Undergo Cell Death Research Articles

Abstract 1435: A GABP dominant negative approach to targeting tumor cell immortality

Abstract Immortality is a hallmark of human cancer cells and therapeutic reversal is of great interest. Telomerase Reverse Transcriptase promoter (TERTp) mutations reactivate TERT expression, the rate limiting step in telomerase activity and cellular immortality. While prior telomerase blocking therapies lack tumor selectivity and were poorly tolerated, TERTp mutations and their regulation offer a unique opportunity for tumor specific reversal of cellular immortality. TERTp mutations are the most common non-coding mutation across all cancers, including glioblastoma (GBM), melanoma, urothelial carcinoma, and many others. The mutations generate a de novo E26 Transformation Specific (ETS) binding motif that recruits the GA-Binding Protein (GABP) multimeric complex to reactivate TERT expression. While GABP can form both a dimeric and tetrameric complex, we found that the tetrameric complex is responsible for reactivation of the mutant TERTp, suggesting selectively targeting the tetramer may induce telomere shortening and tumor cell death. However, through knocking out the tetramer, we found that the GABP dimer is sufficiently upregulated to maintain TERT expression. In the absence of the dimer and the tetramer isoform, many tumor cells senesce or undergo cell death. However, some escape this fate via upregulation of a second tetramer forming GABP paralogue that maintains TERT expression. Therefore, we devised a transactivation domain null GABP subunit that we hypothesized would act in a dominant negative manner to block the GABP dimer and both GABP tetramers. Introduction of the dominant negative construct into TERTp mutant GBM, meningioma, medulloblastoma, oligodendroglioma, and urothelial carcinoma cells significantly reduced TERT expression while not altering expression in TERTp wildtype GBM cells. Furthermore, the dominant negative is able to shorten telomere length in TERTp mutant GBM cells. While therapeutic targeting of the three GABP complexes seems particularly challenging, our initial dominant negative appears to do so. Ultimately the dominant negative could be delivered directly into tumors via clinically approved viral delivery systems such as retroviral replicating vectors. Citation Format: Nicholas O. Stevers, Sara A. Collins, Samuel H. Wu, Noriyuki Kasahara, Joseph F. Costello. A GABP dominant negative approach to targeting tumor cell immortality [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1435.

P53 and Myofibroblast Apoptosis in Organ Fibrosis.

Organ fibrosis represents a dysregulated, maladaptive wound repair response that results in progressive disruption of normal tissue architecture leading to detrimental deterioration in physiological function, and significant morbidity/mortality. Fibrosis is thought to contribute to nearly 50% of all deaths in the Western world with current treatment modalities effective in slowing disease progression but not effective in restoring organ function or reversing fibrotic changes. When physiological wound repair is complete, myofibroblasts are programmed to undergo cell death and self-clearance, however, in fibrosis there is a characteristic absence of myofibroblast apoptosis. It has been shown that in fibrosis, myofibroblasts adopt an apoptotic-resistant, highly proliferative phenotype leading to persistent myofibroblast activation and perpetuation of the fibrotic disease process. Recently, this pathological adaptation has been linked to dysregulated expression of tumour suppressor gene p53. In this review, we discuss p53 dysregulation and apoptotic failure in myofibroblasts and demonstrate its consistent link to fibrotic disease development in all types of organ fibrosis. An enhanced understanding of the role of p53 dysregulation and myofibroblast apoptosis may aid in future novel therapeutic and/or diagnostic strategies in organ fibrosis.

Abstract P2-16-01: The Kinesin-like protein Kif11 is essential for the Survival of TP53-mutant Triple Negative Breast Cancer Cells

