shRNA Targeting Research Articles

Chronic IL1β Exposure Enhances Self-Renewal Ability and Myeloid Priming in TET2 Deficient Stem and Progenitor Cells

Background: Clonal hematopoiesis (CH) increases risk for the development of hematological malignancy (HM). Patients with CH have high levels of circulatory cytokines (such as IL1b, IL6, TNFa) leading to increased inflammatory stress. Inflammation is associated with increased risk for the development of HM. However, how inflammation alters population dynamics of hematopoietic stem and progenitor cells (HSPCs) in CH, is not well understood. Here we modelled inflammation using the proinflammatory cytokine IL1β in TET2 mediated CH. Methods and Results: To identify the impact of chronic IL1β exposure on HSPCs in CH, we used pan-hematopoietic Tet2 loss-of-function mice (VavCre Tet2fl/fl, referred as Tet2 KO). We treated WT and Tet2 KO mice with IL1β for five weeks. Tet2 KO mice treated with IL1β exhibited splenomegaly and elevated neutrophils in the spleen and bone marrow (BM) compared to vehicle treated or WT mice. In a parallel study, we used a murine competitive transplantation model with a doxycycline inducible shRNA targeting Tet2 (shTet2) BM cells competing against WT. Similar to Tet2 KO, shTet2 cells were expended in the upon IL1β stimulation for six weeks compared to vehicle (85.6% vs 61.8%, p <0.05) with significant myeloid bias in PB, BM and spleen. Specifically, IL1β stimulation led to the significant expansion (3-4 fold) of ly6chi monocyte/macrophages in the BM and spleen of Tet2 KO mice relative to WT and with no change in anti-inflammatory ly6clo monocyte/macrophages in the spleen and 13-fold (p <0.001) reduction in the BM. Further, Myeloid biased MPP2/3 were elevated upon IL1β treatment relative to lymphoid biased MPP4s in shTet2 and Tet2 KO compared to WT mice. The ratio of myeloid biased GMPs to megakaryocyte/erythroid biased MEPs was increased within Tet2 KO mice in all conditions. Notably, LT-HSCs were elevated in frequency in the BM of both shTet2 (9.5-fold, p <0.01) and Tet2 KO (1.9-fold, p <0.05) relative to WT upon IL1β treatment. Colony formation assays with serial replating demonstrated IL1β further enhanced the self-renewal ability of Tet2 KO progenitor cells (LSK, Lin-Sca1+ckit+) 10.6-fold by the quaternary plating (p <0.0001) over that of untreated Tet2 KO LSKs. To identify the mechanisms by which IL1β promotes myeloid bias and self-renewal potential of Tet2 KO HSCs, we used scRNAseq and whole genome bisulfite sequencing (WGBS) analyses. scRNAseq identified that the signatures for inflammatory pathways and chronically stressed old HSCs were upregulated in Tet2 KO HSCs relative to WT at steady state and upon IL1β stimulation for 5 weeks in vivo. Additionally, seven gene sets and genes (Erg, Meis1, and Egr1) associated with enhanced self-renewal ability were upregulated in Tet2 KO HSCs relative to WT upon IL1β stimulation, whereas only a single gene set was significant at steady state. WGBS identified that IL1β stimulation for 5 weeks significantly reduced global methylation in WT LSKs, CMPs, and GMPs, but not in Tet2 KO progenitors. Using published ChIPseq data for 35 proteins, we identified 4 transcription factors known to drive terminal hematopoietic differentiation (ELF4, PU.1, CEBPB, JUN) among the top hypermethylated factors specifically in Tet2 KO LSKs relative to WT upon IL1β stimulation, thus reducing terminal differentiation. Finally, to understand the therapeutic potential of IL1 signaling in CH, we examined the PB of aged Tet2 KO, WT, interleukin 1 receptor 1 knockout (Il1r1-/-), and Il1r1-/- Tet2 KO (double knockout, DKO) mice. We found DKO was protected from the myeloid expansion present in Tet2 KO mice. Accordingly, IL1-driven myeloid expansion is blocked by anakinra, a clinically approved IL1 antagonist, in a murine Tet2 KO model in vivo or TET2 edited human progenitors in a colony formation assay. Conclusions: IL1β mediated chronic inflammatory stress promotes expansion of Tet2 KO HSCs, myeloid biased progenitors, pro-inflammatory monocytes and neutrophils. IL1β drastically increased self-renewal capacity of Tet2 KO HSPCs with potent upregulation for transcriptional signatures driving self-renewal and hypermethylation of transcription factor binding sites driving terminal differentiation to preserve the stem cell pool. Finally, genetic or pharmacological mitigation of IL1 signaling reduced myeloid expansion, providing strong evidence for therapeutic targeting of IL1 signaling to prevent or delay premalignant myeloid expansion.

Induction of Fetal Hemoglobin and Reduction of Clinical Manifestations in Patients with Severe Sickle Cell Disease Treated with Shmir-Based Lentiviral Gene Therapy for Post-Transcriptional Gene Editing of BCL11A: Updated Results from Pilot and Feasibility Trial

