Transient Inhibition Research Articles

MTORC1 regulates the metabolic switch of postnatal cardiomyocytes during regeneration

The metabolic switch from glycolysis to fatty acid oxidation in postnatal cardiomyocytes contributes to the loss of the cardiac regenerative potential of the mammalian heart. However, the mechanisms that regulate this metabolic switch remain unclear. The protein kinase complex mechanistic target of rapamycin complex 1 (mTORC1) is a central signaling hub that regulates cellular metabolism and protein synthesis, yet its role during mammalian heart regeneration and postnatal metabolic maturation is undefined. Here, we use immunoblotting, rapamycin treatment, myocardial infarction, and global proteomics to define the role of mTORC1 in postnatal heart development and regeneration. Our results demonstrate that the activity of mTORC1 is dynamically regulated between the regenerating and the non-regenerating hearts. Acute inhibition of mTORC1 by rapamycin or everolimus reduces cardiomyocyte proliferation and inhibits neonatal heart regeneration following injury. Our quantitative proteomic analysis demonstrates that transient inhibition of mTORC1 during neonatal heart injury did not reduce protein synthesis, but rather shifts the cardiac proteome of the neonatal injured heart from glycolysis towards fatty acid oxidation. This indicates that mTORC1 inhibition following injury accelerates the postnatal metabolic switch, which promotes metabolic maturation and impedes cardiomyocyte proliferation and heart regeneration. Taken together, our results define an important role for mTORC1 in regulating postnatal cardiac metabolism and may represent a novel target to modulate cardiac metabolism and promote heart regeneration.

Transient CSF1R Inhibition in the Early Stage of AD Retains Homeostatic Microglia with Reduced Inflammasomes and Increased autophagy in the 5XFAD Model

AbstractBackgroundChronic neuroinflammation is one of the most conspicuous features of Alzheimer’s disease (AD). While microglia play a vital role in phagocytosing amyloid beta (Aβ) and restraining plaque extension, disease‐associated microglia are known to perpetuate neuroinflammation. Previous studies have shown that ∼80% microglia elimination through a month‐long colony‐stimulating factor 1 receptor (CSF1R) inhibition in the advanced stage of AD in 5X Familial AD (5XFAD) mice does not reduce Aβ levels or plaques but restrains synapse loss and neurodegeneration. On the other hand, prolonged CSF1R inhibition has been shown to prevent parenchymal plaque development. However, the immediate effects of transient CSF1R inhibition in the early stage of AD on residual microglial morphology and their metabolic fitness are unknown.MethodWe investigated the impact of transient CSF1R inhibition in three‐month‐old 5XFAD mice, a stage at which neuroinflammation has commenced, but Aβ plaques were minimal. Microglia from the hippocampus and the somatosensory cortex (SSC) of 5XFAD mice receiving a diet containing either the CSF1R inhibitor PLX5622 or a standard diet for ten days were examined for morphology, markers of NOD‐, LRR‐, and pyrin domain‐containing protein 3 (NLRP3) inflammasome complex, autophagy, and mechanistic target of rapamycin (mTOR) signaling.ResultTen days of CSF1R inhibition resulted in 67‐70% depletion of microglia in the hippocampus and SSC. The residual microglia displayed a noninflammatory phenotype, typified by highly branched and ramified processes, reduced incidence of NLRP3 inflammasome complexes, and increased autophagy. Interestingly, Aβ plaque load was reduced, and mTOR signaling was unaltered in microglia but reduced in neurons. However, transient CSF1R inhibition did not impact astrocyte hypertrophy or neurogenesis in the hippocampus.ConclusionThe results showed that transient CSF1R inhibition in the early stage of AD promotes the retention of homeostatic microglia with reduced inflammasomes and better metabolic fitness but with no changes in mTOR signaling, which can restrain the perpetuation of neuroinflammation and improve phagocytosis of Aβ by microglia.

