Wild-type Control Cells Research Articles

Enhanced Gαq Signaling in TSC2-deficient Cells Is Required for Their Neoplastic Behavior.

Inherited or sporadic loss of the TSC2 gene can lead to pulmonary lymphangioleiomyomatosis (LAM), a rare cystic lung disease caused by protease-secreting interstitial tumor nodules. The nodules arise by metastasis of cells that exhibit features of neural crest and smooth muscle lineage ('LAM cells'). Their aberrant growth is attributed to increased activity of 'mechanistic target of rapamycin complex 1' (mTORC1), an anabolic protein kinase that is normally suppressed by the TSC1-TSC2 protein complex. The mTORC1 inhibitor rapamycin slows the progression of LAM, but fails to eradicate disease, indicating a role for mTORC1-independent mechanisms in LAM pathogenesis. Our previous studies revealed G-protein coupled urotensin-II receptor (UT) signaling as a candidate mechanism, but how it promotes oncogenic signaling in TSC2-deficient cells remained unknown. Using a human pluripotent stem cell-derived in vitro model of LAM, we now show hyperactivation of UT, which was required for their enhanced migration and pro-neoplastic signaling in a rapamycin-insensitive mechanism that required heterotrimeric Gαq/11 (Gαq). Bioluminescence resonance energy transfer assays in HEK 293T cells lacking TSC2 demonstrated selective and enhanced activation of Gαq and its RhoA-associated effectors compared to wild-type control cells. By immunoprecipitation, recombinant UT was physically associated with Gαq and TSC2. The augmented Gαq signaling in TSC2-deleted cells was independent of mTOR activity, and associated with increased endosomal targeting of p63RhoGEF, a known RhoA-activating effector of Gαq. These studies identify potential mTORC1-independent pro-neoplastic mechanisms that can be targeted for prevention or eradication of pulmonary and extrapulmonary LAM tumors.

DIFFERENTIAL CELL SURFACE PROTEOMICS OF FLOTILLIN-1, HMGB1 AND AMINOPEPTIDASE N IN H3 MUTATED PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract AIMS High-grade gliomas (HGG) are central nervous system tumour in children, where treatment options remain elusive, leaving children with a dismal prognosis. Histone H3.3 mutations, K27M and G34V/R have been identified as the two most common recurrent somatic mutations in paediatric HGG (pHGG). These mutations are present in 50% of pHGG compared to only 1% in adult gliomas. Our previous proteomic research on cell membrane protein through protein MS data analyses have identified different signatures of differentially expressed cell membrane proteins in H3-G34R/V mutated pHGG cells, in comparison to H3 wild type tumour cells in culture. Among these proteins we have identified flotillin-1, HMGB1 (High mobility group box 1) and aminopeptidase N. Flotillin-1 is associated with numerous malignancies and HMGB1 also functions as an oncogene and aminopeptidase N is a cell surface metalloprotease involved in tumour progression. Our aim is to study the expression and localisation of these cell surface proteins in H3 mutated and wild type tumour cells, to validate them as targets for developing novel therapeutics. METHOD Expression and localisation of flotillin-1, HMGB1 and aminopeptidase N in H3.3 mutated pHGG cell lines in comparison to H3 wildtype glioma and control cells was analysed, using different protein expression assays including immunofluorescence microscopy and western blotting. RESULTS Results showed statistically significant differential protein expression and localisation of these proteins. Flotillin-1 had increased cell membrane localization in H3 mutated cells. HMGB1 expression was increased, while aminopeptidase N expression was decreased in the H3 mutated cells in comparison to wildtype pHGG. CONCLUSION This new knowledge of the cell membrane proteomics of H3 mutated pHGG may help us utilise Flotillin- 1,HMGB1 and aminopeptidase N, as potential therapeutic targets for these unique H3 mutated children’s brain tumours.

9304 Integration of Glucocorticoid and Mineralocorticoid Receptor Signaling in Triple-negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

7873 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

9304 Integration Of Glucocorticoid And Mineralocorticoid Receptor Signaling In Triple-Negative Breast Cancer Models

Abstract Disclosure: S. Posani: None. C.A. Lange: None. Glucocorticoid receptors (GR) sense a convergence of host and cellular stress signaling to orchestrate an array of advanced cancer phenotypes associated with triple-negative breast cancer (TNBC) progression. The Lange lab reported p38 MAP kinase mediated phosphorylation of GR at serine 134 (Ser134) in response to tumor microenvironment-derived cytokines, such as TGF-beta-1. Phospho-Ser134-GR (p-Ser134-GR) upregulated gene sets that promote cancer cell survival, chemoresistance, altered metabolism, and migratory/invasive behavior in TNBC models. In addition to acting as ligands for GR, glucocorticoids also bind to and activate another closely related steroid hormone receptor - the mineralocorticoid receptor (MR). The elucidation of functional responses upon activation of MR remains an untapped area in breast cancer research. Our probe into public datasets (METABRIC, and TCGA) demonstrated significantly higher expression of MR transcripts in TNBC relative to luminal breast cancer. High expression of MR predicted worse overall survival in ERα-negative breast cancer patients compared to the low expressing cohort. Silencing of MR was carried out by siRNA-mediated knockdown and treatment with spironolactone, a competitive MR antagonist. Interestingly, MR knockdown significantly reduced the TGF-beta-1 induced migratory capacity of breast cancer cells compared to the wild-type control cells expressing endogenous MR. This biological phenotype of decreased TGF-beta-1 induced migration upon MR knockdown parallels the reduced migration we observed when the p38 MAPK site (Ser134 on GR was mutated to Ala in TNBC models. We therefore hypothesized that MR and GR may cooperate under the influence stress signaling in response to growth factors and cytokines abundantly present in the tumor-microenvironment. Preliminary data suggest that cytoplasmic p-Ser134-GR/MR complexes associate upon TGF-beta-1 treatment, while GR and/or MR ligands stimulate the formation of nuclear GR/MR complexes. We are further investigating cytokine- and stress-signal mediated regulation of GR/MR complexes and their functional role in TNBC biology. The overarching goal of this study is to elucidate if MR is a required GR binding-partner and a potential therapeutic target in GR- and cytokine-driven metastatic TNBC processes. Presentation: 6/2/2024

