Correspondingly the mRNA of the RTKs and transcription factors change with similar kinetics as that of the proteins and become less reversible with duration of inhibition. Moreover, the persister cells treated with PI3K inhibitors for 2 weeks or longer grow faster than the treatment naive cells upon drug removal both in vitro and in vivo and the degree of reversibility of the increased proliferation rate is inversely proportional to the duration of inhibition. Persister cells treated with PI3K inhibitors for more than two weeks have higher levels of expression of a set of transcription factors, RTKs, Mcl1 and of pAKT after one week of drug wash-out and are less sensitive to re-inhibition of PI3K. Combined inhibition of PI3K with Mcl1 inhibitors lead to greater reduction of cell proliferation and increased cell death.This suggests that continuous long-term inhibition of PI3K leads to a chronic adaptive state driven by relief of negative feedback which becomes less reversible with increasing duration of inhibition. Cells undergoing long term inhibition have higher steady state levels of components of the pathway leading to increased cell growth upon drug removal as compared to untreated cells. The data suggests that induction of adaptive resistance is in part responsible for the continued persistence of tumor masses after the initial response to targeted therapy. We hypothesize that pulsatile targeted inhibition with short intervals off therapy could avoid development of chronic adaptation. Intermittent administration of PI3K inhibitor combinations that cause potent induction of cell death may result in improved therapeutic outcomes. Citation Format: Radha Mukherjee, Elisa de Stanchina, Sarat Chandarlapaty, Neal Rosen. Reversibility of tumor adaptations to PI3K inhibition decreases as a function of time [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 411.

Abstract 1346: T cell receptor repertoire sequencing reveals chemotherapy-driven clonal expansion in colorectal liver metastases

Abstract Background: Colorectal liver metastasis (CLM) is a leading cause of cancer-related mortality in the industrialized world, and only a small fraction of patients are eligible for curative resections. While immune checkpoint inhibition (ICI) has resulted in major breakthroughs in some cancers, the response in microsatellite stable (MSS) colorectal cancer has been disappointing. High density of infiltrating T cells has been associated with improved overall survival and response to ICI. We previously found that T cell infiltration varied considerably in a cohort of MSS CLM cases, which could reflect varying sensitivity to ICI treatment. The aim of this study was to quantify and characterize T cell infiltration in CLM using T cell receptor (TCR) repertoire sequencing (TCRR-seq). Methods: Resection specimens were collected from 85 patients with CLM included in the Oslo CoMet trial (NCT01516710). Of these, 37 and 13 patients had received neoadjuvant chemotherapy (NACT) within a short or long interval, respectively, prior to resection, while 35 had not (no-NACT group). TCRR-seq libraries were prepared from DNA extracted from CLM tissue using the hsTCRB kit from Adaptive Biotechnologies, which allows both quantitative and qualitative analysis of TCR repertoires. The T cell fraction relative to the total number of cells was estimated for each CLM sample. Repertoire clonality was analyzed using Hill diversity profiles, a mathematical framework for determining species diversity derived from the ecological literature. Network analyses were conducted to analyze convergence of clonal T cell receptor amino acid sequences in each repertoire. Results: The mean CLM T cell fraction was 8.1% (0.4-31.3%), and tumors with high T cell fraction had more monoclonal repertoires (p=1.86E-6). Interestingly, CLM from patients in the short interval group had increased T cell infiltration compared to the no-NACT and long interval groups (mean 10.5% and 6.3%, respectively; p=0.04), and these repertoires were also more monoclonal (p=0.003). Network analysis revealed that clonally expanded T cells tended to exhibit little overall receptor similarity to other T cells in the repertoire. Clonally expanded T cells were overall also private (unique) to each patient. Conclusion: A short interval between exposure to cytotoxic chemotherapy and surgical resection was associated with increased T cell infiltration in CLM samples, and the increase was associated with clonal expansion. The findings suggest that chemotherapy could be a driver of clonal expansion in this setting, possibly by induction of immunogenic cell death. Furthermore, the CLM neo-antigen landscape appears to be diverse and private to the individual patient. Citation Format: Eirik Høye, Vegar Johansen Dagenborg, Annette Torgunrud, Christin Lund-Andersen, Åsmund Avdem Fretland, Susanne Lorenz, Bjørn Edwin, Eivind Hovig, Anne Hansen Ree, Kjersti Flatmark. T cell receptor repertoire sequencing reveals chemotherapy-driven clonal expansion in colorectal liver metastases [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 1346.