Abstract B023: The next-generation farnesyltransferase inhibitor KO-2806 constrains compensatory signaling reactivation to deepen responses to KRASG12D inhibition

Abstract The landmark approval of KRASG12C inhibitors toppled the decades-held notion that KRAS was undruggable, fueling the design and development of small molecules targeting other oncogenic KRAS mutants, including the most prevalent, KRASG12D. MRTX1133 is the first KRASG12D-selective inhibitor to reach the clinic, providing a much-needed therapeutic option for patients with KRASG12D-mutant tumors. Nevertheless, both preclinical MRTX1133 data and early clinical results from KRASG12C inhibitor trials suggest that responses to KRASG12D inhibition will vary widely, at least in part due to incomplete and/or transient inhibition of MAPK and mTOR signaling. We previously demonstrated that farnesyltransferase inhibitors (FTIs) can prevent adaptive resistance to PI3K inhibition by blunting compensatory reactivation of these signaling nodes. We therefore hypothesized that farnesyltransferase inhibition may similarly enhance the antitumor activity of MRTX1133 through synergistic blockade of mitogenic and survival signaling. In this study, we leveraged a panel of in vivo cell line (CDX) and patient-derived xenograft (PDX) models, in parallel with in vitro mechanistic studies, to assess the efficacy of combined KO-2806, a FTI, and MRTX1133, in controlling KRASG12D-mutant tumor growth. KO-2806 is a next-generation FTI with increased potency and improved pharmacokinetic properties relative to earlier FTI candidates and is poised to enter a first-in-human clinical trial. Given that reactivation of phospho-ERK and phospho-S6 is linked to diminished response to MRTX1133, we first probed the impact of KO-2806 on cell signaling in vitro. In KRASG12D mutant cell lines, MRTX1133 transiently inhibited both ERK and mTOR, as evidenced by decreased ERK, p90 RSK, p70 S6K, S6, and 4EBP1 phosphorylation. However, phosphorylation of these proteins rebounded partially within one day and completely by 48 hours post treatment. In contrast, when cells were co-exposed to MRTX1133 and KO-2806, robust inhibition of these pathways was maintained at 48 hours and corresponded with greater induction of apoptosis (PARP and caspase cleavage) and cell cycle arrest (ablation of Rb phosphorylation) compared to MRTX1133. These results are consistent with prior reports suggesting that durable MAPK and mTOR inhibition is required for optimal response to KRAS inhibitors. The mechanistic synergy we observed in vitro provided a strong rationale for evaluating combined MRTX1133 and KO-2806 in vivo. Across a panel of KRASG12D-mutant CDX and PDX solid tumor models, the MRTX1133/KO-2806 doublet led to deeper tumor growth inhibition compared to the single agents while remaining well tolerated in mice. We posit that KO-2806 augments the MAPK-mTOR inhibitory effects of MRTX1133 by blocking compensatory pathway reactivation, resulting in superior antitumor efficacy of the combination without amplifying toxicity. Based upon these results, combined farnesyltransferase and KRASG12D inhibition represents a promising therapeutic strategy for the treatment of KRASG12D0-mutant solid tumors. Citation Format: Alison Smith, Hetika Patel, Stacia Chan, Francis Burrows, Shivani Malik. The next-generation farnesyltransferase inhibitor KO-2806 constrains compensatory signaling reactivation to deepen responses to KRASG12D inhibition [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B023.

Transient CDK9 Inhibition with AZD4573 Effectively Induces Apoptosis in Burkitt Lymphoma As a Monotherapy in Pre-Clinical Models

Burkitt Lymphoma (BL) accounts for almost two thirds of all B-cell non-Hodgkin lymphoma cases in children and adolescents. It may affect the jaw, bones of the face, bowel, kidney, ovaries and in some cases spread to the central nervous system. Nearly all types of BL are characterized by c-MYC gene translocation of chromosome 8 with immunoglobulin genes at chromosome 2, 14 or 22 resulting in high levels of the transcription factor. Recently, MCL-1 was shown to be overexpressed in BL and is a critical survival factor in pre-clinical BL models using genetic and chemical approaches. CDK9, a serine/threonine kinase, regulates transcription elongation through phosphorylation of RNA polymerase II at serine 2 (pSer2-RNAPII). AZD4573 is a highly potent and selective CDK9 inhibitor that results in downregulation of short-lived transcripts and labile proteins such as c-MYC and key survival proteins including MCL-1 and BFL-1, resulting in apoptosis in several hematological malignancies (Cidado et al. 2020 & Boiko et al. 2021) We selected several preclinical BL models to assess the rapid apoptogenic potential of AZD4573 in vitro and in vivo. To measure apoptosis, we evaluated cleaved caspase-3 (CC3) induction following acute treatment (6h) using Caspase-Glo 3/7. Using this assay, we found that 3 BL models were sensitive to CDK9 inhibition (EC50 < 100nM; max. CC3 > 50%) and 2 were resistant (EC50 > 100nM; max. CC3 < 50%). Treatment with AZD4573 in these sensitive BL cell line models decreased pSer2-RNAPII, and downregulation of MCL-1 and c-MYC within 6 hours. In AZD4573 resistant lines we observed relatively higher levels of anti-apoptotic proteins BCL2 and BCL-xL, compared to the sensitive cell lines. These increased levels potentially lead to the decreased sensitivity to AZD4573, which does not reduce levels of BCL-2 and BCL-xL due to their longer half-life. Using CRISPR genetic knockouts we demonstrate that BL cell lines are dependent on c-MYC and MCL-1 and that loss of these protein decreases caspase induction after AZD4573 treatment, highlighting the importance of these proteins in driving the efficacy of AZD4573. In vivo AZD4573 dosed on a once weekly schedule resulted in tumor growth inhibition in aggressive BL cell line xenografts Namalwa and Ramos (40-60%) and decreased pSer2-RNAPII, MCL-1 and c-MYC and increased CC3 8 hours after treatment. AZD4573 is currently in a phase 2 study (NCT05140382) to assess the efficacy, safety, and PK of AZD4573 in patients with relapsed or refractory Peripheral T-cell lymphomas (pTCL). Our findings demonstrate that targeting CDK9 with AZD4573 can effectively induce apoptosis in pre-clinical BL models and could be an effective therapy for BL patients.