Background Induction of fetal hemoglobin (HbF) by repressing BCL11A could reduce or eliminate sickle cell disease (SCD) clinical manifestations. The BCH-BB694 lentiviral vector (LVV) encodes a shRNA targeting BCL11A embedded in a microRNA scaffold (shmiR) allowing erythroid-specific knockdown to induce γ-globin expression and concomitantly and coordinately repress β-sickle globin expression (Brendel et al. JCI 2016). The safety and efficacy of gene therapy (GT) with BCH-BB694-transduced autologous CD34+ cells in patients with SCD is being investigated in a pilot and feasibility trial (NCT03282656), which has now completed enrollment of all 10 planned patients. Preliminary data in the first 6 patients described robust HbF induction and a favorable safety profile (Esrick et al. NEJM 2021). Additional follow-up data including all ten pilot trial patients is discussed here with longest follow-up now > 4 years. Methods Patients with severe SCD were screened for eligibility. Autologous CD34+ cells were collected via apheresis after plerixafor mobilization (Esrick and Manis et al. Blood Adv 2018) and then transduced ex vivo with the BCH-BB694 shmiR LVV. Gene modified cells were infused into subjects who had received myeloablative busulfan conditioning. Patients were monitored for adverse events (AEs), hemoglobin (Hb), HbF fraction, percent F cells, hemolysis markers, and SCD clinical manifestations. Results Ten patients (7-25 years of age at enrollment) received GT, with median follow-up of 30.5 (2-50) months. There were no Grade 3 or 4 AEs associated with mobilization, collection or infusion. Median product VCN, % transduced cells, and CD34+ cell dose in the 10 patients were 3.5 (1.8-6.9) copies/diploid genome (c/dg), 96% (62-100%), and 5.7 (3.6-8.8) x 106 CD34+ cells/kg, respectively. Engraftment of neutrophils occurred on day 22 (18-30) and of platelets on day 32 (25-62). One patient (patient 10) has low post-GT peripheral VCN, HbF, and percent F cells; most recently 0.23 c/dg, 11.2%, and 37.3% respectively. Although improved, this was considered a GT failure and the patient has been started on new SCD-directed therapies due to ongoing SCD clinical manifestations. Subsequent data described here exclude patient 10. At the most recent study visit, the 7 untransfused patients with follow up > 6 months had mean total Hb of 10.5 (8.8-12.2) g/dL, HbF/(F+S) of 30.3% (21.4 - 42.4%), percent F cells of 70.6% (58.8-79.8%), and HbF per F cell of 12 (10.1-14.4) pg (Table 1). The lowest Hb response was seen in patient 11 who has 2-gene deletion alpha thalassemia in combination with SCD. Five patients with follow-up > 6 months (patients 4, 6, 8, 9, and 11) had frequent severe vaso-occlusive events (ACS, priapism, or pain requiring inpatient or ED opioids) as indication for eligibility, with a median of 6 (3-13) events (Table 2). From 6 months after GT through present, these 5 patients have had a median of 1 (0-1) event. Three patients with follow-up > 6 months (patients 2, 3, and 7) were on chronic transfusion regimens pre-GT. Patient 3 has continued chronic transfusions due to pre-existing moyamoya with goal of maintaining HbS at pre-GT levels. Patients 2 and 7 have received no post-GT transfusions. Patients 2, 3, and 7 have had no SCD clinical events. Mean lactate dehydrogenase and absolute reticulocyte count at last study visit were 346 (215-389) U/L and 241 (200-360) 109/L, respectively, indicating decreased but persistent mild hemolysis in all patients. There have been no instances of graft failure, replication competent lentivirus detection, or clonal dominance in any patient. Six patients with ≥2 years post GT have had bone marrow biopsies at the 2 year timepoint without evidence of myelodysplasia. Targeted sequencing of ~95 genes frequently mutated in hematologic malignancies has been performed on peripheral blood samples every 6 months since early 2021 in patients with ≥6 months from GT. One patient had a pathogenic mutation detected in the DNMT3A gene, which was present in a stored pre-GT sample and has been present at a stable VAF <4% at 6, 9, and 12 months post-GT. Summary Post-transcriptional gene editing of BCL11A appears safe with stable induction of broadly distributed HbF and significant mitigation of VOE events in severe SCD in all but one treated patient. Based on the results of this pilot study, a phase 2, multi-institutional trial is now open for enrollment using the same vector and approach (NCT05353647). Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

TMET-12. HYPERPOLARIZED Δ- [1-13C] GLUCONOLACTONE IMAGING VISUALIZES TERT-ASSOCIATED CHANGES IN DYNAMIC PENTOSE PHOSPHATE PATHWAY METABOLISM IN GLIOBLASTOMA

Abstract BACKGROUND TERT promoter mutations are a hallmark of glioblastoma (GBM). We recently reported that the expression of TERT and its upstream transcriptional factor, GABPB1, are strongly correlated with redox in GBM with TERT promoter mutations. However, further imaging biomarkers which visualize redox-associated metabolism and TERT expression are needed. Here we demonstrate that 13C magnetic resonance spectroscopy (MRS) of hyperpolarized δ-[1-13C] gluconolactone metabolism is a useful imaging tool to visualize changes in dynamic pentose phosphate pathway (PPP) metabolism that reflect TERT-associated changes in redox in GBM. METHODS U251 human GBM cells stably expressing shRNA targeting TERT or GABPB1 were compared to controls. Doxycyclin-inducible shTERT or shGABPB1 U251 cells were also examined. For in vivo studies, cells were injected into immunodeficient rat brains and tumors confirmed by T2-weighted MRI. δ-[1-13C] gluconolactone was polarized using a Hypersense DNP polarizer and injected into live cells or tumor-bearing rats. For cells 13C-MRS was acquired using a 500MHz Agilent spectrometer and analyzed using Mnova and Matlab software. For in vivo13C-MRS studies, spectra were acquired using a 3T Bruker scanner and a spectral spatial echo-planar spectroscopic imaging sequence. Spectral signal to noise was improved using Tensor denoising. Spectra were processed using a custom-written Matlab script. RESULTS Hyperpolarized 6-phosphogluconolactone (6PG), the metabolic product from δ-[1-13C] gluconolactone via the PPP, was significantly reduced in TERT or GABPB1 silenced cells compared to control cells in both U251 models. The positive correlation between TERT expression and 6PG level was confirmed by linear regression analysis. Hyperpolarized 6PG production in both tumor models was also significantly reduced in TERT or GABPB1-silenced tumors compared to controls. CONCLUSION We successfully visualized TERT-associated changes in dynamic PPP metabolism in a GBM model in cells and in vivo. Hyperpolarized δ-[1-13C] gluconolactone is a potential tool for monitoring TERT expression in GBM with TERT promoter mutations.