Abstract Background: Breast cancer is the most commonly diagnosed non-cutaneous malignancy in American women and one of the leading causes of cancer deaths. Breast cancer can be divided into several subtypes, the most aggressive of which is Triple-Negative Breast Cancer (TNBC), a disease with few targeted therapies. TP53 mutations are found in 80% or more of TNBCs. However, direct targeting of mutant TP53 has been difficult. To identify drugs that can specifically induce the death of TP53-mutant breast cancers, we conducted a drug screen in TP53-mutant and TP53-wild type breast cancer cells. Through this combined in silico and in vitro drug screen, we discovered that TP53 mutant TNBC cells have an increased sensitivity to KIF11 inhibition as compared to TP53 wild-type cells. Hypothesis: We hypothesize that TP53 mutational status confers sensitivity of triple-negative breast cancer cells to KIF11 inhibition. Methods: We obtained data on TP53 mutational status, KIF11 mRNA expression levels, and clinical characteristics from publicly-available TCGA, METABRIC, and CCLE datasets. To demonstrate the effect of KIF11 inhibition on cell growth, we treated TP53 mutant and wild-type breast cancer cell lines with the small molecule KIF11 inhibitor SB-743921 and KIF11 siRNAs. Cell counts were obtained through staining with DAPI or Hoechst nuclear stains and imaging on the MetaXPress PICO instrument. To determine protein expression of p53 and Kif11 across various cell lines, western blotting was performed. We then investigated the biological mechanisms of growth inhibition by DRAQ7 staining and flow cytometry analysis following Annexin V-PI staining. Results: Using cell growth assays we demonstrated that TP53 mutant cells are more sensitive to KIF11 inhibition. We next utilized cell lines in which TP53 mutations had been introduced into TP53 wild-type cells to show that overexpression of a TP53 mutant gene can sensitize cells to KIF11 inhibition. Using assays of proliferation and apoptosis, we showed that TP53 mutant breast cancer cells treated with a KIF11 inhibitor undergo cell death. Using publicly-available datasets of breast cancers, we showed that KIF11 is upregulated in TNBCs and TP53 mutant cancers, and that high expression of KIF11 in breast cancer is correlated with poorer clinical prognosis. Conclusions: Our results show that the Kif11 protein is essential for the survival of TP53 mutant TNBC cells. Inhibitors of Kif11 induce death of TP53 mutant TNBCs and thus, this kinesin-like protein involved in cell spindle mechanics is a potential target for the treatment of these aggressive cancers. Research reported in this publication was supported by the National Center for Advancing Translational Sciences of the National Institutes of Health under Award Numbers TL1TR003169 and UL1TR003167. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This work was also supported by the John Charles Cain Endowment. Citation Format: Amanda Lanier, Abhijit Mazumdar, William Tahaney, Powel Brown. The Kinesin-like protein Kif11 is essential for the Survival of TP53-mutant Triple Negative Breast Cancer Cells [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-01.

Relationship Between Malignant Brain Tumors and Values of Homocysteine, Folic Acid and Vitamin B12

Abstract Glioblastoma (GBM) is the most common primary malignant brain tumor in adults. Homocysteine (Hcy) has a detrimental influence on human neurons, considering that human GBM cells undergo cell death already at D,L-Hcy concentrations in culture medium of 50 μM. This data demonstrate that Hcy is a potent gliotoxic agent capable of inducing the death of human glial cells already at concentrations reached in brain during hyperhomocysteinemia. The one retrospective study found that the serum vitamin B12 level can be used to predict survival time in metastatic cancer patients including neurological cancer. Cancer risk increases with elevated vitamin B12 level, mostly within the first year of the follow-up period, suggesting that vitamin B12 level could be used as a cancer diagnostic marker. In addition, the relationship between elevated vitamin B12 level and poor cancer survival time has been reported. Previous investigation suggests that the folate supplementation could be used as an adjuvant in antiglioma therapy to limit the low DNA methylation level because this confers a poor prognosis in glioblastoma multiforme patients. Taking into account all presented data, it can be concluded that effect of homocystein, folic acid and vitamin B12 on formation, development and outcome of treatment in patients with carcinoma is very intriguing question, whose response requires additional both experimental and clinical research. There lack of data in the literature on the incidence of elevated levels of Hcy in the blood, as well as the disorders of folic acid and vitamin B12, at malignant tumors of the brain.