Interactions among Merlin, Arkadia, and SKOR2 mediate NF2-associated Schwann cell proliferation in human.

NF2-Related Schwannomatosis (previously referred to as Neurofibromatosis Type 2, or NF2) is a genetic-associated disease resulting from mutations in the gene, NF2. NF2 encodes the merlin protein, which acts as a tumor suppressor. Bilateral vestibular schwannoma (VS) is a hallmark of NF2. Although the exactly molecular mechanism mediating NF2-driven schwannomatosis remain unclear, it is known that defective Merlin protein functionality leads to abnormal cell proliferation. Herein, we utilized a human induced pluripotent stem cell (hiPSC)-based Schwann cell (SC) model to investigate the role of merlin in human SCs. SCs were derived from hiPSCs carrying a NF2 mutation (c.191 T > C; p. L64P), its isogenic wild-type control cell line, and a NF2 patient-derived hiPSC line. NF2 mutant SCs showed abnormal cellular morphology and proliferation. Proteomic analyses identified novel interaction partners for Merlin - Arkadia and SKOR2. Our results established a new model in which merlin interacts with Arkadia and SKOR2 and this interaction is required for the proper activation of the SMAD-dependent pathway in TGFβ signaling.

Nuclear receptor corepressor 1 levels differentially impact the intracellular dynamics of mutant thyroid hormone receptors associated with resistance to thyroid hormone syndrome

Thyroid hormone receptor α1 (TRα1) undergoes nucleocytoplasmic shuttling and mediates gene expression in response to thyroid hormone (T3). In Resistance to Thyroid Hormone Syndrome α (RTHα), certain TRα1 mutants have higher affinity for nuclear corepressor 1 (NCoR1) and may form stable complexes that are not released in the presence of T3. Here, we examined whether NCoR1 modulates intranuclear mobility and nuclear retention of TRα1 or RTHα-associated mutants in transfected human cells, as a way of analyzing critical structural components of TRα1 and to further explore the correlation between mutations in TRα1 and aberrant intracellular trafficking. We found no significant difference in intranuclear mobility, as measured by fluorescence recovery after photobleaching, between TRα1 and select RTHα mutants, irrespective of NCoR1 expression. Nuclear-to-cytoplasmic fluorescence ratios of RTHα mutants, however, varied from TRα1 when NCoR1 was overexpressed, with a significant increase in nuclear retention for A263V and a significant decrease for A263S and R384H. In NCoR1-knockout cells, nuclear retention of A263S, A263V, P389R, A382P, C392X, and F397fs406X was significantly decreased compared to control (wild-type) cells. Luciferase reporter gene transcription mediated by TRα1 was significantly repressed by both NCoR1 overexpression and NCoR1 knockout. Most RTHα mutants showed minimal induction regardless of NCoR1 levels, but T3-mediated transcriptional activity was decreased for R384C and F397fs406X when NCoR1 was overexpressed, and also decreased for N359Y in NCoR1-knockout cells. Our results suggest a complex interaction between NCoR1 and RTHα mutants characterized by aberrant intracellular localization patterns and transcriptional activity that potentially arise from variable repressor complex stability, and may provide insight into RTHα pathogenesis on a molecular and cellular level.

HGG-24. DIFFERENTIAL CELL SURFACE PROTEOMICS OF FLOTILLIN-1, HMGB1 AND AMINOPEPTIDASE N IN H3 MUTATED PAEDIATRIC HIGH-GRADE GLIOMAS

Abstract High-grade gliomas (HGG) are the deadliest central nervous system tumour in children. Despite this, treatment options remain elusive, leaving children with a dismal prognosis. Histone H3.3 mutations, K27M and G34V/R have been identified as the two most common recurrent somatic mutations in paediatric HGG (pHGG). These mutations are present in 50% of pHGG compared to only 1% in adult gliomas. Our previous proteomic research on cell membrane protein through protein MS data analyses have identified different signatures of differentially expressed cell membrane proteins in H3-G34R/V mutated pHGG cells, in comparison to H3 wild type tumour cells in culture. Among these proteins we have identified flotillin-1, HMGB1 (High mobility group box 1) and aminopeptidase N. Flotillin-1 is associated with numerous malignancies and HMGB1 also functions as an oncogene and aminopeptidase N is a cell surface metalloprotease involved in tumour progression. We have now verified the expression and localisation of these cell surface proteins in H3 mutated and wild type tumour cells, in order to validate as targets for developing novel therapeutics. Expression and localisation of flotillin-1, HMGB1 and aminopeptidase N in H3.3 mutated pHGG cell lines in comparison to H3 wildtype glioma and control cells was analysed, using different protein expression assays including immunofluorescence microscopy and western blotting. Experimental analyses showed statistically significant differential protein expression and localisation of these proteins. Flotillin-1 had increased cell membrane localization in H3 mutated cells.HMGB1 expression was increased, while aminopeptidase N expression was decreased in the H3 mutated cells in comparison to wildtype pHGG. This new knowledge of the cell membrane proteomics of H3 mutated pHGG may help us utilise Flotillin-1, HMGB1 and aminopeptidase N, as potential therapeutic targets for these unique H3 mutated children’s brain tumours.

