Acute Cell Death Research Articles

Retinal prolactin isoform PRLΔE1 sustains rod disease in inherited retinal degenerations

PRLΔE1, a retina-specific isoform of prolactin, is expressed in multiple and diverse forms of canine inherited retinal degeneration (IRD). We find that while PRLΔE1 expression in rods is not associated with the initial phase of disease characterized by acute photoreceptor cell death, it is associated with the protracted phase of slow cell loss. Restoration of photoreceptors to a healthy state by gene-specific replacement therapy of individual IRDs successfully suppresses PRLΔE1 expression. Moreover, short-term PRLΔE1 silencing using shRNA results in preservation of outer nuclear layer thickness, suggesting PRLΔE1 drives retinal disease. However, longer-term observations reveal off-target toxic effects of the PRLΔE1 shRNA, precluding determination of its full therapeutic potential. Future research efforts aimed at enhancing the safety and specificity of PRLΔE1-targeting strategies may identify a potential universal intervention strategy for sustaining photoreceptors during the prolonged phase of multiple IRDs.

Cytotoxicity by endocrine disruptors through effects on ER Ca2+ transporters, aberrations in Ca2+ signalling pathways and ER stress.

Concerns regarding man-made organic chemicals pervading our ecosystem and having adverse and detrimental effects upon organisms, including man, have now been studied for several decades. Since the 1970s, some environmental pollutants were identified as having endocrine disrupting affects. These endocrine disrupting chemicals (EDC) were initially shown to have estrogenic or anti-estrogenic properties and some were also shown to bind to a variety of hormone receptors. However, since the 1990s it has also been identified that many of these EDC additionally, have the ability of causing abnormal alterations in Ca2+ signalling pathways (also commonly involved in hormone signalling), leading to exaggerated elevations in cytosolic [Ca2+] levels, that is known to cause activation of a number of cell death pathways. The major emphasis of this review is to present a personal perspective of the evidence for some types of EDC, specifically alkylphenols and brominated flame retardants (BFRs), causing direct effects on Ca2+ transporters (mainly the SERCA Ca2+ ATPases), culminating in acute cytotoxicity and cell death. Evidence is also presented to indicate that this Ca2+ATPase inhibition, which leads to abnormally elevated cytosolic [Ca2+], as well as a decreased luminal ER [Ca2+], which triggers the ER stress response, are both involved in acute cytotoxicity.

Hemin-Induced Transient Senescence Via DNA Damage Response: A Neuroprotective Mechanism Against Ferroptosis in Intracerebral Hemorrhage.

Intracerebral hemorrhage (ICH) poses acute fatality and long-term neurological risks due to hemin and iron accumulation from hemoglobin breakdown. Our observation that hemin induces DNA double-strand breaks (DSBs), prompting a senescence-like phenotype in neurons, necessitating deeper exploration of cellular responses. Using experimental ICH models and human ICH patient tissue, we elucidate hemin-mediated DNA damage response (DDR) inducing transient senescence and delayed expression of heme oxygenase (HO-1). HO-1 co-localizes with senescence-associated β-Galactosidase (SA-β-Gal) in ICH patient tissues, emphasizing clinical relevance of inducible HO-1 expression in senescent cells. We reveal a reversible senescence state protective against acute cell death by hemin, while repeat exposure leads to long-lasting senescence. Inhibiting early senescence expression increases cell death, supporting the protective role of senescence against hemin toxicity. Hemin-induced senescence is attenuated by a pleiotropic carbon nanoparticle that is a catalytic mimic of superoxide dismutase, but this treatment increased lipid peroxidation, consistent with ferroptosis from hemin breakdown released iron. When coupled with iron chelator deferoxamine (DEF), the nanoparticle reduces hemin-induced senescence and upregulates factors protecting against ferroptosis. Our study suggests transient senescence induced by DDR as an early potential neuroprotective mechanism in ICH, but the risk or iron-related toxicity supports a multi-pronged therapeutic approach.

Short-term neural and glial response to mild traumatic brain injury in the hippocampus

Traumatic brain injury (TBI) is an established risk factor for developing neurodegenerative disease. However, how TBI leads from acute injury to chronic neurodegeneration is limited to postmortem models. There is a lack of connections between in vitro and in vivo TBI models that can relate injury forces to both macroscale tissue damage and brain function at the cellular level. Needle-induced cavitation (NIC) is a technique that can produce small cavitation bubbles in soft tissues, which allows us to relate small strains and strain rates in living tissue to ensuing acute cell death, tissue damage, and tissue remodeling. Here, we applied NIC to mouse brain slices to create a new model of TBI with high spatial and temporal resolution. We specifically targeted the hippocampus, which is a brain region critical for learning and memory and an area in which injury causes cognitive pathologies in humans and rodent models. By combining NIC with patch-clamp electrophysiology, we demonstrate that NIC in the cornu ammonis 3 region of the hippocampus dynamically alters synaptic release onto cornu ammonis 1 pyramidal neurons in a cannabinoid 1 receptor-dependent manner. Further, we show that NIC induces an increase in extracellular matrix protein GFAP associated with neural repair that is mitigated by cannabinoid 1 receptor antagonism. Together, these data lay the groundwork for advanced approaches in understanding how TBI impacts neural function at the cellular level and the development of treatments that promote neural repair in response to brain injury.