Experience-dependent functional plasticity and visual response selectivity of surviving subplate neurons in the mouse visual cortex

Subplate neurons are early-born cortical neurons that transiently form neural circuits during perinatal development and guide cortical maturation. Thereafter, most subplate neurons undergo cell death, while some survive and renew their target areas for synaptic connections. However, the functional properties of the surviving subplate neurons remain largely unknown. This study aimed to characterize the visual responses and experience-dependent functional plasticity of layer 6b (L6b) neurons, the remnants of subplate neurons, in the primary visual cortex (V1). Two-photon Ca2+ imaging was performed in V1 of awake juvenile mice. L6b neurons showed broader tunings for orientation, direction, and spatial frequency than did layer 2/3 (L2/3) and L6a neurons. In addition, L6b neurons showed lower matching of preferred orientation between the left and right eyes compared with other layers. Post hoc 3D immunohistochemistry confirmed that the majority of recorded L6b neurons expressed connective tissue growth factor (CTGF), a subplate neuron marker. Moreover, chronic two-photon imaging showed that L6b neurons exhibited ocular dominance (OD) plasticity by monocular deprivation during critical periods. The OD shift to the open eye depended on the response strength to the stimulation of the eye to be deprived before starting monocular deprivation. There were no significant differences in visual response selectivity prior to monocular deprivation between the OD changed and unchanged neuron groups, suggesting that OD plasticity can occur in L6b neurons showing any response features. In conclusion, our results provide strong evidence that surviving subplate neurons exhibit sensory responses and experience-dependent plasticity at a relatively late stage of cortical development.

GRK5-mediated inflammation and fibrosis exert cardioprotective effects during the acute phase of myocardial infarction.

During myocardial infarction (MI), cardiac cells at the infarcted area undergo cell death. In response, cardiac myofibroblasts, which are mainly differentiated from resident fibroblasts upon inflammation, produce extracellular matrix proteins such as collagen to fill the damaged areas of the heart to prevent cardiac rupture. In this study, we identified a cardioprotective role of G-protein-coupled receptor kinase 5 (GRK5) in MI. GRK5 expression was found to increase in the mouse heart after MI and was highly expressed in cardiac fibroblasts/myofibroblasts. In fibroblasts/myofibroblasts, GRK5 promoted the expression of inflammation-related genes through nuclear factor-κB activation, leading to an increase in the expression levels of fibrosis-related genes. Bone marrow transfer experiments confirmed that GRK5 in fibroblasts/myofibroblasts, but not in infiltrated macrophages in the infarcted area, is mainly responsible for GRK5-mediated inflammation in infarcted hearts. In addition, inflammation and fibrosis at the infarcted area were significantly suppressed in GRK5 knockout mice, resulting in increased mortality compared with that in wild-type mice. These data indicate that GRK5 in cardiac fibroblasts/myofibroblasts promotes inflammation and fibrosis to ameliorate the damage after MI.

Distinct Transcriptional and Functional Differences of Lung Resident and Monocyte-Derived Alveolar Macrophages During the Recovery Period of Acute Lung Injury.

In acute lung injury, two subsets of lung macrophages exist in the alveoli: tissue-resident alveolar macrophages (AMs) and monocyte-derived alveolar macrophages (MDMs). However, it is unclear whether these 2 subsets of macrophages have different functions and characteristics during the recovery phase. RNA-sequencing of AMs and MDMs from the recovery period of LPS-induced lung injury mice revealed their differences in proliferation, cell death, phagocytosis, inflammation and tissue repair. Using flow cytometry, we found that AMs showed a higher ability to proliferate, whereas MDMs expressed a larger amount of cell death. We also compared the ability of phagocytosing apoptotic cells and activating adaptive immunity and found that AMs have a stronger ability to phagocytose, while MDMs are the cells that activate lymphocytes during the resolving phase. By testing surface markers, we found that MDMs were more prone to the M1 phenotype, but expressed a higher level of pro-repairing genes. Finally, analysis of a publicly available set of single-cell RNA-sequencing data on bronchoalveolar lavage cells from patients with SARS-CoV-2 infection validated the double-sided role of MDMs. Blockade of inflammatory MDM recruitment using CCR2-/- mice effectively attenuates lung injury. Therefore, AMs and MDMs exhibited large differences during recovery. AMs are long-lived M2-like tissue-resident macrophages that have a strong ability to proliferate and phagocytose. MDMs are a paradoxical group of macrophages that promote the repair of tissue damage despite being strongly pro-inflammatory early in infection, and they may undergo cell death as inflammation fades. Preventing the massive recruitment of inflammatory MDMs or promoting their transition to pro-repairing phenotype may be a new direction for the treatment of acute lung injury.