The mechanistic role of connexin 43 in maturation of hiPSC cardiomyocytes

Abstract Funding Acknowledgements Type of funding sources: Public grant(s) – EU funding. Main funding source(s): ECR (European research council) and ReNew (the Novo Nordisk Foundation Center for Stem Cell Medicine supported by Novo Nordisk Foundation grants) Introduction Cardiomyocytes (CMs) differentiated from human induced pluripotent stem cells (hiPSCs) are functionally immature and this limits the application of these cells to cardiovascular research. Maturation can be improved by aggregating hiPSC-CMs into three-dimensional (3D) microtissues together with hiPSC-cardiac fibroblasts (hiPSC-CFs) and hiPSC-endothelial cells (hiPSC-ECs). These cardiac microtissues showed improved structural (increase of sarcomere length and organisation) and functional (more mature action potential profile) properties. This effect was only observed when the cardiac microtissues contained hiPSC-CFs. Since connexin 43 (CX43), the most ubiquitous connexin isoform in the heart, was significantly upregulated in hiPSC-CMs from cardiac microtissues, we hypothesise that mechanisms underlying hiPSC-CMs maturation involves the coupling of hiPSC-CMs with hiPSC-CFs through this protein. Purpose The aim of this study is to unravel the role of CX43 in the maturation of hiPSC-CMs in the context of 3D multicellular cardiac tissues. Methods We used the CRISPR/Cas9 technology to generate a CX43 knock-out (KO) hiPSC line. We designed two sgRNAs annealing to exon 2 of GJA1 gene, encoding CX43; indels generated by the non-homologous end joining after the Cas9 double strand break will result in a non-functional protein. Cells were differentiated in vitro into CMs, epicardial cells (EPI) and then CFs and characterized for the expression of cell-specific markers and compared with the wild-type parental control cells. Results and conclusions We screened 31 hiPSC clones and selected those in which both GJA1 alleles contained a deletion. Copy number variation analysis by ddPCR of the flanking regions confirmed that the deletion was specific and confined. The lack of GJA1 expression in the KO clones was confirmed by qPCR, while the lack of the CX43 protein was confirmed by immunofluorescence and Western Blot. This did not affect CX43-KO hiPSCs pluripotency state shown by the presence of pluripotent markers such as NANOG, OCT3/4, SSEA4. Additionally, CX43-KO hiPSCs were able to differentiate into the three germ layers. CX43-KO hiPSCs were successfully differentiated into hiPSC-CMs, -EPI, and -CFs. CX43-KO hiPSC-CMs and controls showed comparable gene expression of cardiac troponin T. Since immunofluorescent staining showed that CX43-KO hiPSC-EPI expressed the epicardial markers WT1 and ZO1, we continued to differentiate them into hiPSC-CFs, which expressed markers such as VIM and COL1A1, similar to the control. Differentiated CX43-KO hiPSC-CMs and CFs will be assembled into cardiac microtissues. Functional and structural assays that include patch-clamp and immunofluorescence will be performed on these microtissue to assess the effect of the CX43 KO on the maturation of hiPSC-CMs. Our results show that the lack of CX43 expression does not impede hiPSCs to differentiate into various cell types originating from the mesoderm germ layer.

Upregulated selenoprotein I during lipopolysaccharide-induced B cell activation promotes lipidomic changes and is required for effective differentiation into IgM-secreting plasma B cells.

The mechanisms driving metabolic reprogramming during B cell activation are unclear, particularly roles for enzymatic pathways involved in lipid remodeling. We found that murine B cell activation with lipopolysaccharide (LPS) led to a 1.6-fold increase in total lipids that included higher levels of phosphatidylethanolamine (PE) and plasmenyl PE. Selenoprotein I (SELENOI) is an ethanolamine phospholipid transferase involved in the synthesis of both PE and plasmenyl PE, and SELENOI expression was also upregulated during activation. Selenoi knockout (KO) B cells exhibited decreased levels of plasmenyl PE, which plays an important antioxidant role. Lipid peroxidation was measured and found to increase ∼2-fold in KO vs. wild-type (WT) B cells. Cell death was not impacted by KO in LPS-treated B cells and proliferation was only slightly reduced, but differentiation into CD138 + Blimp-1+ plasma B cells was decreased ∼2-fold. This led to examination of B cell receptors important for differentiation that recognize the ligand B cell activating factor, and levels of TACI (transmembrane activator, calcium-modulator, and cytophilin ligand interactor) (CD267) were significantly decreased on KO B cells compared with WT control cells. Vaccination with ovalbumin/adjuvant led to decreased ovalbumin-specific immunoglobulin M (IgM) levels in sera of KO mice compared with WT mice. Real-time polymerase chain reaction analyses revealed a decreased switch from surface to secreted IgM in spleens of KO mice induced by vaccination or LP-BM5 retrovirus infection. Overall, these findings detail the lipidomic response of B cells to LPS activation and reveal the importance of upregulated SELENOI for promoting differentiation into IgM-secreting plasma B cells.