Disruption of polyunsaturated fatty acid biosynthesis drives STING-dependent acute myeloid leukemia cell maturation and death

The role of polyunsaturated fatty acid (PUFA) biosynthesis in acute myeloid leukemia (AML) remains largely undefined. A comparative expression analysis of 35 genes encoding fatty acid biosynthesis enzymes showed that fatty acid desaturase 1 (FADS1) was highly expressed across multiple AML subtypes relative to healthy controls and that elevated FADS1 expression correlates with worse overall AML patient survival. Functionally, shRNA-mediated inhibition of FADS1 reduced AML cell growth invitro and significantly delayed leukemia onset in an AML mouse model. AML cell lines depleted of FADS1 arrested in the G1/S-phase of the cell cycle, acquired characteristics of myeloid maturation and subsequently died. To understand the molecular consequences of FADS1 inhibition, a combination of mass spectrometry-based analysis of complex lipids and gene expression analysis (RNA-seq) was performed. FADS1 inhibition caused AML cells to exhibit significant lipidomic remodeling, including depletion of PUFAs from the phospholipids, phosphatidylserine, and phosphatidylethanolamine. These lipidomic alterations were accompanied by an increase induction of inflammatory and stimulator of interferon genes (STING)-mediated type-1 interferon signaling. Remarkably, genetic deletion of STING largely prevented the AML cell maturation and death phenotypes mediated by FADS1 inhibition. Highlighting the therapeutic implications of these findings, pharmacological blockade of PUFA biosynthesis reduced patient-derived AML cell numbers exvivo but not that of healthy donor cells. Similarly, STING agonism attenuated patient-derived-AML survival; however, STING activation also reduced healthy granulocyte numbers. Collectively, these data unveil a previously unrecognized importance of PUFA biosynthesis in leukemogenesis and that imbalances in PUFA metabolism can drive STING-mediated AML maturation and death.

Pulsed Photothermal Therapy of Solid Tumors as a Precondition for Immunotherapy.

Photothermal therapy (PTT) refers to the use of plasmonic nanoparticles to convert electromagnetic radiation in the near infrared region to heat and kill tumor cells. Continuous wave lasers have been used clinically to induce PTT, but the treatment is associated with heat-induced tissue damage that limits usability. Here, the engineering and validation of a novel long-pulsed laser device able to induce selective and localized mild hyperthermia in tumors while reducing the heat affected zone and unwanted damage to surrounding tissue are reported. Long-pulsed PTT induces acute necrotic cell death in heat affected areas and the release of tumor associated antigens. This antigen release triggers maturation and stimulation of CD80/CD86 in dendritic cells in vivo that primes a cytotoxic T cell response. Accordingly, long-pulsed PTT enhances the therapeutic effects of immune checkpoint inhibition and increases survival of mice with bladder cancer. Combined, the data promote long-pulsed PTT as a safe and effective strategy for enhancing therapeutic responses to immune checkpoint inhibitors while minimizing unwanted tissue damage.

Inhibition of NRF2 enhances the acute myeloid leukemia cell death induced by venetoclax via the ferroptosis pathway

Venetoclax, an inhibitor that selectively targets B cell lymphoma-2 (BCL-2) that has been approved for treating adult acute myeloid leukemia (AML) in combination with hypomethylating agents. However, its short duration of response and emergence of resistance are significant issues. In this study, we found that the sensitivity of AML cells to venetoclax was considerably enhanced by ML385, an inhibitor of the ferroptosis factor nuclear transcription factor erythroid 2-related factor 2 (NRF2). Using AML samples, we verified that NRF2 and its target gene ferritin heavy chain 1 (FTH1) were highly expressed in patients with AML and correlated with poor prognosis. Downregulation of NRF2 could inhibit FTH1 expression and significantly enhance the venetoclax-induced labile iron pool and lipid peroxidation. By contrast, NRF2 overexpression or administration of the reactive oxygen species inhibitor N-acetylcysteine and vitamin E could effectively suppress the anti-AML effects of ML385+venetoclax. Furthermore, the ferroptosis inducer erastin increased the anti-AML effects of venetoclax. Our study demonstrated that NRF2 inhibition could enhance the AML cell death induced by venetoclax via the ferroptosis pathway. Thus, the combination of ML385 with venetoclax may offer a favorable strategy for AML treatment.