Pediatric Fulminant Hepatic Failure: Systematic Review

Abstract: Fulminant hepatic failure FHF in children is a rare but often fatal condition. Our understanding of this disorder is limited due to the disorder's rarity. The majority of FHF cases in children have no known cause. The mechanisms by which hepatocytes undergo cell death are unknown. The aetiology and degree of CNS involvement influence the outcome. The study aims to summarize current evidence on the prevalence, risk factors and management approaches of irritable bowel syndrome in Saudi Arabia. For article selection, the PubMed database and EBSCO Information Services were used. All relevant articles relevant to our topic and other articles were used in our review. Other articles that were not related to this field were excluded. The data was extracted in a specific format that the group members reviewed. Seven studies were included and affirmed that mortality of fulminant hepatic failure is high, and the commonest aetiology is viral infections, particularly HAV. The peak level of total serum bilirubin, the rate of change of the prothrombin time/daily and ammonia level were significant predictors of mortality.

Differential Cytotoxic Activity of a New Cationic Pd(II) Coordination Compound with N4-Tetradentate Hybrid Ligand in Cancer Cell Lines

Objective: Successful cancer treatment still requires the discovery of novel compounds that hold promise for chemotherapeutics. The objective of this study was to examine the effectiveness of a newly synthesized cationic palladium(II) coordination compound that functions via several pathways to provide an efficient therapeutic option for various cancer cells. Materials and Methods: A new cationic palladium(II) coordination compound, [Pd(L)]Cl2·H2O, denoted as Complex 1, where the ligand L is the compound 6,6'-bis(NH-benzimidazol-2-yl)-2,2'-bipyridine), was synthesized and characterized by the attenuated total reflectance (ATR) - fourier-transform infrared spectroscopy (FT-IR), proton nuclear magnetic resonance (1 H NMR), electrospray ionization mass spectrometry (ESI-MS), and carbon-hydrogen-nitrogen (CHN) analyses. The density functional theory (DFT) calculations show the coordination sphere around the metal center in Complex 1 to be made up of tertiary N atoms of the pyridine (py) and benzimidazole (bim) rings completing the square-planar geometry with significant distortion. The anti-growth/cytotoxic activity of the complex was determined using the sulforhodamine B (SRB) and adenosine triphosphate (ATP) viability assays for 24 and 48 h in vitro. The study evaluates the determinations for annexin V-propidium iodide (PI) positivity, mitochondrial membrane potential loss, Bcl-2 protein inactivation, and deoxyribonucleic acid (DNA) damage to investigate the cell death mode and its partial mechanism. Results: Complex 1 caused cytotoxicity in a dose-dependent manner in all the cell lines used, with IC50 values ranging from 2.6-8.8 μM for 48 h. Among the cancer models, colon and breast cancer cell lines underwent cell death by well-described apoptosis through the intrinsic pathway involving the mitochondria. However, the other cell lines did not show such a cell death modality. This implies that differential cell death modes operate based on the cancer type. Conclusion: For the treatment of breast and colon cancers, the complex 1 appears to be a unique, promising complex. Therefore, complex 1 deserves further attention for proof of concept in animal models.

Histone-deacetylase-inhibitory effects of periodontopathic-bacterial metabolites induce human gingival epithelial Ca9-22 cell death.