Abstract 11876: The Selenoprotein, Vimp, Selectively Regulates a Newly Defined Non-Canonical Form of Proteasomal Degradation at the ER to Modulate Cardiac Hypertrophy

Introduction: Canonical endoplasmic reticulum (ER) associated degradation (ERAD) adaptively degrades misfolded potentially toxic ER proteins that can accumulate in cells under a variety of stresses. However, roles for ERAD in vivo in potentially pathological conditions have not been well studied. Hypothesis: Here, we examined a functional component of ERAD, VCP-interacting membrane protein (VIMP), positing that decreasing VIMP would decrease canonical ERAD, which would be maladaptive in a mouse model of pathological cardiac hypertrophy. Methods: Mice were injected with AAV9 to transduce cardiomyocytes with either a shRNA targeting VIMP (shVIMP), or ectopic wild-type or an ERAD-deficient AAV-FLAGVIMP and subjected to pressure overload-induced cardiac hypertrophy. Mice were also injected with a novel AAV expressing a canonical ERAD substrate to monitor the rate of ERAD in vivo for the first time in any organ system. Results: Unexpectedly, both VIMP knockdown and overexpression in mouse hearts decreased canonical ERAD while only VIMP knockdown adaptively decreased pathological cardiac hypertrophy. We evaluated the effects of VIMP gain and loss of function on ERAD-mediated degradation of a cytosolic pro-growth kinase, serum/glucocorticoid-regulated kinase 1, SGK1, which we showed here to contribute to pathological cardiac hypertrophy and to be a non-canonical, i.e non-ER localized substrate for ERAD. In contrast to its effect on canonical ERAD, VIMP overexpression decreased noncanonical ERAD-mediated degradation of SGK1, which accounted for the maladaptive effects of increased VIMP in the heart. We found that DERLIN1 was responsible for increasing ERAD-mediated SGK1 degradation, which accounted for the suppression of pathological cardiac hypertrophy when VIMP was knocked down. Conclusions: Thus, the relative levels of VIMP and DERLIN1 comprise a molecular switch mechanism that regulates a newly defined non-canonical ERAD-mediated degradation of SGK1, a critical signaling kinase contributing to pathological cardiac hypertrophy. Furthermore, this noncanonical ERAD is of unique importance in cardiomyocytes as these effects of VIMP gain and loss of function were not observed in cardiac fibroblasts from the same mouse hearts.

ILGBMSH: an interpretable classification model for the shRNA target prediction with ensemble learning algorithm.

Short hairpin RNA (shRNA)-mediated gene silencing is an important technology to achieve RNA interference, in which the design of potent and reliable shRNA molecules plays a crucial role. However, efficient shRNA target selection through biological technology is expensive and time consuming. Hence, it is crucial to develop a more precise and efficient computational method to design potent and reliable shRNA molecules. In this work, we present an interpretable classification model for the shRNA target prediction using the Light Gradient Boosting Machine algorithm called ILGBMSH. Rather than utilizing only the shRNA sequence feature, we extracted 554 biological and deep learning features, which were not considered in previous shRNA prediction research. We evaluated the performance of our model compared with the state-of-the-art shRNA target prediction models. Besides, we investigated the feature explanation from the model's parameters and interpretable method called Shapley Additive Explanations, which provided us with biological insights from the model. We used independent shRNA experiment data from other resources to prove the predictive ability and robustness of our model. Finally, we used our model to design the miR30-shRNA sequences and conducted a gene knockdown experiment. The experimental result was perfectly in correspondence with our expectation with a Pearson's coefficient correlation of 0.985. In summary, the ILGBMSH model can achieve state-of-the-art shRNA prediction performance and give biological insights from the machine learning model parameters.

Prognostic Analysis of LncRNA MCM3AP-AS1 in Colorectal Cancer and the Mechanism of Its Effect on Tumor Cell Activity.

To determine the clinical prognostic significance of lncRNA MCM3AP-AS1 in colorectal cancer (CRC) and its preliminary mechanism, 43 CRC patients and 48 healthy individuals were analyzed. Peripheral blood MCM3AP-AS1 was quantified via qRT–PCR in CRC patients at admission and 2 h after surgery and in healthy individuals. Human colon cancer cells (HCT116 and SW480) were transfected with shRNAs targeting upregulation of MCM3AP-AS1 expression (named as sh-MCM3AP-AS1 group) and corresponding negative RNAs (named as sh-MCM3AP-AS1 group). Additionally, the cells were then treated either with 50 mM of the VEGF-specific inhibitor PTK787 (Selleck, USA) (named as inhibition group) or normal saline as a control (named as control group). Before therapy, CRC patients presented a higher MCM3AP-AS1 level than healthy individuals (P < 0.05), and the sensitivity and specificity of MCM3AP-AS1 in predicting the occurrence of CRC were 65.12% and 83.33%, respectively (P < 0.001). After therapy, CRC patients presented a decrease in MCM3AP-AS1 levels, and recurrence was higher in patients who died (P < 0.05). Additionally, the high MCM3AP-AS1 expression group presented a higher mortality than the low MCM3AP-AS1 expression group (P < 0.05). In an in vitro assay, CRC cells showed a higher MCM3AP-AS1 level than CCD-18Co cells, and the sh-MCM3AP-AS1 group presented decreased cell proliferation and invasiveness, whereas the levels apoptosis-associated proteins were increased (P < 0.05). Moreover, the VEGF and VEGFR2 mRNA levels were increased in CRC cells, and VEGF/VEGFR2 pathway-associated proteins were inhibited in the sh-MCM3AP-AS1 group (P < 0.05). Moreover, treatment with PTK787 decreased cell proliferation and invasivness but increased the levels of apoptosis-associated proteins (P < 0.05).