Unique Pathologic Findings in the Heart of a Red-Eared Turtle (Trachemys Scripta Elegans)

The necropsy of a turtle (Trachemys scripta elegans), which was euthanized after rapid loss of vitality, revealed multiple unique abnormalities in the heart. We found areas of atrophy in the wall of the ventricle. These may be connected with aneurysmata at the same locations. A prominent hypertrophic, disc-like cartilago cordis was present. It may have disturbed the flow of blood leading to local atrophies. Gradually, the excessive size may have impeded the flow of blood by compression of large blood vessels at the heart base resulting in ischemia and acute cell death in the myocardium on one side of the ventricle. In one of the larger blood vessels there was an ulcus covered with a thrombus. Thrombi were found on various places and filled the larger part of the ventricular chamber. It can be assumed that these abnormalities were linked and fatal in the end.

Repurposing GSK3B Small Molecule Inhibitors for Refractory Lymphoid Malignancies

Background: Small molecule GSK3B inhibitors were developed for solid tumors and neurological conditions, including Alzheimer's and Parkinson disease. Five GSK3B inhibitors achieved favorable safety and PK/PD profiles in a total of 22 Phase 1 and 2 clinical trials but no clinical response for any of these indications. GSK3B is the kinase that phosphorylates β-catenin to initiate its degradation, hence GSK3B small molecule inhibition results in rapid nuclear accumulation of β-catenin protein. Significance: Activating mutations in the Wnt/β-catenin pathway are common oncogenic drivers throughout all main types of cancer (n=64,812) but not found in B- and T-lymphoid malignancies (n=2,137). Studying tissue microarrays by β-catenin immunohistochemistry, lung, colon, breast, and melanoma specimen (n=57) showed consistently high levels of nuclear β-catenin, whereas B- and T-cell lymphomas (n=84) lacked β-catenin signal. Despite comparable mRNA levels, proteomic analyses revealed 60-400-fold lower β-catenin levels in lymphoid compared to epithelial tumors (n=1,389) suggesting that lymphoid malignancies rely on powerful mechanisms for β-catenin degradation, e.g. by GSK3B. Results: To study the effects of β-catenin activation in human cells, we induced expression of stabilized form of β-catenin in 18 lymphoid (B- and T-ALL, MCL, DLBCL, PTCL) and four myeloid leukemia cell lines. In B- and T-lymphoid cells, β-catenin accumulation compromised clonal fitness, colony formation and induced cell death, but increased competitive fitness and colony formation in myeloid leukemia cells. CEBPα-mediated reprogramming of B-ALL cells into a myeloid phenotype reversed the deleterious effects highlighting that effects of β-catenin-accumulation strictly depend on lymphoid lineage-identity. RNA-seq studies upon inducible activation of β-catenin in lymphoid cells, revealed transcriptional repression of Myc and Myc target genes as principal gene expression change. Unlike activating β-catenin:TCF7 complexes in epithelial cells, our global interactome studies in B- and T-ALL and lymphoma cells identified repressive β-catenin complexes with lymphoid-specific Ikaros zinc finger (IKZF1/2/3) proteins and several components of the NuRD complex including Mta1/2, Gatad2a/b, Chd4, HDAC1/2. Interactome and ChIP-seq analyses revealed that β-catenin together with lymphoid-specific Ikaros factors recruited repressive NuRD complex members to deacetylate H3K27 and repress MYC superenhancer (SE) regions. Genetic deletion of both IKZF1 and IKZF3 or HDRT-based mutation of Ikaros binding motifs within the Myc-SE enabled resistance to β-catenin accumulation. To leverage this previously unrecognized vulnerability of lymphoid malignancies, we examined five clinically approved GSK3B small molecule inhibitors that achieved favorable safety profiles at micromolar plasma concentrations in clinical trials for neurological disorders and solid tumors. Strikingly, LY2090314 and CHIR99021 were effective at low nanomolar concentrations in B- and T-cell malignancies that expressed lymphoid Ikaros factors. At IC50 concentrations of <5 nM, GSK3B-inhibitors induced massive accumulation of β-catenin, repression of MYC and acute cell death. In B-ALL cells with IKZF1-deletion, expression of the remaining IKZF3 factor was sufficient to retain full sensitivity to GSK3B-inhibition. Deletion of β-catenin, however, abolished the effects of GSK3B inhibitors and relieved suppression of MYC, demonstrating accumulation of β-catenin represents as central mechanism of action. Preclinical in vivo treatment experiments of refractory B-ALL and mantle cell lymphoma PDX validated small molecule GSK3B-inhibition as strategy to overcome conventional mechanisms of drug-resistance. Conclusions: B- and T-lymphoid malignancies not only lacked expression of β-catenin but critically depend on GSK3B for effective β-catenin degradation. Our interactome studies in lymphoid tumors revealed that β-catenin formed repressive complexes with lymphoid-specific Ikaros and NuRD complex factors. Strikingly, GSK3B small molecule inhibitors engage this new pathway at low nanomolar concentrations and demonstrated favorable safety profiles in Phase 1 and Phase 2 trials. Safety and efficacy of GSK3B-inhibitors opens up an immediate path for repurposing these agents towards refractory B-cell malignancies.