Dental plaque bacteria produce high concentrations of short-chain fatty acids (SCFAs), as bacterial metabolites. SCFA-treated gingival epithelial cells undergo cell death. Our previous reports demonstrated that butyrate-induced cell death depends on autophagy and reactive oxygen species (ROS). However, the precise mechanisms underlying SCFA-induced gingival epithelial cell death is poorly understood. Butyrate is a strong histone deacetylase (HDAC) inhibitor. Therefore, we determined the involvement of HDAC inhibitory activity in SCFA-induced gingival epithelial cells. Ca9-22 cells were used as an in vitro counterpart of gingival epithelial cells. Ca9-22 cells were treated with HDAC inhibitors in the presence or absence of C646, aP300 histone acetyltransferase (HAT) inhibitor, and compared the number of dead cells, which are measured using SYTOX Green dye. Acetylation levels of histone H3 were examined using western blotting. Changes in transcriptomes during the butyrate and C646 treatment were examined using RNA sequencing analysis. The butyrateor propionate-treatment of Ca9-22 cells induced acetylation of histone H3, while the C646 treatment strongly reduced the elevated acetylation levels. Accordingly, butyrate or propionate-induced cell death was inhibited by the C646 treatment. Similar results were obtained when other HDAC inhibitors were used. Whole transcriptome analysis revealed that the expression of numerous genes was altered by butyrate-induced histone acetylation. Moreover, some autophagy and ROS-related genes found in the altered genes might induce cell death. This study suggests the need for HDAC-inhibitory activity of bacterial metabolites to induce cell death, and the effects might enhance autophagy and ROS production.

SRSF6 balances mitochondrial-driven innate immune outcomes through alternative splicing of BAX.

To mount a protective response to infection while preventing hyperinflammation, gene expression in innate immune cells must be tightly regulated. Despite the importance of pre-mRNA splicing in shaping the proteome, its role in balancing immune outcomes remains understudied. Transcriptomic analysis of murine macrophage cell lines identified Serine/Arginine Rich Splicing factor 6 (SRSF6) as a gatekeeper of mitochondrial homeostasis. SRSF6-dependent orchestration of mitochondrial health is directed in large part by alternative splicing of the pro-apoptosis pore-forming protein BAX. Loss of SRSF6 promotes accumulation of BAX-κ, a variant that sensitizes macrophages to undergo cell death and triggers upregulation of interferon stimulated genes through cGAS sensing of cytosolic mitochondrial DNA. Upon pathogen sensing, macrophages regulate SRSF6 expression to control the liberation of immunogenic mtDNA and adjust the threshold for entry into programmed cell death. This work defines BAX alternative splicing by SRSF6 as a critical node not only in mitochondrial homeostasis but also in the macrophage's response to pathogens.

CSIG-05. A GABP DOMINANT NEGATIVE APPROACH TO THE REVERSAL OF TUMOR IMMORTALITY

Abstract Immortality is a fundamental hallmark of human cancer cells and therapeutic reversal is of great interest. Telomerase Reverse Transcriptase promoter (TERTp) mutations reactivate TERT expression, the rate limiting step in telomerase activity and cellular immortality. TERTp mutations are the most common non-coding mutation across all cancer types, including glioblastoma (GBM), oligodendroglioma, medulloblastoma, and high-grade meningioma. While prior telomerase therapies lack tumor selectivity and are poorly tolerated, TERTp mutations and their regulation offer a unique opportunity for tumor specific reversal of cellular immortality. The mutations generate a de novo E26 Transformation Specific (ETS) binding motif that recruits the GA-Binding Protein (GABP) multimeric complex to reactivate TERT expression. Initially we found a GABP tetramer reactivates the mutant TERTp, suggesting targeting only the tetramer may induce telomere shortening and tumor cell death. However, through knocking out this tetramer in cell cultures, we found that the GABP dimer is upregulated and maintains TERT expression. In the absence of both the dimer and one tetramer isoform, many tumor cells senesce or undergo cell death. However, some tumor cells escape this fate via upregulation of a second GABP tetrameric paralogue that maintains TERT expression. Therefore, to block all GABP complexes with a single approach, we devised a transactivation domain null, putative dominant negative GABP subunit that ultimately could be delivered to patients via a replicating retroviral system. Introduction of the construct into TERTp mutant glioma cells significantly reduced TERT expression while not altering expression in TERTp wildtype GBM cells, suggesting tumor selectivity. TERT expression was also reduced in a second TERTp mutant tumor type. We are currently investigating the long-term effects of the dominant negative GABP on telomere length and tumor immortality. Dominant negative GABP is a promising candidate for tumor selective reversal of cellular immortality by inhibiting mutant TERTp activation by all GABP multimeric forms.

Promoting regeneration while blocking cell death preserves motor neuron function in a model of ALS.