ADAM33 Silencing Inhibits Vascular Smooth Muscle Cell Migration and Regulates Cytokine Secretion in Airway Vascular Remodeling via the PI3K/AKT/mTOR Pathway.

Introduction Vascular smooth muscle cells (VSMCs) are highly involved in airway vascular remodeling in asthma. Objectives This study aimed to investigate the mechanisms underlying the effects of a disintegrin and metalloproteinase-33 (ADAM33) gene on the migration capacity and inflammatory cytokine secretion of VSMCs. Methods Human aortic smooth muscle cells (HASMCs) were transfected with lentiviral vectors carrying short hairpin RNA (shRNA) targeting ADAM33 or negative control vectors. The migration capacity of HASMCs was evaluated by a transwell assay. The levels of secreted inflammatory cytokines were measured using enzyme-linked immunosorbent assay (ELISA) kits. Reverse transcription-quantitative polymerase chain reaction and Western blot assays were performed to detect mRNA and protein expression levels. Results Silencing of ADAM33 significantly inhibited the migration of HASMCs. The expression of tumor necrosis factor alpha (TNF-α) in the supernatant of HASMCs was decreased, while that of interferon gamma (IFN-γ) was increased after the transfection of shRNA targeting ADAM33. Insufficient ADAM33 expression also suppressed the expression levels of phosphatidylinositol 3-kinase (PI3K), phospho-protein kinase B (AKT), phospho-mammalian target of rapamycin (mTOR), Rho-associated protein kinases, phospho-forkhead box protein O1 (FOXO1), and cyclin D1, but it did not affect the levels of AKT, mTOR, or Rho. Conclusion Silencing of the ADAM33 gene inhibited HASMC migration and regulated inflammatory cytokine secretion via targeting the PI3K/AKT/mTOR pathway and its downstream signaling. These data contribute to a better understanding of the regulatory mechanisms of airway vascular remodeling in asthma.

Downregulation of glycoprotein non-metastatic melanoma protein B prevents high glucose-induced angiogenesis in diabetic retinopathy.

Diabetic retinopathy (DR), a microvascular complication characterized by abnormal angiogenesis, is the most common reason for irreversible blindness. Glycoprotein non-metastatic melanoma protein B (GPNMB), as a transmembrane protein, was found to be associated with angiogenesis. This study aims to investigate the role of GPNMB in DR. The levels of GPNMB and Integrin β1 were detected by real-time PCR and western blot and were found to be increased in human retinal microvascular endothelial cells (HRMECs) with high glucose (HG, 25mmol/L) treatment. Knockdown of GPNMB was mediated by lentivirus carrying shRNA targeting GPNMB in vivo and in vitro. Functional experiments, including cell counting kit-8 (CCK-8), scratch, and tube formation assays, showed the anti-proliferative, anti-migrative, and anti-angiogenic roles of GPNMB knockdown in HRMECs using the lentivirus system following HG challenge. Additionally, increased GPNMB levels were detected in the retina of DR rats induced by a single intraperitoneal injection of streptozotocin (60mg/kg) using real-time PCR, western blot, and immunofluorescence assays. Downregulation of GPNMB inhibited the angiogenesis and vascular endothelial growth factor production in the retina of rats with DR. Furthermore, overexpression of Integrin β1 led to increased angiogenesis in DR. Integrin β1 was indicated as a target protein of GPNMB. Upregulated-Integrin β1 restored GPNMB knockdown-induced inhibition of cell viability, migration, and tube formation in HRMECs. In conclusion, we provide evidence for the angiogenic role of GPNMB and demonstrate that silencing GPNMB may represent a therapeutic potential in the treatment of DR.

Interleukin-23 Mediates Osteoclastogenesis in Collagen-Induced Arthritis by Modulating MicroRNA-223.

Interleukin-23 (IL-23) plays a pivotal role in rheumatoid arthritis (RA). IL-23 and microRNA-223 (miR-223) are both up-regulated and mediate osteoclastogenesis in mice with collagen-induced arthritis (CIA). The aim of this study was to examine the association between IL-23 and miR-223 in contributing to osteoclastogenesis and arthritis. Levels of IL-23p19 in joints of mice with CIA were determined. Lentiviral vectors expressing short hairpin RNA (shRNA) targeting IL-23p19 and lisofylline (LSF) were injected intraperitoneally into arthritic mice. Bone marrow-derived macrophages (BMMs) were treated with signal transducers and activators of transcription 4 (STAT4) specific shRNA and miR-223 sponge carried by lentiviral vectors in response to IL-23 stimulation. Treatment responses were determined by evaluating arthritis scores and histopathology in vivo, and detecting osteoclast differentiation and miR-223 levels in vitro. The binding of STAT4 to the promoter region of primary miR-223 (pri-miR-223) was determined in the Raw264.7 cell line. IL-23p19 expression was increased in the synovium of mice with CIA. Silencing IL-23p19 and inhibiting STAT4 activity ameliorates arthritis by reducing miR-223 expression. BMMs from mice in which STAT4 and miR-223 were silenced showed decreased osteoclast differentiation in response to IL-23 stimulation. IL-23 treatment increased the expression of miR-223 and enhanced the binding of STAT4 to the promoter of pri-miR-223. This study is the first to demonstrate that IL-23 promotes osteoclastogenesis by transcriptional regulation of miR-223 in murine macrophages and mice with CIA. Furthermore, our data indicate that LSF, a selective inhibitor of STAT4, should be an ideal therapeutic agent for treating RA through down-regulating miR-223-associated osteoclastogenesis.