Extracellular Vesicle Trafficking Regulates Hematopoietic Stem and Progenitor Cell Fitness

Among regulatory pathways critical to hematopoietic stem and progenitor cell (HSPC) function, sphingolipid metabolism has recently been recognized for its role in safeguarding essential HSPC properties of self-renewal and lineage fate. In particular, de novo ceramide (Cer) synthesis appears to regulate a coordinated stress response that results in enhanced self-renewal of human HSPCs. However, the fundamental molecular mechanisms underlying these regenerative stress responses have yet to be uncovered and broadly validated. Moreover, alternative synthetic pathways regulating Cer and sphingomyelin (SM) levels may serve additional regulatory roles in HSPCs. Here we target neutral sphingomyelinase-2 (nSMase2/SMPD3)-dependent SM hydrolysis through pharmacologic (GW4869) and genetic (Cas9-mediated) interruption, and similarly confirm an adaptive ATF4-mediated integrated stress response (ISR). Functionally, transient inhibition led to durable improvements in HSPC reconstitution after primary (>8 fold at steady-state, p<0.001) and secondary (>20 fold at steady-state, p<0.001) transplantation. Surprisingly, lipidomic profiling of GW4869-treated HSPCs demonstrated neither global Cer depletion nor SM accumulation, suggesting an alternative mechanism underlying the observed gains in HSPC fitness. Sphingolipid metabolism also regulates the biogenesis of select subclasses of secreted extracellular vesicles (EVs). To segregate metabolic regulation of HSPCs by sphingolipids from their role in EV formation and secretion, we began to systematically modulate and compare several core EV biogenesis pathways by Cas9/ribonucleoprotein-mediated knockout (KO) of SMPD3, CD63 and TSG101 in HSPC ex vivo expansion cultures . Similar to pharmacologic nSMase2 inhibition, loss of all 3 genes suppressed EV secretion, whereas only depletion of SMPD3 or ESCRT protein TSG101, but not the tetraspanin protein CD63, led to ISR activation in HSPCs (p<0.05). As a functional corollary, clonogenicity and long-term transplantation outcomes were enhanced by KO of SMPD3 (1.5-fold; p<0.01)and TSG101 (2-fold; p<0.001), but reduced after CD63-KO (0.6-fold; p<0.05). One function of EVs is the export of protein and nucleic acid cargo from the cell. We reasoned the observed differences in stress response and HSPC fitness may reflect uniquely altered secretomes. Quantitative proteomic analysis of the vesicle secretome across KO cells revealed impaired secretion of mitochondrial (n=160, FDR=8.3E-22) and oxidative stress-related (n=82, FDR=2.8E-12) proteins from SMPD3- and TSG101- KO, but not CD63-KO HSPCs, highlighting the heterogeneity and specificity of EV cargo trafficking. We validated this observation by pharmacologic blockade of SMPD3 with GW4869, which similarly led to transient decreases in mitochondrial DNA (0.36-fold, p<0.01) and protein (0.6-fold, p<0.05) export. Finally, we confirmed that impaired mitochondrial cargo secretion into EVs (mtEV) after SMPD3 or TSG101, but not CD63, depletion led to transient accumulation of mitochondrial-sourced reactive oxygen species (p<0.01) that specifically triggered the observed ISR activation and downstream mTOR inhibition. Our study reveals a novel mechanism by which secretory cargo trafficking into EVs guards HSPC homeostasis and fitness. We further demonstrate that transient modulation of this pathway, wherein EV cargo retention activates a molecularly-defined stress response pathway, can durably improve long-term transplantation outcomes. Sphingolipid biology inextricably links cellular metabolism and EV biogenesis, making it likely that prior descriptions of Cer-dependent adaptive stress response involve modulation of EV secretion. Finally, our work underscores the high degree of specificity that governs EV secretion in regulating intrinsic cell function. In aggregate, we show that differential EV biogenesis pathways regulate HSPC fate and fitness, with important translational implications in safeguarding stemness and regenerative capacity during ex vivo expansion and therapeutic transplantation.