Tweak/Fn14 system is involved in rhabdomyolysis-induced acute kidney injury

BackgroundRhabdomyolysis is a severe clinical syndrome associated to acute kidney injury (AKI) and chronic kidney disease (CKD). TWEAK/Fn14 signaling axis regulates renal inflammation and tubular cell death. However, the functional role of TWEAK/Fn14 in rhabdomyolysis remains unknown. MethodsRhabdomyolysis was induced in wild-type, TWEAK- and Fn14-deficient mice or mice treated with TWEAK blocking antibody. Renal injury, inflammation, fibrosis and cell death were assessed. Additionally, we performed in vivo and in vitro studies to explore the possible signalling pathways involved in Fn14 regulation. FindingsFn14 renal expression was increased in mice with rhabdomyolysis, correlating with decline of renal function. Mechanistically, myoglobin (Mb) induced Fn14 expression via ERK and p38 pathway, whereas Nrf2 activation diminished Mb-mediated Fn14 upregulation in cultured renal cells. TWEAK or Fn14 genetic depletion ameliorated rhabdomyolysis-associated loss of renal function, histological damage, tubular cell death, inflammation, and expression of both tubular and endothelial injury markers. Deficiency of TWEAK or Fn14 also decreased long-term renal inflammation and fibrosis in mice with rhabdomyolysis. Finally, pharmacological treatment with a blocking TWEAK antibody diminished the expression of acute renal injury markers and cell death and lessened residual kidney fibrosis and chronic inflammation in rhabdomyolysis. InterpretationTWEAK/Fn14 axis participates in the pathogenesis of rhabdomyolysis-AKI and subsequent AKI-CKD transition. Blockade of this signaling pathway may represent a promising therapeutic strategy for reducing rhabdomyolysis-mediated renal injury. FundingSpanish Ministry of Science and Innovation, ISCIII and Junta de Andalucía.

Necroptosis in Organ Transplantation: Mechanisms and Potential Therapeutic Targets.

Organ transplantation remains the only treatment option for patients with end-stage organ dysfunction. However, there are numerous limitations that challenge its clinical application, including the shortage of organ donations, the quality of donated organs, injury during organ preservation and reperfusion, primary and chronic graft dysfunction, acute and chronic rejection, infection, and carcinogenesis in post-transplantation patients. Acute and chronic inflammation and cell death are two major underlying mechanisms for graft injury. Necroptosis is a type of programmed cell death involved in many diseases and has been studied in the setting of all major solid organ transplants, including the kidney, heart, liver, and lung. It is determined by the underlying donor organ conditions (e.g., age, alcohol consumption, fatty liver, hemorrhage shock, donation after circulatory death, etc.), preservation conditions and reperfusion, and allograft rejection. The specific molecular mechanisms of necroptosis have been uncovered in the organ transplantation setting, and potential targeting drugs have been identified. We hope this review article will promote more clinical research to determine the role of necroptosis and other types of programmed cell death in solid organ transplantation to alleviate the clinical burden of ischemia-reperfusion injury and graft rejection.

Targeted hyperactivation of AKT through inhibition of ectopic expressed SHIP1 induces cell death in colon carcinoma cells and derived metastases

Current therapeutic approaches for colorectal cancer (CRC) focus on the suppression of oncogenic kinase signaling. Here, we test the hypothesis that targeted hyperactivation of the PI3K/AKT-signaling may lead to trigger CRC cell death. Recently we found that hematopoietic SHIP1 is ectopically expressed in CRC cells. Here we show that SHIP1 is more strongly expressed in metastatic cells than in the primary cancer cells, which allows for an increase in AKT signaling in metastatic cells, giving them an advantage from an evolutionary point of view. Mechanistically, the increased SHIP1 expression reduces the activation of the PI3K/ AKT signaling to a value that is below the threshold that leads to cell death. This mechanism gives the cell a selection advantage. We show that genetic hyperactivation of PI3K/AKT-signaling or blocking the activity of the inhibitory phosphatase SHIP1, induces acute cell death in CRC cells, because of excessive accumulation of reactive oxygen species. Our results demonstrate that CRC cells critically depend on mechanisms to fine-tune PI3K/AKT activity and show SHIP1 inhibition as an unexpectedly promising concept for CRC therapy.