The flavonoid luteolin reduces mutant huntingtin aggregation and cytotoxicity in huntingtin-mutated neuroblastoma cells

BackgroundHuntington's disease is an inherited progressive neurodegenerative disorder caused by an expansion of the polyglutamine tract leading to malformation and aggregation of the mutant huntingtin protein in the cell cytoplasm and nucleus of affected brain regions. The development of neuroprotective agents from plants has received considerable research attention. ObjectiveOur study aims to investigate the neuroprotective effects of luteolin and the mechanisms that underline its potential mediated protection in the mutant htt neuroblastoma cells. MethodsThe mutant htt neuroblastoma cells were transfected with 160Q, and the control wild-type neuroblastoma cells were transfected with 20Q htt for 24 h and later treated with luteolin. Cell viability was determined by MTT and PI staining in both groups, while western blotting was used to evaluate caspase 3 protein expression. Aggregation formation was assessed via immunofluorescence microscopy. Also, western blotting was utilized to measure the protein expression of mutant htt aggregated and soluble protein, Nrf2 and HO-1. The impact of Nrf2 on luteolin-treated neuroblastoma cells was assessed using small interfering RNAs. ResultsOur study reports that luteolin can protect cultured cells from mutant huntingtin cytotoxicity, evidenced by increased viability and decreased apoptosis. Also, luteolin reduced the accumulation of soluble and insoluble mutant huntingtin aggregates in mutant htt neuroblastoma cells transfected with 160Q compared to the control wild-type. The mutant htt aggregate reduction mediated by luteolin appeared to be independent of the Nrf2 –HO-1 antioxidant pathway. ConclusionLuteolin presents a new potential therapeutic and protective agent for the treatment and decreasing the cytotoxicity in neurodegenerative diseases such as Huntington's disease.

Plasmodium falciparum exploits CD44 as a coreceptor for erythrocyte invasion

AbstractThe malaria parasite Plasmodium falciparum invades and replicates asexually within human erythrocytes. CD44 expressed on erythrocytes was previously identified as an important host factor for P falciparum infection through a forward genetic screen, but little is known about its regulation or function in these cells, nor how it may be used by the parasite. We found that CD44 can be efficiently deleted from primary human hematopoietic stem cells using CRISPR/Cas9 genome editing, and that the efficiency of ex vivo erythropoiesis to enucleated cultured red blood cells (cRBCs) is not affected by lack of CD44. However, the rate of P falciparum invasion was reduced in CD44-null cRBCs relative to isogenic wild-type control cells, validating CD44 as an important host factor for this parasite. We identified 2 P falciparum invasion ligands as binding partners for CD44, erythrocyte binding antigen 175 (EBA-175) and EBA-140 and demonstrated that their ability to bind to human erythrocytes relies primarily on their canonical receptors, glycophorin A and glycophorin C, respectively. We further show that EBA-175 induces phosphorylation of erythrocyte cytoskeletal proteins in a CD44-dependent manner. Our findings support a model in which P falciparum exploits CD44 as a coreceptor during invasion of human erythrocytes, stimulating CD44-dependent phosphorylation of host cytoskeletal proteins that alter host cell deformability and facilitate parasite entry.

Synthesis and Evaluation of Clinically Translatable Targeted Microbubbles Using a Microfluidic Device for In Vivo Ultrasound Molecular Imaging.

The main aim of this study is to synthesize contrast microbubbles (MB) functionalized with engineered protein ligands using a microfluidic device to target breast cancer specific vascular B7-H3 receptor in vivo for diagnostic ultrasound imaging. We used a high-affinity affibody (ABY) selected against human/mouse B7-H3 receptor for engineering targeted MBs (TMBs). We introduced a C-terminal cysteine residue to this ABY ligand for facilitating site-specific conjugation to DSPE-PEG-2K-maleimide (M. Wt = 2.9416 kDa) phospholipid for MB formulation. We optimized the reaction conditions of bioconjugations and applied it for microfluidic based synthesis of TMBs using DSPE-PEG-ABY and DPPC liposomes (5:95 mole %). The binding affinity of TMBs to B7-H3 (MBB7-H3) was tested in vitro in MS1 endothelial cells expressing human B7-H3 (MS1B7-H3) by flow chamber assay, and by ex vivo in the mammary tumors of a transgenic mouse model (FVB/N-Tg (MMTV-PyMT)634Mul/J), expressing murine B7-H3 in the vascular endothelial cells by immunostaining analyses. We successfully optimized the conditions needed for generating TMBs using a microfluidic system. The synthesized MBs showed higher affinity to MS1 cells engineered to express higher level of hB7-H3, and in the endothelial cells of mouse tumor tissue upon injecting TMBs in a live animal. The average number (mean ± SD) of MBB7-H3 binding to MS1B7-H3 cells was estimated to be 354.4 ± 52.3 per field of view (FOV) compared to wild-type control cells (MS1WT; 36.2 ± 7.5/FOV). The non-targeted MBs did not show any selective binding affinity to both the cells (37.7 ± 7.8/FOV for MS1B7-H3 and 28.3 ± 6.7/FOV for MS1WT cells). The fluorescently labeled MBB7-H3 upon systemic injection in vivo co-localized to tumor vessels, expressing B7-H3 receptor, as validated by ex vivo immunofluorescence analyses. We have successfully synthesized a novel MBB7-H3 via microfluidic device, which allows us to produce on demand TMBs for clinical applications. This clinically translatable MBB7-H3 showed significant binding affinity to vascular endothelial cells expressing B7-H3 both in vitro and in vivo, which shows its potential for clinical translation as a molecular ultrasound contrast agent for human applications.