Casein kinase 2 activity is a host restriction factor for AAV transduction

So far, the mechanisms that impede AAV transduction, especially in the human heart, are poorly understood, hampering the introduction of new, effective gene therapy strategies. Therefore, the aim of this study was to identify and overcome the main cellular barriers to successful transduction in the heart, using induced pluripotent stem cell (iPSC)-derived cardiomyocytes (iPSC-CMs), iPSC-derived cardiac fibroblasts (iPSC-CFs), and primary endothelial cells to model vector-host interactions. Through phosphoproteome analysis we established that casein kinase 2 (CK2) signaling is one of the most significantly affected pathways upon AAV exposure. Transient inhibition of CK2 activity substantially enhanced the transduction rate of AAV2, AAV6, and AAV9 in all tested cell types. In particular, CK2 inhibition improved the trafficking of AAVs through the cytoplasm, impaired DNA damage response through destabilization of MRE11, and altered the RNA processing pathways, which were also highly responsive to AAV transduction. Also, it augmented transgene expression in already transduced iPSC-CFs, which retain AAV genomes in a functional, but probably silent form. In summary, the present study provides new insights into the current understanding of the host-AAV vector interaction, identifying CK2 activity as a key barrier to efficient transduction and transgene expression, which may translate to improving the outcome of AAV-based therapies in the future.

Oocyte quality is enhanced by hypoglycosylated FSH through increased cell-to-cell interaction during mouse follicle development.

Macroheterogeneity in follicle-stimulating hormone (FSH) β-subunit N-glycosylation results in distinct FSH glycoforms. Hypoglycosylated FSH21 is the abundant and more bioactive form in pituitaries of females under 35 years of age, whereas fully glycosylated FSH24 is less bioactive and increases with age. To investigate whether the shift in FSH glycoform abundance contributes to the age-dependent decline in oocyte quality, the direct effects of FSH glycoforms on folliculogenesis and oocyte quality were determined using an encapsulated in vitro mouse follicle growth system. Long-term culture (10-12 days) with FSH21 (10 ng/ml) enhanced follicle growth, estradiol secretion and oocyte quality compared with FSH24 (10 ng/ml) treatment. FSH21 enhanced establishment of transzonal projections, gap junctions and cell-to-cell communication within 24 h in culture. Transient inhibition of FSH21-mediated bidirectional communication abrogated the positive effects of FSH21 on follicle growth, estradiol secretion and oocyte quality. Our data indicate that FSH21 promotes folliculogenesis and oocyte quality in vitro by increasing cell-to-cell communication early in folliculogenesis, and that the shift in in vivo abundance from FSH21 to FSH24 with reproductive aging may contribute to the age-dependent decline in oocyte quality.

CD4 + T cells reverse surface antigen persistence in a mouse model of HBV replication

ABSTRACT Hepatitis B virus (HBV) is responsible for over 800,000 deaths yearly worldwide. While chronic HBV infection currently has no highly effective cure, immunotherapy has generated interest as a potential therapeutic solution due to the connection between viral chronicity and the magnitude of HBV-specific immune responses. Suitable animal models for studying successful immune responses to HBV are limited due to the lack of natural viral tropism for rodents. Using a mouse model of HBV replication, we found that CD4 + T cells are essential for the antibody-mediated clearance of hepatitis B surface antigen (HBsAg) and that early transient inhibition of the CD4 + T cell-B cell axis imparts HBsAg persistence through CD4 + T cell functional impairment. HBsAg resolution in BALB/c mice transduced with an adeno-associated virus encoding the HBV genome (AAV-HBV) depended on B cells, MHCII, and CD4 + T cells, but not CD8 + T cells, indicative of antibody-mediated control. Furthermore, disrupting the CD40-CD40L interaction blunted HBsAg clearance and the generation of HBsAg-specific B cells. Finally, HBsAg persistence induced by transient CD4 depletion was reversible by the transfer of total splenocytes or purified CD4 + T cells from immunized mice. These findings demonstrate the T cell-dependent nature of HBsAg-resolving antibody responses as well as the ability of CD4 + T cells to reverse HBsAg persistence. IMPORTANCE Hepatitis B virus (HBV) is a leading causative agent of viral hepatitis. A preventative vaccine has existed for decades, but only limited treatment options are available for people living with chronic HBV. Animal models for studying HBV are constrained due to narrow viral tropism, impeding understanding of the natural immune response to the virus. Here, using a vector to overcome the narrow host range and establish HBV replication in mice, we identified the role of helper T cells in controlling HBV. We show that helper T cells promote the B cell’s ability to generate antibodies that remove HBV and its associated surface antigen from the blood and that transfer of purified helper T cells from HBV-immunized mice can reverse the accumulation of virus and antigen, furthering our understanding of the immune response to HBV.