Overexpression of NCAPG in ovarian cancer is associated with ovarian cancer proliferation and apoptosis via p38 MAPK signaling pathway

BackgroundNon-SMC condensin I complex subunit G (NCAPG), a member of the subunit of condensin complex, is significantly overexpressed in various cancers and involved in the pathogenesis of cancers. However, the roles of NCAPG in ovarian cancer remain unclear.MethodsThe mRNA expression, overall survival, and disease-free survival of NCAPG in ovarian cancer were analyzed by GEPIA and KM plotter database, and the expression levels of NCAPG in OC tissues and cell lines were determined by qPCR and immunohistochemistry analysis. shRNA targeting NCAPG gene (sh-NCAPG) was utilized to knock down NCAPG expression in OVCAR3 and SKOV3 cells. Subsequently, CCK-8 assay, colony formation assay, transwell invasion assay and flow cytometric analysis were performed to detect the effect of NCAPG on OC cell proliferation, apoptosis, and invasion. Finally, western blot assays were performed to detect the mechanism of NCAPG in ovarian cancer.ResultsAnalysis using GEPIA and KM plotter database showed NCAPG was upregulated in ovarian cancer and negatively associated with the survival of OC patients. qPCR and immunohistochemistry analysis confirmed it was highly expressed in both ovarian cancer tissues and cells. The silencing of NCAPG inhibited OC cell proliferation and invasion, and induced cell apoptosis. Additionally, flow cytometric analysis revealed that NCAPG knockdown arrested the cell cycle at G2 and S phases. Furthermore, we also found that downregulation of NCAPG could suppress OC cell proliferation and invasion via activating the p38 MAPK signaling pathway.ConclusionOur results suggest that NCAPG exhibits an important role in the development and progression of ovarian cancer and implicates NCAPG as a potential therapeutic target in ovarian cancer.

IGFBP5 antisense and short hairpin RNA (shRNA) constructs improve erectile function by inducing cavernosum angiogenesis in diabetic mice.

The incidence of diabetic erectile dysfunction (ED) is rapidly increasing, and due to the severe angiopathy caused by diabetes, current drugs are ineffective at treating ED. Insulin-like growth factor-binding protein 5 (IGFBP5) promotes cell death and induces apoptosis in various cell types. To evaluate the effectiveness of IGFBP5 knockdown in improving erectile function in diabetic mice. Diabetes was induced by injecting streptozotocin (STZ) intraperitoneally into male 8-week-old C57BL/6 mice. Eight weeks after diabetes induction, mice were divided into four groups: a nondiabetic control group and three STZ-induced diabetic mice groups, which were administered intracavernous injections of phosphate buffered saline, scrambled control shRNA, or shRNA targeting mouse IGFBP5 (shIGFBP5) lentivirus particles. Two weeks later, we measured erectile function by electrically stimulating the bilateral cavernous nerve. To mimic diabetic angiopathy, primary cavernous endothelial cells (MCECs) from healthy mice were cultured and treated with glucose. IGFBP5 expression in MCECs or cavernous tissues were significantly increased under diabetic conditions, and knockdown of IGFBP5 induced MCECs angiogenic activity under high-glucose conditions. STZ-induced diabetic mice had reduced erectile function, but shIGFBP5 treatment resulted in significant improvements (to 90% of the nondiabetic control group level). Furthermore, in diabetic mice, numbers of cavernous endothelial cells, pericytes, and neuronal cells were increased by shIGFBP5 treatment, which also increased eNOS Ser1177 phosphorylation, decreased permeability and apoptosis of cavernous endothelial cells. In addition, IGFBP5 was found to mediate the AKT, ERK, p38 signaling pathways. Knockdown of IGFBP5 improved erectile function in diabetic mice by promoting cell proliferation and reducing apoptosis and permeability. Local inhibition of IGFBP5 expression may provide a new treatment strategy for diabetic ED and other ischemic vascular or neurological diseases.

Effects of smoking on the tissue regeneration-associated functions of human endometrial stem cells via a novel target gene SERPINB2

BackgroundSmokers directly inhale mainstream cigarette smoke, which contains numerous known and potential toxic substances, and thus, smoking is expected to have broad harmful effects that cause tissue injury and dysfunction. Interestingly, many studies have suggested that the recent decline in female fertility and increased rate of spontaneous abortion could be associated with increased smoking rates. Indeed, women that smoked for 10 years or more were reported to have a ~ 20% higher infertility rate than women that had never smoked. However, the reasons for the underlying harmful aspects of smoking on female fertility remain a matter of debate. Importantly, a previous study revealed that resident endometrial stem cell deficiency significantly limits the cyclic regeneration potential of endometrium, which, in turn, decreases successful pregnancy outcomes. In this context, we postulated that exposure to mainstream cigarette smoke extracts might decrease female fertility by inhibiting the functions of resident endometrial stem cells.MethodsWe investigated whether cigarette mainstream smoke exposure directly inhibits various tissue regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, pluripotency, and differentiation capacity in vitro. Next, we determined whether SERPINB2 mediates cigarette smoke-induced suppressive effects on various tissue regeneration-associated functions by depleting SERPINB2 expression with specific shRNA targeting SERPINB2. Mice were injected intraperitoneally with low (0.5 mg/kg) or high (1 mg/kg) doses of cigarette smoke extract (10 times for two weeks), and endometrial stem cells were then isolated from mice uterine tissues.ResultsWe found that exposure to cigarette smoke extracts remarkably suppressed various tissue regeneration-associated functions of endometrial stem cells, such as self-renewal, migration, multilineage differentiation ability, and pluripotency in vitro and in vivo by activating the SERPINB2 gene. Indeed, cigarette smoke-induced inhibitory effects on various endometrial stem cell functions were significantly abolished by SERPINB2 knockdown.ConclusionsThese findings provide valuable information on the harmful effects of cigarette smoking on resident endometrial stem cells and hopefully will facilitate the developments of promising therapeutic strategies for subfertile or infertile women that smoke cigarettes.