Abstract 2366: Inhibition of Ref-1/APE1 redox activity with APX3330 enhances Ref-1/APE1 protein unfolded conformation in human PDAC cells

Abstract Ref-1/APE1 (Redox factor-1, Ref-1; Apurinic/apyrimidinic endonuclease 1, APE1) is a redox signaling enzyme that biochemically converts several transcription factors (TFs), such as NF-kB, STAT3, HIF-1α from an oxidized to a reduced state, allowing TFs to bind DNA and activate various biological responses such as cell growth and inflammation. Ref-1 and its transcriptional targets are highly active in Pancreatic Ductal Adenocarcinoma (PDAC) which often presents with tumor metastasis and therapy resistance. The redox activity of Ref-1 is determined by the redox status of the relevant cysteine residues (Cys), particularly Cys residue 65 (C65), which is critical among seven Cys residues for Ref-1’s redox activity. Using CRISPR editing in PDAC cells, we changed the C65 to Ala (C65A) in Ref-1 to evaluate the effects of loss of Ref-1 redox activity on cell signaling pathways and how Ref-1 activity regulates critical transcription factors. Using Ref-1 redox inhibitor, APX3330, we previously investigated interactions between Ref-1 and APX3330 in a cell-free system in which Ref-1 underwent conformational changes in the presence of APX3330. This resulted in complete exposure of all seven Cys residues causing oxidation of the Ref-1 protein and redox signaling inactivity. However, how these events translate in the cell has not been completely established. Using a gel-based mobility shift assay, we examined the redox-modified Cys residues of Ref-1 in response to APX3330 in PDAC cell lines over time. We observed a gradual increase in labeling of Cys in Ref-1 following APX3330 treatment between 10min to 6h which returned to control levels at 48h in both wild-type control (Cas9) and C65A-expressing cells. This reduction in the redox status of Ref-1 was likely due to unfolding alteration like the cell-free system. To confirm this, both cell lines were treated with N-ethylmaleimide (NEM) prior to cell lysing, which blocks free thiols in the protein. We detected dramatic decreases in reduced forms of Ref-1 after APX3330 treatment (between 10min-6h) in both cell lines, demonstrating that C65 is not essential for the unfolded conformational changes of Ref-1. However, C65A-Ref-1 protein demonstrated a distinctly different redox pattern upon APX3330 treatment in comparison to WT Ref-1 in the Cas9 control line. C65A-expressing cells also had reduced mitochondrial function compared to Cas9 control PDAC cells. In vivo studies demonstrated significant reduction in tumor size, weight, and growth in C65A PDAC cells compared to the Cas9 control lines, further demonstrating the critical role of C65 in the Ref-1 redox signaling and downstream pathways including metabolism and proliferation. In conclusion, we demonstrate direct interactions between Ref-1 and APX3330 in PDAC cells and subsequent biological results confirm a critical involvement of Ref-1-C65 in the redox signaling and tumor phenotype. Citation Format: Mahmut Mijit, Olivia Babb, Silpa Gampala, Randall Wireman, Millie M. Georgiadis, Melissa L. Fishel, Mark R. Kelley. Inhibition of Ref-1/APE1 redox activity with APX3330 enhances Ref-1/APE1 protein unfolded conformation in human PDAC 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 2366.

Abstract 1575: Knocking out alpha synuclein causes decreased release of extracellular vesicles in melanoma cells

Abstract The purpose of this study is to understand the role of α-synuclein in the regulation of the trafficking and release of modulators of metastasis in melanoma. Metastasis causes melanoma-induced death and 5-year survival rates, subsequent to metastasis to regional lymph nodes or distal organs, of 9% and 4%, respectively, hence novel therapeutic targets are required to control metastasis. Emerging evidence suggests that α-synuclein, a 14.4 KDa protein, which is a key player in Parkinson’s disease, is significantly correlated with melanoma progression. Our hypothesis is that α-synuclein positively regulates the release of extracellular vesicles. Extracellular vesicles (EVs) have been shown to be important in tumor metastasis, immune modulation and chemoresistance. The rationale for this hypothesis is that α-synuclein is a unique membrane-associated catalyst of endocytosis and exocytosis, and, specifically, synuclein regulates neurotransmitter release through SNARE complex assembly and the dilation of the fusion pore. The objective of the study is to measure the concentration of extracellular vesicles in the conditioned media from wild type SK-MEL-28 melanoma cells, SNCA knock out cells, and rescued SNCA-KO cells (where α-synuclein is re-expressed in KO cells via lentivirus) using Nanoparticle Tracking Analysis. The expected result was that knocking out SNCA would decrease the number of EVs released per cell relative to wild type control cells. To this end, we grew wild type, SNCA-KO and rescued SNCA-KO cells for 48 hours at 37°C and collected conditioned media. EVs were extracted from each conditioned medium using the ExoQuick-TC kit (Systems Bioscience) and stored at -80°C. EVs were characterized by PAGE followed by western blotting for common EV-markers and EV samples were shipped to the University of Colorado for Nanoparticle Tracking. Here we report a significant reduction in the concentration of EVs in SNCA-KO cell lines compared to control cells (P=0.0123), an effect that was rescued upon re-expression of α-synuclein in knock-out cells (P<0.01). Unpublished data from our lab suggests that invasion and migration are significantly reduced in SNCA-KO melanoma compared to control cells, a pathological outcome that returns in rescued SNCA-KO cells. These observations point to considering α-synuclein as a therapeutic target for regulating the release of pro-metastatic factors in extracellular vesicles, an intervention that would have important health implications as a novel therapeutic approach in melanoma. Work is ongoing in our lab to elucidate the mechanistic details of how α-synuclein promotes EV release. Citation Format: Nirjhar M. Aloy, Michael W. Graner, Christina Coughlan, Sergey V. Slepenkov, Sahar Shekoohi, Stephan N. Witt. Knocking out alpha synuclein causes decreased release of extracellular vesicles in melanoma 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 1575.