Lufenuron treatment temporarily represses gene expression and affects the SUMO pathway in liver of Atlantic salmon.

Lufenuron is a benzoylurea insecticide currently in use to combat sea lice infestation in salmon aquaculture in Chile. With pending approval in Norway, the aim of this work was to study the uptake and toxicity of lufenuron in liver tissue of Atlantic salmon. Juvenile salmon weighing 40 g were given a standard 7-day oral dose, and bioaccumulation and transcriptional responses in the liver were examined 1 day after the end-of-treatment (day 8) and after 1 week of elimination (day 14). Bioaccumulation levels of lufenuron were 29 ± 3 mg/kg at day 8 and 14 ± 1 mg/kg at day 14, indicating relatively rapid clearance. However, residues of lufenuron were still present in the liver after 513 days of depuration. The exposure gave a transient inhibition of transcription in the liver at day 8 (2437 significant DEGs, p-adj < .05), followed by a weaker compensatory response at day 14 (169 significant DEGs). Pathways associated with RNA metabolism such as the sumoylation pathway were most strongly affected at day 8, while the apelin pathway was most profoundly affected at day 14. In conclusion, this study shows that lufenuron easily bioaccumulates and that a standard 7-day oral dose induces a transient inhibition of transcription in liver of salmon.

Transient inhibition of lysosomal functions potentiates nucleic acid vaccines.

Nucleic acid vaccines have shown promising results in the clinic against infectious diseases and cancers. To robustly improve the vaccine efficacy and safety, we developed an approach to increase the intracellular stability of nucleic acids by transiently inhibiting lysosomal function in targeted tissues using sucrose. To achieve efficient and localized delivery of sucrose in animals, we designed a biomimetic lipid nanoparticle (LNP) to target the delivery of sucrose into mouse muscle cells. Using this approach, viral antigen expression in mouse muscle after DNA vaccination was substantially increased and prolonged without inducing local or systemic inflammation or toxicity. The same change in antigen expression would be achieved if the vaccine dose could be increased by 3,000 folds, which is experimentally and clinically impractical due to material restrictions and severe toxicity that will be induced by such a high dose of nucleic acids. The increase in antigen expression augmented the infiltration and activation of antigen-presenting cells, significantly improved vaccine-elicited humoral and T cell responses, and fully protected mice against the viral challenge at a low dose of vaccine. Based on these observations, we conclude that transient inhibition of lysosome function in target tissue by sucrose LNPs is a safe and potent approach to substantially improve nucleic acid-based vaccines.

Effects of reversible SERCA inhibition on catecholamine exocytosis and intracellular [Ca2+] signaling in chromaffin cells from normotensive Wistar Kyoto rats and spontaneously hypertensive rats