PTPRO knockdown protects against inflammation in hemorrhage shock-induced lung injury involving the NF-κB signaling pathway

BackgroundHemorrhage shock (HS) is characterized by decreased tissue oxygenation and organ damage due to severe blood loss. Protein tyrosine phosphatase receptor type O (PTPRO) is abnormally up-regulated in the rat lungs after trauma/HS.MethodsTo elucidate the regulatory mechanism of PTPRO in lung inflammation following HS, we established a rat model of HS via withdrawing blood by a catheter inserted into the femoral artery followed by resuscitation. The rats were infected with lentivirus harboring short hairpin RNA (shRNA) targeting PTPRO by intratracheal instillation.ResultsPTPRO was significantly up-regulated in rat lungs after HS. PTPRO knockdown enhanced epithelial integrity and reduced capillary leakage by up-regulating tight junction proteins zonula occludens-1 (ZO-1) and occludin (OCC) in the lungs. Besides, HS-induced myeloperoxidase activity and inflammatory cell infiltration was mitigated by PTPRO knockdown. The expression of inflammatory cytokines/chemokines (TNF-α, IL-6, MIP-2, MCP-1, and KC) in the lungs and bronchoalveolar lavage fluid was regressed after PTPRO knockdown. The nuclear factor kappa B (NF-κB) pathway was involved in HS-induced lung inflammation. PTPRO down-regulation inhibited the NF-κB pathway activation by suppressing the phosphorylation of NF-κB and its translocation from the cytoplasm into the nucleus in HS.ConclusionTaken together, we demonstrated that PTPRO knockdown may contribute to attenuating inflammation in HS-induced lung injury via inhibiting NF-κB pathway activation.

622 Regulation of XPC binding dynamics and global nucleotide excision repair by p63 and vitamin D receptor

p63 and the vitamin D receptor (VDR) play important roles in epidermal development and differentiation, but their roles and relationship in the response to ultraviolet (UV) radiation are unclear. Using TERT-immortalized human keratinocytes expressing shRNA targeting p63 in concert with exogenously applied siRNA targeting VDR, we assessed p63 and VDR’s separate and combined effect on nucleotide excision repair (NER) of UV-induced 6-4 photoproducts (6-4PP) with a focus on the XPC DNA damage recognition protein.

KNTC1 as a putative tumor oncogene in pancreatic cancer

PurposeRecent studies have demonstrated that kinetochore-associated protein 1 (KNTC1) plays a significant role in the carcinogenesis of numerous types of cancer. This study aimed to explore the role and possible mechanisms of KNTC1 in the development of pancreatic cancer.Methods and resultsWe analyzed differentially expressed genes by RNA sequencing in three paired pancreatic cancer and para-cancerous tissue samples and found that the expression of KNTC1 was significantly upregulated in pancreatic cancer. A Cancer and Tumor Gene Map pan-analysis showed that high expression of KNTC1 was related to poor prognosis in 9499 tumor samples. With immunohistochemical staining, we found that the high expression of KNTC1 in pancreatic cancer was related to pathological grade and clinical prognosis. Similarly, RT-PCR results indicated that the expression of KNTC1 was higher in three groups of pancreatic cancer cell lines (BxPC-3, PANC-1, and SW1990) than in normal pancreatic ductal cells. We introduced lentivirus-mediated shRNA targeting KNTC1 into PANC-1 and SW1990 cells and found that KNTC1 knockdown significantly decreased cell growth and increased cell apoptosis compared to the control group cells. Bioinformatic analysis of the cell expression profile revealed that differential genes were mainly enriched in the cell cycle, mitosis, and STAT3 signaling pathways, and co-immunoprecipitation confirmed an interaction between KNTC1 and cell division cycle associated 8.ConclusionsKNTC1 could be linked to the pathophysiology of pancreatic cancer and may be an early diagnostic marker of cervical precancerous lesions.

Targeting the SREBP-1/Hsa-Mir-497/SCAP/FASN Oncometabolic Axis Inhibits the Cancer Stem-like and Chemoresistant Phenotype of Non-Small Cell Lung Carcinoma Cells.

Background: Lung cancer remains a leading cause of cancer-related death, with an annual global mortality rate of 18.4%. Despite advances in diagnostic and therapeutic technologies, non–small cell lung carcinoma (NSCLC) continues to be characterized by a poor prognosis. This may be associated with the enrichment of cancer stem cells (CSCs) and the development of chemoresistance—a double-edged challenge that continues to impede the improvement of long-term outcomes. Metabolic reprogramming is a new hallmark of cancer. Sterol regulatory element-binding proteins (SREBPs) play crucial regulatory roles in the synthesis and uptake of cholesterol, fatty acids, and phospholipids. Recent evidence has demonstrated that SREBP-1 is upregulated in several cancer types. However, its role in lung cancer remains unclear. Objective: This study investigated the role of SREBP-1 in NSCLC biology, progression, and therapeutic response and explored the therapeutic exploitability of SREBP-1 and SREBP-1-dependent oncometabolic signaling and miRNA epigenetic regulation. Methods: We analyzed SREBP-1 levels and biological functions in clinical samples and the human NSCLC cell lines H441 and A549 through shRNA-based knock down of SREBP function, cisplatin-resistant clone generation, immunohistochemical staining of clinical samples, and cell viability, sphere-formation, Western blot, and quantitative PCR assays. We conducted in-silico analysis of miRNA expression in NSCLC samples by using the Gene Expression Omnibus (GSE102286) database. Results: We demonstrated that SREBP-1 and SCAP are highly expressed in NSCLC and are positively correlated with the aggressive phenotypes of NSCLC cells. In addition, downregulation of the expression of tumor-suppressing hsa-miR-497-5p, which predictively targets SREBP-1, was observed. We also demonstrated that SREBP-1/SCAP/FASN lipogenic signaling plays a key role in CSCs-like and chemoresistant NSCLC phenotypes, especially because the fatostatin or shRNA targeting of SREBP-1 significantly suppressed the viability, cisplatin resistance, and cancer stemness of NSCLC cells and because treatment induced the expression of hsa-miR-497. Conclusion: Targeting the SREBP-1/hsa-miR-497 signaling axis is a potentially effective anticancer therapeutic strategy for NSCLC.