Abstract LB035: The role of nucleophosmin1 mediated caspase2 activation in acute myeloid leukemia cell death and cell survival

Abstract Nucleophosmin 1 (NPM1) mutation is the primary genetic lesion in adult acute myeloid leukemia (AML). Typically, a nucleolar protein, NPM1 mutations cause cytoplasmic delocalization of NPM1(NPM1c+), triggering leukemogenesis. Interestingly, NPM1c+ mutation confers enhanced chemosensitivity. However, the mechanism underlying NPM1c+ mutation-induced oncogenesis or chemosensitivity remains unclear. We previously reported that, in response to genotoxic stimuli, NPM1 forms a platform for caspase-2 activation in the nucleolus. Caspase-2 is a pro-apoptotic protein and a known tumor suppressor. Our results indicate that nucleolar caspase-2 activation is specific to NPM1wt cells. In the NPM1c+ cells, caspase-2 activation is exclusively cytosolic, suggesting that caspase-2 is activated in the same sub-cellular compartment as NPM1 localization. Loss of caspase-2 rescued the increased sensitivity of NPM1c+ cells to apoptotic cell death. This is accompanied by reduced cleavage of the apoptotic substrate, caspase-3. Notably, caspase-2 deficient NPM1wt cells showed no significant difference in the percentage of apoptotic cell death or endogenous substrate cleavage compared to its caspase-2 wild-type counterpart. This suggests that caspase-2 may contribute to the chemosensitivity of NPM1c+ cells. Strikingly, in unstimulated cells, loss of caspase-2 leads to prolonged G1 arrest and progressive loss of cell viability. Immunophenotyping of NPM1c+ cells revealed a remarkable increase in the CD14+population representing monocyte/macrophage-like features, suggesting that loss of caspase-2 results in terminal differentiation. Exogenous expression of NPM1c+ in caspase-2 deficient NPM1wt cells impaired viability, confirming that NPM1c+ cells require caspase-2 for its growth and proliferation. Transcriptomic analysis revealed that loss of caspase-2 significantly downregulates pathways involved in stem cell pluripotency. In particular, FGF2, IGFR, WNT, LIF, and one of the core members of the self-renewal transcription factor, OCT 4, were downregulated in the absence of caspase-2 in NPM1c+ cells. This reveals a novel role for caspase-2 in maintaining NPM1c+ AML stemness. Taken together, our study shows that NPM1c+ mediated caspase-2 activation regulates AML cell death and survival cascades, a key determinant of chemosensitivity and leukemogenesis. Citation Format: Dharaniya Sakthivel, Alexandra Brown-Suedel, Francesca Keane, F. Kenneth McSherry, Chloe I. Charendoff, Kevin Dunne, Dexter Robichaux, Jonathan M. Flanagan, Lisa Bouchier-Hayes. The role of nucleophosmin1 mediated caspase2 activation in acute myeloid leukemia cell death and cell survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 2 (Clinical Trials and Late-Breaking Research); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(8_Suppl):Abstract nr LB035.

Defective Mitochondrial Dynamics and Protein Degradation Pathways Underlie Cadmium-Induced Neurotoxicity and Cell Death in Huntington's Disease Striatal Cells.

Exposure to heavy metals, including cadmium (Cd), can induce neurotoxicity and cell death. Cd is abundant in the environment and accumulates in the striatum, the primary brain region selectively affected by Huntington's disease (HD). We have previously reported that mutant huntingtin protein (mHTT) combined with chronic Cd exposure induces oxidative stress and promotes metal dyshomeostasis, resulting in cell death in a striatal cell model of HD. To understand the effect of acute Cd exposure on mitochondrial health and protein degradation pathways, we hypothesized that expression of mHTT coupled with acute Cd exposure would cooperatively alter mitochondrial bioenergetics and protein degradation mechanisms in striatal STHdh cells to reveal novel pathways that augment Cd cytotoxicity and HD pathogenicity. We report that mHTT cells are significantly more susceptible to acute Cd-induced cell death as early as 6 h after 40 µM CdCl2 exposure compared with wild-type (WT). Confocal microscopy, biochemical assays, and immunoblotting analysis revealed that mHTT and acute Cd exposure synergistically impair mitochondrial bioenergetics by reducing mitochondrial potential and cellular ATP levels and down-regulating the essential pro-fusion proteins MFN1 and MFN2. These pathogenic effects triggered cell death. Furthermore, Cd exposure increases the expression of autophagic markers, such as p62, LC3, and ATG5, and reduces the activity of the ubiquitin-proteasome system to promote neurodegeneration in HD striatal cells. Overall, these results reveal a novel mechanism to further establish Cd as a pathogenic neuromodulator in striatal HD cells via Cd-triggered neurotoxicity and cell death mediated by an impairment in mitochondrial bioenergetics and autophagy with subsequent alteration in protein degradation pathways.