Intracellular Ca2+ ([Ca2+]i) signaling and catecholamine (CA) exocytosis from adrenal chromaffin cells (CCs) differ between mammalian species. These differences partly result from the different contributions of Ca2+-induced Ca2+-release (CICR) from internal stores, which boosts intracellular Ca2+ signals. Transient inhibition of the sarcoendoplasmic reticulum (SERCA) Ca2+ pump with cyclopiazonic acid (CPA) reduces CICR. Recently, Martínez-Ramírez et al. found that CPA had contrasting effects on catecholamine secretion and intracellular Ca2+ signals in mouse and bovine CCs, where it enhanced and inhibited exocytosis, respectively. After CPA withdrawal, exocytosis diminished in mouse CCs and increased in bovine CCs. These differences can be explained if mouse CCs have weak CICR and strong Ca2+ uptake, and the reverse is true for bovine CCs. Surprisingly, CPA slightly reduced the amplitude of Ca2+ signals in both mouse and bovine CCs. Here we examined the effects of CPA on stimulated CA exocytosis and Ca2+ signaling in rat CCs and investigated if it alters differently the responses of CCs from normotensive (WKY) or hypertensive (SHR) rats, which differ in the gain of CICR. Our results demonstrate that CPA application strongly inhibits voltage-gated exocytosis and Ca2+ transients in rat CCs, regardless of strain (SHR or WKY). Thus, despite the greater phylogenetic distance from the most recent common ancestors, suppression of endoplasmic reticulum (ER) Ca2+ uptake through CPA inhibits the CA secretion in rat CCs more similarly to bovine than mouse CCs, unveiling divergent evolutionary relationships in the mechanism of CA exocytosis of CCs between rodents. Agents that inhibit the SERCA pump, such as CPA, suppress catecholamine secretion equally well in WKY and SHR CCs and are not potential therapeutic agents for hypertension. Rat CCs display Ca2+ signals of varying widths. Some even show early and late Ca2+ components. Narrowing the Ca2+ transients by CPA and ryanodine suggests that the late component is mainly due to CICR. Simultaneous recordings of Ca2+ signaling and amperometry in CCs revealed the existence of a robust and predictable correlation between the kinetics of the whole-cell intracellular Ca2+ signal and the rate of exocytosis at the single-cell level.

Neutral sphingomyelinase blockade enhances hematopoietic stem cell fitness through an integrated stress response

AbstractHematopoietic stem and progenitor cell (HSPC) transplantation serves as a curative therapy for many benign and malignant hematopoietic disorders and as a platform for gene therapy. However, growing needs for ex vivo manipulation of HSPC-graft products are limited by barriers in maintaining critical self-renewal and quiescence properties. The role of sphingolipid metabolism in safeguarding these essential cellular properties has been recently recognized, but not yet widely explored. Here, we demonstrate that pharmacologic and genetic inhibition of neutral sphingomyelinase 2 (nSMase-2) leads to sustained improvements in long-term competitive transplantation efficiency after ex vivo culture. Mechanistically, nSMase-2 blockade activates a canonical integrated stress response (ISR) and promotes metabolic quiescence in human and murine HSPCs. These adaptations result in part from disruption in sphingolipid metabolism that impairs the release of nSMase-2–dependent extracellular vesicles (EVs). The aggregate findings link EV trafficking and the ISR as a regulatory dyad guarding HSPC homeostasis and long-term fitness. Translationally, transient nSMase-2 inhibition enables ex vivo graft manipulation with enhanced HSPC potency.

Antitumor activity of alkylphospholipid edelfosine in prostate cancer models and endoplasmic reticulum targeting

Prostate cancer is the second most frequent cancer and the fifth leading cause of cancer death among men worldwide. While the five-year survival in local and regional prostate cancer is higher than 99%, it falls to about 28% in advanced metastatic prostate cancer. The ether lipid edelfosine is considered the prototype of a family of promising antitumor drugs collectively named as alkylphospholipid analogs. Here, we found that edelfosine was the most potent alkylphospholipid analog in inducing apoptosis in three different human prostate cancer cell lines (LNCaP, PC3, and DU145) with distinct androgen dependency, and differing in tumor suppressor phosphatase and tensin homolog (PTEN) and p53 status. Edelfosine accumulated in the endoplasmic reticulum of prostate cancer cells, leading to endoplasmic reticulum stress and cell death in the three prostate cancer cells. Inhibition of autophagy potentiated the pro-apoptotic activity of edelfosine in LNCaP and PC3 cells, where autophagy was induced as a survival response. Edelfosine induced a slight and transient inhibition of AKT in PTEN-negative LNCaP and PC3 cells, but not in PTEN-positive DU145 cells. Daily oral administration of edelfosine in murine prostate restricted AKT kinase transgenic mice, expressing active AKT in a prostate-specific manner, and in a DU145 xenograft mouse model resulted in significant tumor regression and apoptosis in tumor cells. Taken together, these results show a significant in vitro and in vivo antitumor activity of edelfosine against prostate cancer, and highlight the endoplasmic reticulum as a novel and promising therapeutic target in prostate cancer.