Knockdown of CBX7 inhibits ferroptosis in rats with cerebral ischemia and improves cognitive dysfunction by activating the Nrf2/HO-1 pathway

Iron-dependent lipid peroxidation-induced ferroptosis has been implicated in the pathogenesis of cerebral ischemia. Ischemia-reperfusion (I/R) contributes to cognitive dysfunction. Chromobox7 (CBX7) was upregulated in neural progenitor cells, followed by hypoxia and ischemia, and the suppression of CBX7 protected against renal and cerebral I/R injuries. However, the role of CBX7 in ferroptosis and cognitive dysfunction in rats with cerebral ischemia has not yet been reported. In our study, first, rats with cerebral I/R were established through middle cerebral artery occlusion (MCAO). Intracerebroventricular injection of shRNA targeting CBX7 (sh-CBX7) enhanced brain water content, and reduced neurological deficit scores and infarct size in rats post MCAO. Second, rats were subjected to 4 trials per day for 4 consecutive days in a Morris water maze. The time to find the platform in with 1 min was recorded as escape latency. On day 5, the platform was removed to conduct a spatial probe test. Rats were given 1 min to find the position of the platform, and the times for crossing the target within 1 min were recorded. Crossing the platform time is the time spent in each quadrant. Data from the Morris water maze showed that cognitive dysfunction in ischemic rats was ameliorated by the injection of sh-CBX7, which decreased the swimming distance and escape latency of ischemic rats, increased the times for crossing the target and platform in the maze. Moreover, injection of sh-CBX7 down-regulated levels of MDA and ROS, while it up-regulated SOD and GSH-Px to repress the oxidative stress. Third, knockdown of CBX7 in ischemic rats decreased the concentration of the ferric ions and the protein expression of transferrin receptor 1 (TFR1), but enhanced the solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4), resulting in the inhibition of ferroptosis. Lastly, expressions of Nrf2, HO-1 and NAD(P)H:quinone oxidoreductase 1 (NQO1) were up-regulated in ischemic rats post sh-CBX7 injection. In conclusion, suppression of CBX7 attenuated I/R injury, improved cognitive dysfunction and inhibited ferroptosis through the activation of the Nrf2/HO-1 signaling pathway.

PB1952: EFFECTS OF KNOCKDOWN OF NUPR1 GENE ON THE PROLIFERATION AND APOPTOSIS OF MULTIPLE MYELOMA CELLS AND ITS MECHANISM

Background: Multiple myeloma is easy to relapse and incurable. It is necessary to further explore the pathogenesis to find new drugs Aims: To construct nuclear protein 1-short hairpin RNA (NUPR1-shRNA) expressing lentiviral vector targeting NUPR1 gene and investigate the effect of NUPR1 gene silencing on the proliferation and apoptosis of human multiple myeloma U266 and RPMI8226 cells and its related mechanisms. Methods: The normal human bone cells were used as control and the mRNA expression level of NUPR1 in multiple myeloma cell lines (U266 and RPMI8226) and MM patient specimens were detected by quantitative real-time PCR (qRT-PCR). Then the NUPR1 gene target shRNA plasmid was constructed and screen for the best target for transfected into U266 and RPMI8226 cells. The transfection efficiency was detected by flow cytometry. The interference effect of NUPR1 gene was detected by qRT-PCR and Western blotting. The cell proliferation was analyzed by CCK-8 assay. The cell apoptosis and cell cycle were determined by flow cytometry. Quantitative RT-PCR and Western blot were used to detect the expression of proliferation and apoptosis-related genes and proteins. Results: The mRNA expression levels of NUPR1 in MM cell lines (U266 and RPMI8226) and MM patient specimens were higher than that in normal bone cells. NUPR1-shRNA-expressing lentiviral vector was successfully constructed, and flow cytometry results shows that the transfection efficiency of both cell line reached over 80%. Quantitative RT-PCR and Western blot showed that NUPR1-shRNA2 lentiviral vector most effectively and as the best target. With NUPR1 gene interference, cell proliferation were significantly inhibited in U266 and RPMI8226 cells infected with the NUPR1-shRNA lentiviral compared with the control group. Compared with the control group, the apoptosis rate of U266 and RPMI8226 in shNUPR1 group were significantly increased (P<0.05). Flow cytometer indicated that the U266 and RPMI8226 cells in shRNA group were arrested in G0/G1 phase (P < 0.01). Compared with the control group, qRT-PCR and Western blot showed that the levels of Bcl-2 and PCNA were significantly down-regulated, and the level of PTEN was significantly increased in shNUPR1 group. Meanwhile, the expression of cleaved caspase-3, cleaved caspase-8 and cleaved caspase-9 protein were significantly higher in U266 and RPMI8226 cells infected with the NUPR1-shRNA lentiviral compared with the control group. Summary/Conclusion: The mRNA expression levels of NUPR1 in U266 and RPMI8226 cells were higher than that in normal bone cells, and the NUPR1-shRNA-expressing lentiviral vector was successfully constructed. Down-regulate the expression of NUPR1 in U266 and RPMI8226 cells can inhibit cell proliferation and promote its apoptosis.