Abstract 1384: Stearoyl-CoA desaturase confers cell density-dependent resistance to ferroptosis induced by inhibition of glutathione peroxidase 4 in melanoma cells

Abstract Ferroptosis, one of the programmed cell deaths, is induced by accumulation of lipid peroxidation. To protect from ferroptosis, cells possess a wide range of reduction systems including glutathione peroxidase 4 (GPX4), which reduces lipid peroxides to non-toxic lipid alcohols. Indeed, inhibiting GPX4 leads to ferroptosis in various cancer cells, but cellular sensitivity to GPX4 inhibition often varies depending on cell conditions. Some previous studies reported that an increase in cell density induces resistance to GPX4 inhibition, but the mechanisms of cell density-dependent resistance are not fully understood. In this study, we have also found that several melanoma cell lines become resistant to ferroptosis induced by GPX4 inhibition in a density-dependent manner. For example, a selective GPX4 inhibitor, RSL3, immediately induced cell death at low cell density of A375 within 6-8 hours, but did not at high density even after 24 hours. The acute cell death at low density was rescued by ferroptosis inhibitors (lipid peroxidation inhibitors or iron chelator), but not those of apoptosis and necrosis, supporting that this type of cell death is ferroptosis. To elucidate the mechanisms of ferroptosis resistance at high density, we first conducted transcriptome and metabolome analyses using the difference between low and high densities of A375. The gene ontology analysis on upregulated genes at high density indicated an increase in fatty acid metabolism. In contrast, nutritional stress was not implied from the transcriptional analysis, resulting from frequent changes of culture medium to avoid nutrition starvation. The enrichment analysis based on upregulated metabolites at high density similarly suggested increased de novo lipogenesis. Particularly, gene and protein levels of stearoyl-CoA desaturase (SCD) were induced in a density-dependent manner. Similar induction of SCD was observed in several melanoma cell lines. We further examined the role of SCD in density-dependent ferroptosis resistance. With this aim, pharmacological inhibition or genetic knockout of SCD was conducted on A375 at different densities (low and high) treated with RSL3. The result showed that SCD inhibition reduced RSL3 resistance at high density while having no effect on RSL3 sensitivity at low density. In summary, we found that SCD is upregulated in melanoma cells at high density and protects cells from ferroptosis induced by GPX4 inhibition. It is known that metastasizing melanomas in the blood are sensitive to ferroptosis, resembling cells at low density. This report might provide an explanation for the ferroptosis sensitivity of metastasizing melanomas and a strategy for ferroptosis-based therapy. Citation Format: Hitomi Shirahama, Yuri Tani, Satomi Tsukahara, Yuka Okamoto, Akiko Hasebe, Shingo Dan, Akihiro Tomida. Stearoyl-CoA desaturase confers cell density-dependent resistance to ferroptosis induced by inhibition of glutathione peroxidase 4 in melanoma cells [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 1384.

Abstract 2537: Targeting acute myeloid leukemia cell death via small molecule AP-3-84 binding to the LxCxE pocket of Rb1