Interactions of the male contraceptive target EPPIN with semenogelin-1 and small organic ligands

Novel male contraceptives will promote gender equality in sharing contraceptive responsibility. The sperm-associated protein epididymal protease inhibitor (EPPIN) is a promising target for non-hormonal male contraception. EPPIN interacts with the semen coagulum protein semenogelin-1 (SEMG1) on the sperm surface, leading to transient inhibition of sperm motility after ejaculation. Small organic molecules targeting EPPIN's SEMG1-binding are under development as male contraceptives. Here, we combined computational approaches to uncover key aspects underlying EPPIN binding to SEMG1 and small organic ligands. We generated a human EPPIN model showing a typical arrangement of the WFDC (Whey-acid four disulfide core)-type and Kunitz-type domains, connected by a hinge region. Determining the EPPIN model's intrinsic motion by molecular dynamics simulations and normal mode analysis revealed a conformation, presenting a binding pocket that accommodates SEMG1Glu229-Gln247, EP055, and EP012. EPPIN's residues Phe63 and Lys68 (WFDC domain), Asp71 (hinge region), and Asn113, Asn114, and Asn115 (Kunitz domain) were identified as hot spots for SEMG1, EP055, and EP012 binding. Moreover, hydrophobic and hydrophilic residues in the WFDC and Kunitz domains allow plasma membrane anchoring, orienting the EPPIN binding pocket to the solvent. Targeting EPPIN's essential residues for its biomolecular interactions may improve the rational design of EPPIN ligands as spermiostatic compounds.

Molecular dynamics of genome editing with CRISPR-Cas9 and rAAV6 virus in human HSPCs to treat sickle cell disease

Exvivo gene correction with CRISPR-Cas9 and a recombinant adeno-associated virus serotype 6 (rAAV6) in autologous hematopoietic stem/progenitor cells (HSPCs) to treat sickle cell disease (SCD) has now entered early-phase clinical investigation. To facilitate the progress of CRISPR-Cas9/rAAV6 genome editing technology, we analyzed the molecular changes in key reagents and cellular responses during and after the genome editing procedure in human HSPCs. We demonstrated the high stability of rAAV6 to serve as the donor DNA template. We assessed the benefit of longer HSPC pre-stimulation in terms of increased numbers of edited cells. We observed that the p53 pathway was transiently activated, peaking at 6 h, and resolved over time. Notably, we revealed a strong correlation between p21 mRNA level and rAAV6 genome number in cells and beneficial effects of transient inhibition of p53 with siRNA on genome editing, cell proliferation, and cell survival. In terms of potential immunogenicity, we found that rAAV6 capsid protein was not detectable, while a trace amount of residual Cas9 protein was still detected at 48h post-genome editing. We believe this information will provide important insights for future improvements of gene correction protocols in HSPCs.

Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.

Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.

Topotecan alleviates acetic acid-induced ulcerative colitis in rats via attenuation of the RORγT transcription factor

AimsUlcerative colitis is characterized as a chronic immune-mediated inflammatory condition, affecting the intestinal gastroenteric tissue. Previous studies revealed that Th-17 cells are key players in the pathogenesis of ulcerative colitis. RORγT (Retinoic-acid-receptor-related orphan receptor-gamma T) is a lineage-specific transcription factor of Th-17 cells and thus has a role in their differentiation. Transient inhibition of RORγT has been reported to attenuate the differentiation of Th-17 cells and secretion of interleukin-17 (IL-17). Here, we investigated the efficacy of topotecan in ameliorating ulcerative colitis in rodents, via inhibition of the RORγT transcription factor. Main methods and key findingsExperimental ulcerative colitis was induced in rats by intrarectal acetic acid administration. Topotecan attenuated the severity of ulcerative colitis in rats by revoking neutrophils and macrophage infiltration to the colon. It also alleviated diarrhea and rectal bleeding and improved body weight. Further, attenuation of RORγT and IL-17 expression was observed in topotecan treated animals. Levels of pro-inflammatory cytokines TNF-α, IL-6, and IL-1β in the colon tissue were reduced by topotecan treatment. Significant reduction in malondialdehyde level, elevation of superoxide dismutase (SOD) and catalase activity was observed in the colon tissue of rats treated with topotecan compared to the diseased group. SignificanceThis study shows the therapeutic potential of topotecan in attenuating ulcerative colitis in rats probably via inhibition of the RORγT transcription factor and downstream mediators of Th-17 cells.

Transient inhibition of mitochondrial function by chrysin and apigenin prolong longevity via mitohormesis in C. elegans.