3055 – METALLOTHIONEIN 1 (MT1) AS A TARGETABLE DEPENDENCY IN ACUTE MYELOID LEUKEMIA

Adult de novo acute myeloid leukemia (AML) is a blood cancer with poor prognosis. The heterogeneous nature of this disease motivates identification and understanding of specific dependencies within genetic and/or epigenetic subtypes of AML. The goal of this project is to understand the molecular mechanisms underlying AML development in the context of two recurrent and frequently co-mutated genes in human AML; the DNA methyltransferase DNMT3A and nucleophosmin NPM1. We used single-cell RNA-seq and cellHarmony analysis of our recently developed mouse model replicating DNMT3A and NPM1 mutations found in human AML to identify differentially expressed transcripts. Across four independently derived Dnmt3a;Npm1-mutant AML samples, metallothionein 1 (Mt1) was the top common, significantly upregulated transcript in the most primitive AML stem/progenitor cell compartment. MT-1 functions to protect against heavy metal toxicity, inflammation, oxidative stress, and has been implicated in cancer progression. This led us to hypothesize that upregulation of MT-1 is critical for growth and survival of DNMT3A;NPM1-mutant AML. First, we validated increased Mt1 expression using additional independent Dnmt3a;Npm1-mutant AML bone marrow and spleen samples compared to control wild-type cells. Next, using the DNMT3A;NPM1-mutant human AML cell line OCI-AML3, we found that MT-1 expression was increased 100-fold relative to a control human 293T cell line. To test the extent to which loss of Mt1 can inhibit growth of Dnmt3a;Npm1-mutant AML cells, we performed CRISPR-mediated knockout followed by ex vivo culture. We found that Mt1-knockout Dnmt3a;Npm1-mutant AML cells have reduced proliferation and expansion compared to control-targeted cells. Taken together, our study nominates MT-1 as a target towards the goal of treatment and prevention of DNMT3A;NPM1-mutant AML.

Abstract 1213: PDE10A as a novel target to suppress Wnt/β-catenin signaling and other oncogenic pathways in ovarian cancer

Abstract Objective: A leading theory for ovarian carcinogenesis proposes that the inflammation associated with incessant ovulation is a driver of oncogenesis. Consistent with this theory, epidemiological studies have shown that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease ovarian cancer risk. Previous studies suggest, however, that the antineoplastic activity of NSAIDs does not require the traditional cyclooxygenase (COX) enzyme inhibition, and rather may be exerted through phosphodiesterase (PDE) inhibition. PDEs represent a potentially unique chemopreventive target for ovarian cancer given that ovulation is regulated by cyclic nucleotide signaling. This study evaluates the effects of phosphodiesterase 10A (PDE10A) inhibition as a novel chemopreventive and therapeutic approach for ovarian cancer. Methods: We investigated the effects of PDE10A small molecule inhibitors, Pf-2545920 and MCI-030 (a novel non-COX inhibitory NSAID-derivative), and PDE10A knockout by CRISPR/Cas9 gene editing in various ovarian cancer cell lines using in vitro assays that measured cell proliferation, cell viability, cell cycle arrest, apoptosis, cell migration and invasion. Downstream signaling pathways affected by PDE10A inhibition and gene knockout (KO) were assessed by western-blotting, confocal microscopy, and RNA sequencing. Results: Analysis of The Cancer Genome Atlas (TCGA) ovarian cancer database and an institutional cohort of ovarian cancer patients revealed that high PDE10A mRNA expression was associated with significantly worse overall survival. PDE10A expression was also positively correlated with oncogenic and inflammatory signaling pathways in the TCGA dataset. PDE10A inhibition with Pf-2545920 or MCI-030 decreased ovarian cancer cell proliferation, while inducing cell cycle arrest and apoptosis. Using inhibitors to block PKA (H89) and PKG (KT5823) kinase activity, we demonstrated that the pro-apoptotic effects of PDE10A inhibition were mediated by activation of cGMP/PKG and cAMP/PKA signaling. SKOV3 and OV-90 PDE10A KO cells showed decreased colony formation, cell proliferation, and migration and invasion properties compared to their wild-type (WT) counterparts. Moreover, PDE10A inhibition decreased Wnt-induced β-catenin nuclear translocation as well as decreased EGF-mediated activation of RAS/MAPK and AKT pathways in ovarian cancer cells. RNA sequencing of SKOV3 PDE10A KO clones revealed that pathways associated with cancer, Wnt and TGF-β signaling were downregulated compared to WT control cells. Conclusions: Our data demonstrate that PDE10A has pro-tumorigenic effects in ovarian cancer cells and its high expression in ovarian cancer patients is associated with poor prognosis. Altogether, our results warrant future studies of PDE10A as a novel target for ovarian cancer chemoprevention and/or treatment. Citation Format: Rebecca M. Barber, Elaine Gavin, Alla Musiyenko, Wito Richter, Kevin J. Lee, Annelise Wilhite, Joel F. Andrews, Steve McClellan, Ileana Aragon, Antonio Ward, Xi Chen, Adam Keeton, Kristy Berry, Gary A. Piazza, Jennifer M. Scalici, Luciana Madeira da Silva. PDE10A as a novel target to suppress Wnt/β-catenin signaling and other oncogenic pathways in ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1213.