Amyotrophic lateral sclerosis (ALS) is a devastating and fatal neurodegenerative disease of motor neurons with very few treatment options. We had previously found that motor neuron degeneration in a mouse model of ALS can be delayed by deleting the axon damage sensor MAP3K12 or dual leucine zipper kinase (DLK). However, DLK is also involved in axon regeneration, prompting us to ask whether combining DLK deletion with a way to promote axon regeneration would result in greater motor neuron protection. To achieve this, we used a mouse line that constitutively expresses ATF3, a master regulator of regeneration in neurons. Although there is precedence for each individual strategy in the SOD1G93A mouse model of ALS, these have not previously been combined. By several lines of evidence including motor neuron electrophysiology, histology and behaviour, we observed a powerful synergy when combining DLK deletion with ATF3 expression. The combinatorial strategy resulted in significant protection of motor neurons with fewer undergoing cell death, reduced axon degeneration and preservation of motor function and connectivity to muscle. This study provides a demonstration of the power of combinatorial therapy to treat neurodegenerative disease.

A New Drug-Free Cancer Therapy Using Ultraviolet Pulsed Irradiation. PDT (PhotoDynamic Therapy) to PPT (Pulsed Photon Therapy)

Pulsed ultraviolet (UV) irradiation can be used to generate a broad UV-C spectrum. The pulsing nature of such a spectrum helps increase the damage to cancer cells, leading to their injury and death. In contrast, non-tumor cells repair the damage and survive the same pulsed UV irradiation energy. Herein, we describe the development of a pulsed UV irradiation method for cancer cell dysfunction that irradiates cells with pulsed light by generating tremendous instantaneous UV energy-tens of thousands of times greater than that generated by UV lamps-to cause specific cell injury and dysfunction of cancer cells. A newly developed pulsed ultraviolet irradiation device was used. Features of the device used in this study. This device employs a quartz discharge xenon lamp. Cultured tumor cells and non-tumor cells were irradiated with pulsed light at different irradiation doses, and their reactions were observed using optical, electron, and laser microscopes. Cancer cells have more FAS (CD95) receptor domains than non-cancer cells, and pulsed UV irradiation stimulates the production of reactive oxygen species (ROS) and OH, which exceeds the oxidative stress removal function, resulting in cell injury and death. That is, at low UV doses, only cancer cells underwent cell death, whereas non-cancer cells did not. The pulsed UV irradiation technique directly destroys cancer cells and minimizes the number of residual cancer cells while allowing minimum invasion into non-tumor cells, thereby improving their survival. This suggests the possibility of activating the host's local immune response to eliminate residual cancer cells. A newly developed pulsed UV radiation system shows potential for use in the development of a drug-free cancer treatment system that selectively kills tumor cells by irradiating them with high-intensity pulsed UV rays over a broad UV-C range of 230-280 nm.

TAK1 Limits Death Receptor Fas-Induced Proinflammatory Cell Death in Macrophages.

Fas, a member of the death receptor family, plays a central role in initiating cell death, a biological process crucial for immune homeostasis. However, the immunological and pathophysiological impacts to which enhanced Fas signaling gives rise remain to be fully understood. Here we demonstrate that TGF-β-activated kinase 1 (TAK1) works as a negative regulator of Fas signaling in macrophages. Upon Fas engagement with high concentrations of FasL, mouse primary macrophages underwent cell death, and, surprisingly, Fas stimulation led to proteolytic cleavage of gasdermin (GSDM) family members GSDMD and GSDME, a hallmark of pyroptosis, in a manner dependent on caspase enzymatic activity. Remarkably, TAK1-deficient macrophages were highly sensitive to even low concentrations of FasL. Mechanistically, TAK1 negatively modulated RIPK1 kinase activity to protect macrophages from excessive cell death. Intriguingly, mice deficient for TAK1 in macrophages (TAK1mKO mice) spontaneously developed tissue inflammation, and, more important, the emergence of inflammatory disease symptoms was markedly diminished in TAK1mKO mice harboring a catalytically inactive RIPK1. Taken together, these findings not only revealed an unappreciated role of TAK1 in Fas-induced macrophage death but provided insight into the possibility of perturbation of immune homeostasis driven by aberrant cell death.