Design, synthesis and evaluation of novel tetrahydropyridothienopyrimidin-ureas as cytotoxic and anti-angiogenic agents

The novel derivatives of tetrahydropyridothienopyrimidine-based compounds have been designed and efficiently synthesized with good yields through seven steps reaction. The anticancer activity of compounds 11a-y has been evaluated against MCF-7, PC-3, HEPG-2, SW-480, and HUVEC cell lines by MTT assay. The target compounds showed IC50 values between 2.81–29.6 μg/mL and were compared with sorafenib as a reference drug. Among them, compound 11n showed high cytotoxic activity against four out of five examined cell lines and was 14 times more selective against MRC5. The flow cytometric analysis confirmed the induction of apoptotic cell death by this compound against HUVEC and MCF-7 cells. In addition, 11n caused sub-G1 phase arrest in the cell cycle arrest. Besides, this compound induced anti-angiogenesis in CAM assay and increased the level of caspase-3 by 5.2 fold. The western-blot analysis of the most active compound, 11n, revealed the inhibition of VEGFR-2 phosphorylation. Molecular docking study also showed the important interactions for compound 11n.

Assessment of antitumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone in subcutaneous xenografts of A-549 tumor cell culture.

e20520 Background: The aim of the study was an assessment of antitumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone for subcutaneous xenografts of A-549 lung tumor cells in immunodeficient Balb/c Nude mice. Methods: 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone is a new compound in the series of 2-quinoline-2-yl-1,3-tropolone derivatives. The study included 50 immunodeficient Balb/c Nude mice divided into 5 groups depending on the dosage of the studied substance (0.0055; 0.055; 0.55 and 2.75 mg/g). The A-549 cells of lung cancer were used as the xenograft. Antitumor effect of tropolone was evaluated by the inhibition of tumor growth (ITG%) and the index of tumor growth (I). The experiment lasted for 36 days starting from the first administration of substances. Results: Indicators of the average tumor volume on day 36 of the experiment in experimental groups 1, 2, 3 and 4 and in the control group were 678.9; 952.3; 1746.4; 2150.8 and 2729.5 mm3, respectively. The indices of tumor growth in groups 1, 2, 3 and 4 were statistically significantly lower than in group 5 (controls) starting from days 15, 21, 21 and 24, respectively, and until the end of the experiment. Maximal differences between groups 1 and 5 were observed on days 33 and 36 – by 3.7 (p = 0.01) and 4.1 (p = 0.003) times, respectively. Antitumor effect of 2-(6,8-dimethyl-5-nitro-4-chloroquinoline-2-yl)-5,6,7-trichloro-1,3-tropolone demonstrated by the study could be associated with various mechanisms. For example, its closely related compound – hinoktiol, according to numerous studies, has a cytotoxic effect associated with stopping the cell cycle, induction of apoptosis, DNA damage, and autophagic death of tumor cells. Conclusions: The study demonstrated statistically significant differences in xenograft volumes between all experimental groups and the control group. 2.75 mg/g of mouse weight was the most effective dosage of the studied compound leading to a slow decrease in tumor growth rates and a decrease in the volumes of subcutaneous xenografts.

Evaluation of the Anti-Histoplasma capsulatum Activity of Indole and Nitrofuran Derivatives and Their Pharmacological Safety in Three-Dimensional Cell Cultures.

Histoplasma capsulatum is a fungus that causes histoplasmosis. The increased evolution of microbial resistance and the adverse effects of current antifungals help new drugs to emerge. In this work, fifty-four nitrofurans and indoles were tested against the H. capsulatum EH-315 strain. Compounds with a minimum inhibitory concentration (MIC90) equal to or lower than 7.81 µg/mL were selected to evaluate their MIC90 on ATCC G217-B strain and their minimum fungicide concentration (MFC) on both strains. The quantification of membrane ergosterol, cell wall integrity, the production of reactive oxygen species, and the induction of death by necrosis–apoptosis was performed to investigate the mechanism of action of compounds 7, 11, and 32. These compounds could reduce the extracted sterol and induce necrotic cell death, similarly to itraconazole. Moreover, 7 and 11 damaged the cell wall, causing flaws in the contour (11), or changing the size and shape of the fungal cell wall (7). Furthermore, 7 and 32 induced reactive oxygen species (ROS) formation higher than 11 and control. Finally, the cytotoxicity was measured in two models of cell culture, i.e., monolayers (cells are flat) and a three-dimensional (3D) model, where they present a spheroidal conformation. Cytotoxicity assays in the 3D model showed a lower toxicity in the compounds than those performed on cell monolayers. Overall, these results suggest that derivatives of nitrofurans and indoles are promising compounds for the treatment of histoplasmosis.