Phospholipase Cgamma Pathway-Dependent mTOR Signaling Controls IL-7-Mediated Early B Cell Development

Early B cell lymphopoiesis is intricately regulated by signals from various receptors including the pre-B cell receptor (pre-BCR) and the interleukin-7 receptor (IL-7R), but the molecular mechanism underlying the regulation is not fully understood. The Phospholipase Cγ (PLCγ) signaling pathway plays a critical role in antigen receptor-mediated activation of mature B cells, but the role of this pathway in cytokine function and early B cell development is completely unknown. Here we generated inducible PLCγ1/PLCγ2 double-deficient mice and found that the mutant mice exhibited a complete blockage of early B cell development at the pre-pro-B cell stage, similar to IL-7R-deficient but not pre-BCR-deficient mice. Of note, PLCγ1/PLCγ2 double-deficient mice had normal numbers of the HSC-containing Lin-c-Kit+Sca1+ (LSK) cells, myeloid progenitor-containing Lin-c-Kit+Sca1- (LK) cells and myeloid lineage cells compared to wild-type mice. PLCγ1/PLCγ2 double-deficient mice displayed a marked reduction in IL-7-induced rearrangement of the distal VHJ558 family gene segments. In addition, PLCγ1/PLCγ2 double deficiency altered the expression of lymphoid-lineage-affiliated genes in common lymphoid progenitors (CLPs) and the expression of B cell lineage-affiliated genes, IL-7 signaling pathway-regulated genes and cell cycle pathway-related genes in B cell progenitors. Further, PLCγ1/PLCγ2 double-deficient Lin- BM progenitors co-cultured with OP-9 stromal cells plus IL-7 failed to give rise to B cells whereas wild-type control BM progenitors generated B cells. PLCγ1/PLCγ2 double-deficient B progenitors exhibited a decrease in the level of IL-7-induced BrdU uptake compared to the corresponding wild-type control cells. Biochemically, the PLCγ pathway was activated upon IL-7 engagement in Rag1-deficient pro-B cells that lack the pre-BCR. PLCγ1/PLCγ2-double deficiency or inhibition of PLCγ pathway by PLC inhibitors impaired IL-7-induced activation of mTORC1 but not Stat5. Consistently, inhibition of the PLCγ pathway impaired IL-7-mediated metabolism of B cell progenitors. It's known that PLCγ activates the DAG/PKC-dependent and IP3/Ca2+-dependent signaling pathways, the two major downstream signaling cascades. We found that inhibition of the PKC-dependent, but not the Ca2+-dependent, pathway impaired IL-7-mediated B cell production and mTORC1 but not Stat5 activation. In addition, the PKC agonist PDBu induced mTORC1 activation in wild-type B cell progenitors, and stimulated 3H-thymidine incorporation in PLCγ1/PLCγ2 double-deficient B cell progenitors to the same extent as it did in wild-type control cells. Moreover, disruption of conventional Akt/TSC/Rheb signaling axis did not affect IL-7-controlled mTORC1 activation or early B cell development. Taken together, our current study for the first time demonstrates that the PLCγ pathway is critical for cytokine-mediated biological function, such as IL-7R-regulated early B lymphopoiesis, and this pathway directs IL-7R-mediated early B cell development through a novel and thitherto-unknown PLCγ/DAG/PKC-dependent but not conventional Akt/Rheb-dependent activation of mTORC1. DisclosuresNo relevant conflicts of interest to declare.

Normal Hematopoiesis Is Dependent on the Expression of U2af1, a Spliceosome Gene Commonly Mutated in MDS