Mild inhibition of mitochondrial function leads to longevity. Genetic disruption of mitochondrial respiratory components either by mutation or RNAi greatly extends the lifespan in yeast, worms, and drosophila. This has given rise to the idea that pharmacologically inhibiting mitochondrial function would be a workable strategy for postponing aging. Toward this end, we used a transgenic worm strain that expresses the firefly luciferase enzyme widely to evaluate compounds by tracking real-time ATP levels. We identified chrysin and apigenin, which reduced ATP production and increased the lifespan of worms. Mechanistically, we discovered that chrysin and apigenin transiently inhibit mitochondrial respiration and induce an early ROS, and the lifespan-extending effect is dependent on transient ROS formation. We also show that AAK-2/AMPK, DAF-16/FOXO, and SKN-1/NRF-2 are required for chrysin or apigenin-mediated lifespan extension. Temporary increases in ROS levels trigger an adaptive response in a mitohormetic way, thereby increasing oxidative stress capacity and cellular metabolic adaptation, finally leading to longevity. Thus, chrysin and apigenin represent a class of compounds isolated from natural products that delay senescence and improve age-related diseases by inhibiting mitochondrial function and shed new light on the function of additional plant-derived polyphenols in enhancing health and delaying aging. Collectively, this work provides an avenue for pharmacological inhibition of mitochondrial function and the mechanism underlining their lifespan-extending properties.

512-P: Single-Cell RNA-Sequencing Identifies Novel Molecular Targets of Endothelial MicroRNA-200b in the Diabetic Ischemic Wound

Injury-induced transient downregulation of endothelial miR-200b is required to jump-start wound angiogenesis, but the underlying mechanisms remain unclear. Single cell RNA sequencing was performed on ~15000 human endothelial cells under high (mimic treated) miR-200b conditions. Unsupervised clustering using CellRanger identified five cell clusters, three of which were responsive to changes in miR-200b abundance. Stable Isotope Labelling by Amino acids in Cell culture (SILAC) based proteomic analysis was employed to look for novel potential targets of endothelial miR-200b. A total of 3818 proteins were detected which were then filtered using statistical cut-offs (p value &amp;lt; 0.05; % change &amp;gt; 10%) identifying 319 proteins targeted by miR-200b. To determine the potential role of these candidates in regulating vascular function, miR-200b-429fl/fl-Tie2 Cre mice were generated where endothelial miR-200b levels could be specifically depleted in vivo. The therapeutic significance of endothelial miR-200b inhibition was studied using ischemic hindlimb in these mice. The significance of miR-200b depletion was also studied in streptozotocin-induced diabetic miR-200b-429fl/fl-Tie2 Cre mice. Perfusion imaging was performed using Laser Speckle imaging (Perimed Inc.) at different time-points (d3, d7, d10, d14). Inhibition of endothelial miR-200b rescued hindlimb ischemia with improved perfusion (n=11). Such effect of miR-200b inhibition on rescuing HL-ischemia was markedly increased in diabetic animals (n =8). Such rescue was associated with increased abundance of CD31+/vWF+ vasculogenic cells (n=6). This work identified miR-200b regulated endothelial cell clusters, the functional significance of which have been experimentally validated. These findings provide insight into novel mechanisms explaining how transient inhibition of miR-200b in the endothelial compartment improves angiogenic outcomes in the diabetic ischemic limb. Disclosure K.Singh: None. M.Kumar: None. S.K.Mohanty: None. S.Khanna: None. S.Roy: None. C.K.Sen: n/a. Funding National Institutes of Health (DK128845); U.S. Department of Defense (W81XWH-22-1-0146, W81XWH-21-1-0033)

New insights into the disulfide stress response by the Bacillus subtilis Spx system at a single-cell level.

Spx is a global transcriptional regulator that orchestrates the Bacillus subtilis response to disulfide stress. The YjbH (SpxH) protein adapts Spx for ClpXP-mediated degradation, playing a critical role in the regulation of the cellular Spx levels. Upon stress, YjbH forms aggregates by a yet unknown mechanism, resulting in increased Spx levels due to reduced proteolysis. Here, we studied how individual cells use the Spx-YjbH system to respond to disulfide stress. We show, using fluorescent reporters, a correlation between the Spx levels and the amount of YjbH, as well as a transient growth inhibition upon disulfide stress. The in vivo dynamics and inheritance of YjbH aggregates are characterized by a bipolar distribution over time and appear to be entropy-driven by nucleoid exclusion. Moreover, we reveal that the population following disulfide stress is highly heterogenous in terms of aggregate load and that the aggregate load has strong implications for cellular fitness. We propose that the observed heterogeneity could be a mechanism to ensure population survival during stress. Finally, we find that the two YjbH domains (DsbA-like domain and winged-helix domain) contribute to its aggregation function, and show that the aggregation of the DsbA-like domain is conserved among other studied orthologs, whereas important differences are observed for the winged-helix domain.