Triiodothyronine-stimulated dendritic cell vaccination boosts antitumor immunity against murine colon cancer

Given the ability of dendritic cells (DCs) to modulate other immune players, their manipulation holds great potential for inducing efficient antitumor immunity. However, DC vaccine manufacturing deserve optimization since tumor cell cargo and DC functional state induced by maturation signals influence their in vivo immunogenic potential. We reported that triiodothyronine (T3) stimulates mice DCs’ maturation and their ability to promote pro-inflammatory and cytotoxic T-cell responses. This study aimed to evaluate the efficacy of T3-conditioned DC vaccination in a murine model of colon carcinoma, deciphering the molecular players involved; and to examine the effects of T3 on the maturation and activation of human DCs (huDCs). Bone marrow-derived DCs were exposed to T3 and MC38 cancer cells that underwent cell death (MC38-Apo/Nec) by UVB irradiation. Our results showed that MC38-Apo/Nec cells are efficiently uptaken by DCs and that T3 upregulates CD86 expression with increased production of the pro-inflammatory cytokines IL-12, IL-6 and TGF-β. In a colon cancer model, vaccination with T3-stimulated and tumor antigen-loaded DCs inhibited tumor growth in wild type mice, an effect that was eliminated in IL-17-deficient animals. Notably, secretion of high levels of IFN-γ and IL-17, induction of Th1, Th17 and tumor-specific cytotoxic T lymphocytes characterized the antitumor response upon vaccination. Moreover, our initial findings demonstrated a significant increase in CD86 expression and IL-12 production by huDCs induced by T3. Overall, these results reinforce the adjuvant properties of T3-conditioned DCs to potentiate T-cell-mediated antitumor immunity and support promising advances in the translation process to human oncotherapy.

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation.

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

Melanoma Stem Cells Educate Neutrophils to Support Cancer Progression.

Simple SummaryIn melanoma patients, poor prognosis often correlates with high presence of cancer-associated neutrophils, indicating that tumors can recruit these immune cells to specifically sustain their own development and progression. However, the role of cancer stem cells (CSCs) in this dialogue has not been elucidated yet. Our results revealed that melanoma SCs can reshape the immune microenvironment by triggering a pro-tumor N2 phenotype in neutrophils, which in turn are able to confer stemness properties to melanoma cells.Background: It is now well-established that cancer stem cells (CSCs) can support melanoma progression by reshaping the tumor immune microenvironment. However, the molecular mechanisms underlying the crosstalk between melanoma SCs and cancer-associated neutrophils have not been elucidated yet. Methods: The aim of the present study was to unravel the role of melanoma SCs in neutrophil polarization. HL60 neutrophil-like (dHL60) cells were treated with conditioned medium from A375 melanoma SCs (CSC-CM), and their phenotype was investigated. Results: We demonstrated that CSC-CM could specifically activate immune cells by increasing CD66b and CD11b expression. In particular, we revealed that A375 CSCs could release various soluble factors, namely TGF-β, IL-6, and IL-8, able to promote the recruitment of neutrophils and their switch toward an N2 phenotype characterized by the activation of ERK, STAT3, and P38 pathways and the overexpression of CXCR2 and NF-kB. Moreover, after exposure to CSC-CM, dHL60 cells exhibited enhanced ROS production and NET release, without undergoing cell death; increased secretion of MMP-9 and pro-inflammatory cytokines was also observed. Finally, CSC-CM-activated neutrophils endowed A375 cells with stemness traits, stimulating both sphere formation and ABCG2 expression. Conclusion: Collectively, our results suggest that melanoma SCs can prime neutrophils to support cancer progression.