Recently, somatic mutations in genes encoding factors involved in pre-mRNA splicing have been identified in up to 50% of myelodysplastic syndrome (MDS) patients. U2AF1, a spliceosome gene, has recurrent missense mutations in 11% of MDS patients. This gene is a key component of the spliceosome that recognizes the AG dinucleotide in the 3' splice acceptor site of pre-mRNA. U2AF1 mutations are always heterozygous and the wild-type allele is expressed, suggesting that cells may not tolerate biallelic mutations. Mice expressing mutant U2AF1 have altered hematopoiesis and RNA splicing, similar to mutant MDS patients but the role of wild-type U2AF1 in normal hematopoiesis has not been studied. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing is critical to our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse by flanking exon 2 with LoxP sites.We confirmed deletion at the genomic DNA and mRNA level in hematopoietic cells by crossing mice to various Cre-recombinase expressing models. We first generated U2af1flox/flox , U2af1flox/wt and U2af1wt/wt littermate control mice all carrying ERT2- Cre (and U2af1flox/flox without ERT2- Cre as a control). To study the hematopoietic cell-intrinsic effects of U2af1 deletion, we performed a non-competitive bone marrow transplant into lethally irradiated congenic recipient mice. Four weeks after engraftment, we treated mice with tamoxifen to active ERT2- Cre and monitored their survival and blood counts. Homozygous U2af1 KO mice (ERT2- Cre/ U2af1flox/flox ), but not other genotypes, became moribund (and were sacrificed) as early as only 6 doses of tamoxifen (Fig. 1A). The bone marrow cellularity in homozygous U2af1 KO mice was reduced (Fig. 1B). Analysis of mice at day 24 (from 1st tamoxifen dose) revealed anemia and a reduction in blood and bone marrow neutrophils, monocytes, and B-cells (p ≤ 0.01 ).We also generated mice carrying the interferon inducible Mx1- Cre to verify the observed phenotypes using an independent model. After a non-competitive bone marrow transplant into lethally irradiated congenic recipient mice, deletion of U2af1 was induced by polyinosinic:polycytidylic acid (poly I:C). The homozygous U2af1 KO mice, but not other genotypes, became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (decrease >1.7 fold) and a reduction in platelets (>9 fold) and white blood cell counts (>4.7 fold) in homozygous U2af1 KO mice compared to all other genotypes (p ≤ 0.001, n=5) . Peripheral blood neutrophils, monocytes, B-cells, and T-cells were reduced (p ≤ 0.001, n=5). Deletion of U2af1 also led to rapid bone marrow failure and a reduction in bone marrow neutrophils, monocytes, and B-cells as well as a significant depletion hematopoietic progenitor cells [ KL (Lin-, cKit+, Sca1-), and KLS (Lin-, cKit+, Sca1+)], (Fig. 1C). In order to study the hematopoietic cell-intrinsic effects of U2af1 deletion on stem cell function, we mixed equal numbers of whole bone marrow test cells (Ly5.2) with congenic control wild-type bone marrow competitor cells (Ly5.1/5.2) and transplanted them into lethally irradiated congenic recipient mice (Ly5.1). As early as 10 days following Cre-induction we observed a significant decrease in white blood cell count chimerism (Fig. 1D) and complete loss of homozygous U2af1 KO neutrophils and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001 ). Heterozygous U2af1 KO bone marrow had a modest competitive disadvantage compared to controls after 120 days (Figure 1D, p ≤ 0.05 ).Collectively, the data indicate that stem cell function, multilineage hematopoiesis and hematopoietic cell survival are dependent on U2af1 expression. In ongoing studies to understand why U2AF1 mutations are always heterozygous, we will cross the U2af1 KO mouse to existing transgenic mutant U2AF1(S34F)-expressing mice and determine whether mutant U2AF1-expression is sufficient to rescue cell survival and normal hematopoiesis when both endogenous U2af1 wild-type alleles are deleted. Proving that U2AF1 mutant cells are vulnerable to loss of the wild-type U2AF1 allele could provide a therapeutic vulnerability to mutant cells by selectively targeting the wild-type U2AF1 allele in heterozygous mutant cells. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mutated MYD88 Activates the BCR Component SYK and Provides a Rationale Therapeutic Target in Waldenstrom's Macroglobulinemia

Background: Mutations in the Toll receptor (TLR) pathway are highly prevalent in Waldenstrom's Macroglobulinemia (WM) and ABC DLBCL, wherein mutated MYD88 triggers NF-kB pro-survival signaling through BTK/IRAK. Activation of B-cell receptor (BCR) signaling can also be triggered by activating mutations in CD79A/B in ABC DLBCL, though are rare in WM (5-8%). Despite these findings, there is evidence for BCR activation in WM (Argyropoulos et al, Leukemia 2016). We hypothesized that crosstalk between TLR and BCR might account for aberrant BCR signaling in WM. We therefore performed PhosFLow studies on MYD88 and BCR signaling components and observed the hyperactivation of SYK (pY525-pY526) in MYD88 mutated WM (BCWM.1, MWCL-1) cells compared to wild-type MYD88 expressing control cells. We next treated MYD88 mutated WM cells with a peptide inhibitor of MYD88 homodimerization, and observed that the phosphorylation of SYK at tyrosine 525 and 526 (pY525-pY526) together with phosphorylation of BTK at tyrosine 223 (pY223) was markedly reduced. In contrast, the MYD88 inhibitor had no impact on either SYK or BTK activation in MYD88 wild-type Ramos cells. Knockdown of MYD88 by lentiviral transduction in MYD88 mutated BCWM.1 cells confirmed the reduction of SYK (pY525-pY526) by two shRNAs. Cell viability analysis by CellTiter-Glo® assay showed that the SYK inhibitor R406 was cytotoxic to MYD88 mutated WM cells at pharmacologically attainable drug levels (EC50 0.247 uM for BCWM.1; 0.315 uM for MWCL-1), versus >1 uM for MYD88 wild-type control cells. Furthermore, the combination of R406 with ibrutinib produced synergistic cytotoxicity in MYD88 mutated WM cells. Conclusion: Our findings demonstrate that in addition to TLR path activation, mutated MYD88 activates the BCR component SYK tyrosine kinase. These findings provide the rationale for combined therapeutic targeting of BCR and TLR pathways in MYD88 mutated WM. DisclosuresCastillo:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Millennium: Research Funding; Abbvie: Research Funding. Treon:Pharmacyclics: Consultancy, Research Funding.