Abstract 3159: CUX1 dosage-dependent regulation of hematopoietic stem and progenitor cells

Abstract The mechanism by which stem and progenitor cells commit to proliferate, self-renew, or differentiate towards different lineages remains unclear. Understanding this process is essential to decipher both normal tissue development and aberrant differentiation associated with malignant transformation. CUX1 is a non-clustered HOX-family transcription factor that is recurrently mutated in hematopoietic and solid tumours and regulates cell fate in multiple developmental systems. We reported that CUX1 is required for normal hematopoietic homeostasis of both murine and human hematopoietic stem and progenitor cells (HSPCs). To date, the transcriptional role of CUX1 in HSPCs has been unclear, partly because of the complexity of the Cux1 locus. Namely, Cux1 shares exons with a distinct gene, Casp, which encodes a highly expressed golgi-associated protein. As most transcriptomic studies combine Casp and Cux1 transcripts, the nuances of Cux1 expression are concealed. To identify the transcriptional mechanism by which CUX1 regulates HSPC functions, we compared scRNA-seq data of Kit+lin- HSPCs from transgenic mice expressing inducible shRNAs targeting Cux1, Cux1mid and Cux1low, to control counterparts expressing an shRNA targeting renilla luciferase. We demonstrate that separating Cux1 from Casp transcripts provides more power to detect the influence of Cux1 in scRNA-seq data analysis. While cell numbers within most HSPC sub-populations are largely unchanged after five days of Cux1 knockdown, the transcriptomes within multiple clusters are disrupted. The effect size is greatest in Cux1low cells, compared to Cux1mid, consistent with dosage dependent roles of CUX1 in HSPCs. Across sub-populations, Cux1 levels correlated with gene signatures of “stemness,” and Cux1 knockdown disrupted this gene signature in the core hematopoietic stem cell (HSC) population. Concomitantly, low levels of Cux1 led to upregulation of cellular metabolism and G2/M cell cycle transition pathways in core HSCs, indicating that CUX1 regulates stem cell quiescence at the hematopoietic apex. We are currently integrating this data with epigenetic datasets to determine the mechanism by which CUX1 regulates critical hematopoietic transcriptional programs. Overall, understanding the regulatory function of CUX1 will provide insight into how HSCs commit to different fates and may ultimately lead to novel cancer therapeutic interventions. Citation Format: Weihan Liu, Stephanie Konecki, Tanner Martinez, Megan McNerney. CUX1 dosage-dependent regulation of hematopoietic stem and progenitor cells [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 3159.

Abstract 4070: Caveolin-1 functions as a potential predictive biomarker for JNTX-101, an albumin encapsulated gemcitabine prodrug

Abstract Purpose: Human serum albumin (HSA) is an attractive candidate as a carrier in developing novel albumin-bound nanoparticle therapeutic drugs. Gemcitabine has been used in the treatment of solid tumors, including non-small cell lung, pancreatic, bladder, and breast cancers. Our previous work in pancreatic and lung cancer models demonstrates that caveolin-1 (Cav-1) enhances uptake of albumin and albumin-conjugated drugs. The aim of this study is to investigate whether the presence of Cav-1 expression alters therapeutic efficacy of JNTX-101, a novel albumin encapsulated gemcitabine prodrug. Experimental design: We first determined the treatment efficacy of JNTX-101 in a panel of 4 pancreatic/lung cancer cell lines (MIAPaCa-2, PANC-1, H23, and HCC15) by alamarBlue cell viability assay. We further established 4 pairs of Cav-1 isogenic cells stably expressing shRNA targeting Cav-1 (shCav-1) or scrambled control shRNA (shCtrl). We investigated whether the absence of Cav-1 expression alters sensitivity of JNTX-101 using alamarBlue cytotoxicity assay and western blotting in both high and low serum conditions. In addition, we tested whether JNTX-101 treatment can alter expression and cellular distribution of Cav-1 as well as other caveolae resident proteins, such as Cav-2 and Cavin-1. Results: Differences in Cav-1 expression resulted in different uptake of albumin prodrug conjugate and Cav-1 depletion attenuated sensitivity of the cells to JNTX-101. Decreased Cav-1 expression markedly reduced JNTX-101-induced apoptosis in a panel of cells in both high and low serum conditions, but particularly in low serum conditions. In addition, our data showed both serum starvation and JNTX-101 independently increased Cav-1 expression in a time-dependent manner (24hrs, 48hrs, 72hrs) similar to gemcitabine, with peak level at 48 hrs post treatment, while other caveolae resident proteins were minimally affected. Conclusions: Our preliminary results suggest that JNTX-101 effectively inhibits cell viability in pancreatic/lung cancer cells and Cav-1 expression dictates sensitivity to JNTX-101, at least partially through reduced apoptotic cell death. These data indicate that Cav-1 may be a potential predictive biomarker of JNTX-101 sensitivity, especially in nutrient deprived conditions. Further study will be performed to explore the impact of Cav-1 expression on JNTX-101 sensitivity in mouse models of pancreatic/lung cancer. Citation Format: Tiantian Cui, Sergio Corrales-Guerrero, Dan Maneval, Curtis Monnig, Patrick Kearney, Sam Ellis, Terence M. Williams. Caveolin-1 functions as a potential predictive biomarker for JNTX-101, an albumin encapsulated gemcitabine prodrug [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 4070.