Abstract Background: Acute myeloid leukemia (AML) is the most common form of leukemia in adults. In 2017, 120,000 new AML cases of were reported worldwide, with an expectation to reach double the number by 2040. Presently, with an 8.5 months of median survival rate and approximately 60% recurrence rate following chemotherapy treatment, leads to the opportunity to improve overall AML cancer therapy. The Retinoblastoma (Rb1) protein can control canonical cell cycle progression and be a determinant to apoptosis depending on its phosphorylation state, binding partners, and localization (specifically mitochondrial Rb1). We have developed a new small molecule, AP-3-84, which binds the LxCxE domain of Rb1 and can induce cell death in multiple acute myeloid leukemia cell lines by in vitro studies. Methods: AML cell lines (THP-1, AML-193, Kasumi-1) were cultured at different cell concentration (75000/ml, 125,000/ml and 200,000/ml) and exposed to 5, 10, 15, 20 uM concentration of AP-3-84. Cell differentiation was induced by 100 ng/ml PMA over 24 hours. Nonhemopoietic cancer cells (ID8, PDAC) were analyzed after exposure to AP-3-84. Cell death outcomes were measured by Annexin-V using flowcytometry or viability measures by Alamar blue assay. Protein levels were measured by Western blots at intervals between 30 min and 6 hours after exposure to AP-3-84 for total Rb1, phosphorylated Rb1, ATF3, BIM, NOXA, Bak, Bax, cleaved PARP, cleaved Caspase-3. Cytoplasmic and mitochondrial lysates were examined. Confocal immunocytochemistry on Rb1 expression in nucleus versus cytoplasm was performed. Results: Rb1 protein expression was documented by immunocytochemistry in nucleus and cytoplasm of THP1 cells consistent with its detection in nuclear and mitochondrial lysates (Western). AP-3-84 induction of cell death was observed at 10uM or higher concentration, with a 5 minute exposure sufficient to cause cell death in 70-80% of cells 24 hours later. Cell death by AP-3-84 was specific to dividing AML cells as no cell death was induced in PMA differentiated THP1 cells or nonhemopoietic cancer cells. No effect in cell division rate by AP-3-84 was present in nonhemopoitic cancer cells indicating a lack of cell cycle regulation. Mechanistically, AP-3-84 induced higher levels of ER-stress signals (higher ATF4, ATF3) and increased mitochondrial protein levels of NOXA, BIM, cleaved-PARP, and cleaved Caspase-3. Bax and Bak levels were unchanged after AP-3-84 exposure. Conclusion: We identify a new small molecule, AP-3-84, with therapeutic potential in AML by its binding to the LxCxE pocket of Rb1 triggering mitochondria-mediated cell apoptosis. Citation Format: Avishek Bhuniya, Lily Lu, Adi Narayana Reddy Poli, Evgenii N. Tcyganov, Colin Hart, Brijesh Karanam, Paridhima Sharma, Taylor Hill, Joseph Salvino, Luis J. Montaner. Targeting acute myeloid leukemia cell death via small molecule AP-3-84 binding to the LxCxE pocket of Rb1 [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 2537.

MP27-02 MICROARRAY ANALYSIS OF CORPORA CAVERNOSAL TISSUE FROM PEYRONIE'S AND PROSTATECTOMY PATIENTS WITH ERECTILE DYSFUNCTION IN COMPARISON TO A RAT CAVERNOUS NERVE RESECTION MODEL

You have accessJournal of UrologyCME1 Apr 2023MP27-02 MICROARRAY ANALYSIS OF CORPORA CAVERNOSAL TISSUE FROM PEYRONIE'S AND PROSTATECTOMY PATIENTS WITH ERECTILE DYSFUNCTION IN COMPARISON TO A RAT CAVERNOUS NERVE RESECTION MODEL Timothy Searl, Samuel Ohlander, Kevin McVary, and Carol Podlasek Timothy SearlTimothy Searl More articles by this author , Samuel OhlanderSamuel Ohlander More articles by this author , Kevin McVaryKevin McVary More articles by this author , and Carol PodlasekCarol Podlasek More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003255.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Prostatectomy patients are at high risk of erectile dysfunction (ED) that is refractory to PDE5 inhibitors. Cavernous nerve (CN) damage causes loss of innervation, smooth muscle apoptosis, and fibrosis, resulting in ED. We performed microarray and pathway analysis to identify mechanisms that are altered with ED, to find new targets for ED treatment. METHODS: Corpora cavernosal (CC) tissue was obtained from Peyronie’s (control, n=3) and prostatectomy (control, n=3) patients undergoing prosthesis implant to treat ED. For our rat CN injury model, Sprague Dawley rats underwent bilateral CN resection (n=5) or sham surgery (controls, n=5) and C.C. tissue was harvested 2 days post surgery. RNA was extracted from CC tissue using TRIzol, DNase treated, and purified with Qiagen mini kit. Microarray analysis (HG 2.0 ST, RU34), and pathway analysis was performed (ShinyGO, WebGestalt. Ingenuity). A 2-fold change was used as threshold for differentially expressed genes (DEGs). FDR<0.05 was considered significant for enrichment terms. RESULTS: There were 197 DEGs in CC from prostatectomy patients. 112 of the 197 DEGs were non-protein-coding genes. The overall changed expression of these non-protein-coding genes expected to favor a reduction in cell growth compared with Peyronie’s control. In the rat, microarray detected 182 protein coding DEGs. Pathway Analysis identified changes in cell death, stress and cellular development. Examination of the role of individual genes found changes in proliferation and cell death. CONCLUSIONS: Prostatectomy patients display alterations in gene expression in their corpora cavernosa that are consistent with decreased growth, likely impacting tissue repair and regenerative processes. Non-protein-coding RNAs may play a crucial role in ED in prostatectomy patients. In the acute rat CN resection model, cell death and proliferation are followed by apoptosis domination. Prostatectomy patient results show penile remodeling continues in patients long after the acute surgical injury to the CN takes place, offering the opportunity for clinical intervention to reverse penile remodeling and improve erectile function. Source of Funding: NIH/NIDDK DK101536 © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e362 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Timothy Searl More articles by this author Samuel Ohlander More articles by this author Kevin McVary More articles by this author Carol Podlasek More articles by this author Expand All Advertisement PDF downloadLoading ...