Abstract 1832: CHK1 inhibitor prexasertib induces NOXA-dependent apoptosis in ovarian cancer

Abstract Background: The kinases ataxia-telangiectasia-mutated-and-Rad3-related (ATR), checkpoint kinase 1 (CHK1) and WEE1 participate in the response to replication stress. Inhibitors of ATR, WEE1 and CHK1 are being tested for antineoplastic activity as monotherapies and in combination with chemotherapy. Although these agents impair cell cycle checkpoints, fork stabilization, origin firing and homologous recombination (HR), little is known about the apoptotic pathways that are engaged and the critical mediators activating cell death mechanisms. The present study utilizes high grade serous ovarian cancer (HGSOC) cell lines and patient derived xenografts (PDXs) to identify the primary mechanism of cell death in ovarian cancer and assess changes involved in the resistance setting after treatment with these inhibitors. Methods: A panel of HGSOC cell lines was examined for responses to ceralasertib (ATRi), prexasertib (CHK1i) or adavosertib (WEE1i) using colony forming assays, immunoblotting and assays for apoptosis. Mechanisms involved in acquired resistance and downstream changes in cell fate were evaluated by generating prexasertib-resistant HGSOC cell lines. Subsequent NGS analysis was performed in the sensitive and resistant pairs. Results: Irrespective of the homologous recombination (HR) status, ovarian cell lines and PDXs underwent cell death during prolonged exposure at clinically achievable concentrations. Treatment of ovarian cancer cell lines with ATRi, CHK1i or WEE1i activated the replication checkpoint, consistent with previously published reports. The subsequent apoptotic response involved PMAIP1 and BCL2L11 upregulation rather than the TNFa-induced death receptor-mediated apoptosis we recently described in acute leukemia (Cancer Res. PMID: 33414171). Additionally, ovarian lines selected for CHK1i resistance failed to die in response to either ATRi or WEE1i. Conclusions: Prexasertib, ceralasertib, and adavosertib exhibit monotherapy activity in both HR deficient and proficient HGSOC cell lines and PDX models. Unlike AML, death receptor mediated apoptosis was not observed; instead, the primary mechanism of cell killing in HGSOC lines involves activation of the mitochondrial apoptotic pathway. Cross-resistance of these DNA damage repair modulators is likely multifactorial and mainly involves diminished replication stress response. Citation Format: Annapoorna Venkatachalam, Kevin L. Peterson, Cristina Correia, Karen S. Flatten, Xianon Hou, Paula A. Schneider, Emily Balczewski, Cordelia McGehee, Rachel M. Hurley, Xue W. Meng, Chance Sine, Rameen Shah, Nicole Vincelettte, Husheng Ding, Hu Li, Saravut (John) Weroha, Scott H. Kaufmann. CHK1 inhibitor prexasertib induces NOXA-dependent apoptosis in ovarian 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 1832.

Comparisons of cell proliferation and cell death from tornaria larva to juvenile worm in the hemichordate Schizocardium californicum

BackgroundThere are a wide range of developmental strategies in animal phyla, but most insights into adult body plan formation come from direct-developing species. For indirect-developing species, there are distinct larval and adult body plans that are linked together by metamorphosis. Some outstanding questions in the development of indirect-developing organisms include the extent to which larval tissue undergoes cell death during the process of metamorphosis and when and where the tissue that will give rise to the adult originates. How do the processes of cell division and cell death redesign the body plans of indirect developers? In this study, we present patterns of cell proliferation and cell death during larval body plan development, metamorphosis, and adult body plan formation, in the hemichordate Schizocardium californium (Cameron and Perez in Zootaxa 3569:79–88, 2012) to answer these questions.ResultsWe identified distinct patterns of cell proliferation between larval and adult body plan formation of S. californicum. We found that some adult tissues proliferate during the late larval phase prior to the start of overt metamorphosis. In addition, using an irradiation and transcriptomic approach, we describe a genetic signature of proliferative cells that is shared across the life history states, as well as markers that are unique to larval or juvenile states. Finally, we observed that cell death is minimal in larval stages but begins with the onset of metamorphosis.ConclusionsCell proliferation during the development of S. californicum has distinct patterns in the formation of larval and adult body plans. However, cell death is very limited in larvae and begins during the onset of metamorphosis and into early juvenile development in specific domains. The populations of cells that proliferated and gave rise to the larvae and juveniles have a genetic signature that suggested a heterogeneous pool of proliferative progenitors, rather than a set-aside population of pluripotent cells. Taken together, we propose that the gradual morphological transformation of S. californicum is mirrored at the cellular level and may be more representative of the development strategies that characterize metamorphosis in many metazoan animals.