Altered trafficking of miRNAs at mitochondria modulates mitochondrial functions and cell death in brain ischemia.

Stroke is one of the major causes of death and disabilities worldwide. The rapid induction of cell death by necrosis and apoptosis is observed at the ischemic core, while long lasting apoptosis and brain inflammation continue in the penumbra. The emerging evidence suggests a critical role of mitochondria in acute and chronic inflammation and cell death. Mitochondrial dysfunction may result in the release of mitokines and/or mitochondrial DNA into the cytoplasm and activate multiple cytosolic pathways which in turn triggers inflammation. The role of miRNA, specifically mitochondria-associated miRNAs (mitomiRs) in the regulation of mitochondrial functions is emerging. In the current study, we hypothesized that ischemia-induced mitomiRs may modulate the mitochondrial functions and such alterations under stress conditions may lead to mitochondrial dysfunction and cell death. We have demonstrated the specific pattern of miRNAs associated with mitochondria that is altered under ischemic condition induced by transient middle artery occlusion (tMCAo) in rats. The putative targets of altered miRNAs include several mitochondrial proteins which signifies their involvement in maintaining mitochondrial homeostasis. The alterations of selected miRNA in mitochondria was further detected in a cellular models when hypoxia is induced using a chemical agent CoCl2 in three cell lines. Two candidate mitomiRs, hsa-miR-149-3p and hsa-miR-204-5p were further analyzed for their functional role during in vitro hypoxia by transfecting mitomiR mimics into cells and determining critical mitochondrial functions and cell viability. The results here emphasize the role of certain mitomiRs as an important modulator of mitochondrial function under the ischemic condition.

Immune response following traumatic spinal cord injury: Pathophysiology and therapies.

Traumatic spinal cord injury (SCI) is a devastating condition that is often associated with significant loss of function and/or permanent disability. The pathophysiology of SCI is complex and occurs in two phases. First, the mechanical damage from the trauma causes immediate acute cell dysfunction and cell death. Then, secondary mechanisms of injury further propagate the cell dysfunction and cell death over the course of days, weeks, or even months. Among the secondary injury mechanisms, inflammation has been shown to be a key determinant of the secondary injury severity and significantly worsens cell death and functional outcomes. Thus, in addition to surgical management of SCI, selectively targeting the immune response following SCI could substantially decrease the progression of secondary injury and improve patient outcomes. In order to develop such therapies, a detailed molecular understanding of the timing of the immune response following SCI is necessary. Recently, several studies have mapped the cytokine/chemokine and cell proliferation patterns following SCI. In this review, we examine the immune response underlying the pathophysiology of SCI and assess both current and future therapies including pharmaceutical therapies, stem cell therapy, and the exciting potential of extracellular vesicle therapy.

The uptake pattern of 18F-sodium fluoride radioligand in brain tissue after cerebral infarction

Positron emission tomography with 18F-sodium fluoride (NaF) radioligand has been actively investigated in atherosclerosis research because it is known to detect microcalcification activity within atheroma. We studied whether NaF shows any uptake in the brain tissue of patients with acute ischemic stroke. This is a post-hoc analysis of previously reported cerebral atherosclerosis research with positron emission tomography which applied the two radioligands, 18F-fluorodeoxyglucose and NaF for the detection of culprit atheroma among 20 acute cerebral infarction patients (mean age = 75.1 ± 9.0 years; 10 women). In this study, we measured the maximum and mean standardized uptake value (SUVmax and SUVmean) of NaF uptake level in the cerebral infarct region between lesions with and without diffusion weighted image (DWI) positivity, indicating acute ischemic cell death. Correlation analysis was performed between NaF uptake levels and imaging and clinical variables, including neurological severity. The NaF uptake levels were significantly higher in DWI positive lesions than in negative lesions (SUVmax: 2.0 [0.60–4.2] versus 0.20 [0.10–0.40], p = 0.021 by Mann–Whitney U test). The intensity of NaF uptake (SUVmax) was significantly correlated with the initial neurological severity (Spearman's ρ = 0.579, p= 0.007) and white blood cell count (Spearman's ρ = 0.626, p p 0.003). During ischemic stroke NaF was concentrated in brain tissue undergoing acute cell death and its uptake intensity was correlated with neurological severity, suggesting that NaF could reflect acute ischemic cell death after stroke.