A20 Cells Research Articles

Characterization of Vitronectin Effect in 3D Ewing Sarcoma Models: A Digital Microscopic Analysis of Two Cell Lines.

Ewing sarcoma (ES) is an aggressive bone and soft-tissue pediatric cancer. High vitronectin (VN) expression has been associated with poor prognosis in other cancers, and we aimed to determine the utility of this extracellular matrix glycoprotein as a biomarker of aggressiveness in ES. Silk fibroin plus gelatin-tyramine hydrogels (HGs) were fabricated with and without cross-linked VN and cultivated with A673 and PDX73 ES cell lines for two and three weeks. VN secretion to culture media was assessed using ELISA. Morphometric analysis was applied for phenotypic characterization. VN release to culture media was higher in 3D models than in monolayer cultures, and intracellular, intercellular, and pericluster presence was also observed. A673-HGs showed lower density of clusters but a proportion of larger clusters than PDX73-HGs, which presented low cluster circularity. The cluster density of A673-HGs without added VN was higher than with added VN and slightly lower in the case of PDX73-HGs. Furthermore, a culture time of three weeks provided no benefits in cluster growth compared to two weeks, especially in A673-HGs. These advances in 3D modeling and digital quantification pave the way for future studies in ES and other cancers to deepen understanding about intra- and intercellular heterogeneity and anti-adhesion VN therapies.

Formulation and characterization of a novel anti-human bone cancer supplement from silver nanoparticles green-synthesized using Allium sativum l. Leaf aqueous extract

Several studies have recently uncovered the ability of plants to enhance the therapeutic efficacies of Ag NPs on various carcinoma cells, particularly those associated with human bone tumors. Developing and formulating novel chemotherapy supplements or medications for the management of bone tumors in humans is a top research focus for both developing and developed nations. Our current study involved the development of a contemporary chemotherapeutic medication utilizing silver nanoparticles (AgNPs) combined with an aqueous extract of Allium sativum L. for the purpose of treating bone tumors in humans. AgNPs were characterized using XRD, TEM, UV–Vis, and FE-SEM. The peaks detected at 2θ values of 77.4, 65.1, and 37.8 were associated with the crystal planes 311, 220, and 111, respectively. To examine the antioxidant characteristics of AgNPs, the DPPH assay was employed with BHT serving as the positive control. The MTT assay was employed to examine the anti-human bone tumor and cytotoxicity properties of AgNPs on bone cancer cells, including A-673 and HS-729 (rhabdomyosarcoma), MHH-ES1 and CADO-ES1 (Ewing’s sarcoma), MG-63 and HOS (osteosarcoma), as well as CH-3573 and SW-1353 (chondrosarcoma). The DPPH assay demonstrated comparable antioxidant capacities among AgNPs, and butylated hydroxytoluene. The anti-human bone tumor properties and cell viability of silver nanoparticles exhibited a dose-dependent decrease against CH-3573, SW-1353, MG-63, HOS, MHH-ES1, CADO-ES1, HS-729, and A-673 cells, while showing no cytotoxic effects on the normal cell line. The viability of the cells and their response to the nanoparticles varied based on the dosage administered. The silver nanoparticles exhibited IC50 values of 154, 67, 129, 110, 124, 107, 70, and 81 µg/ml against H-3573, SW-1353, MG-63, HOS, MHH-ES1, CADO-ES1, HS-729, and A-673 cell lines, respectively. The HOS revealed the most effective outcome regarding anti-human bone tumor efficacies when treated with AgNPs, surpassing the results observed in other cell lines. Based on the aforementioned results, it is possible to utilize the silver nanoparticles with A. sativum extract for the management of various human bone tumors, particularly human bone rhabdomyosarcoma, human bone chondrosarcoma, human bone osteosarcoma, and human bone Ewing’s sarcoma.

Eco-friendly, highly interpenetrated and slightly swollen pHEMA hydrogel foam for durable underwater superoleophobicity and emulsion separation

Despite the emergence of hydrogels as ideal candidates for preparing the superhydrophilic materials for emulsion separation, their structural stability and swelling still hinder their long-term use, mainly due to structure defects after swelling. Herein, differing from the common modification, the eco-friendly poly 2-hydroxyethyl methacrylate (pHEMA) hydrogel foam was designed and synthesized via a one-step strategy by using the high internal phase emulsion (HIPE) template method, which endowed it with a highly interpenetrated porous structure. Unlike the normal swellable hydrogels such as poly(N-isoproplyacrylamide) (PNIPAM) hydrogel, or modified hydrogel coatings, the pHEMA hydrogel foam displayed stable structure and underwater superoleophobicity after 20 d of immersion in water. The pHEMA hydrogel foam could separate different kinds of highly surfactant-stabilized oil-in-water (O/W) emulsions with a high separation efficiency of 99.3% for liquid paraffin emulsion obtained solely under gravity-driven. Additionally, it exhibited excellent antifouling performance and long-term acid/alkali tolerance over 100 h without decrease in emulsion separation efficiency (98.0%, oil/water ratio of 99:1) and permeation flux (over 2000 L·m−2·h−1) attributed to its stable bulky structure. Moreover, the pHEMA hydrogel foam demonstrated high cell viability of 96.87% and 95.96% after culturing the 3T3 clone A31 cells in the pHEMA hydrogel foam for 24 h and 48 h, respectively, indicating good biocompatibility. Hence, our work provides a new design to develop an eco-friendly bulk hydrogel foam that achieves stable structure and performance for emulsion separation.

Abstract 129: Understanding the molecular drivers that regulate Ewing sarcoma cell fate

Abstract Like other translocation-positive sarcomas, Ewing sarcoma (ES) harbors a pathognomonic fusion protein (FP) capable of widespread epigenetic reprogramming that can lead cells to acquire a high-grade, undifferentiated phenotypic state. To better understand how this might occur, we employed a CRISPR knockout (CRISPR-KO) model of A673, targeting exon 4 of the N-terminus EWSR1 portion of the FP and generated a clonal pool (EWSR1 CRISPR KO). We used single-cell RNA and ATAC sequencing (scMultiome) to identify the transcriptional and chromatin changes after the FP knockout (FP KO). We identified several clusters of ES cell heterogeneity. Since the EWSR1-KO is a pooled population, we used known FP-regulated genes to delineate cells bearing the FP KO from wild-type (FP WT) cells. We observed robust expression of FP-activated genes, PRKCB, and LIPI, in many clusters suggesting regular FP function (FP WT). Conversely, FP-repressed genes, LOX, and IGFBP3 were elevated in a single cluster containing only EWSR1-KO cells. This suggests the FP was successfully knocked out in this cluster (FP KO). That FP KO cluster also lost transcriptional resemblance to ES. A granular identification with cell lines confirmed that all clusters except FP KO were identified as A673 cells. However, the FP KO resembled cell lines with a fibroblastic phenotype. We analyzed enriched motifs and observed no significant difference between the FP KO and FP WT for EWSR1::FLI1. However, the FP KO cells were enriched for motifs in the Fos/Jun family and Smad2/3, which have roles in EMT and mesenchymal differentiation. The FP-KO cells significantly expressed the Fos/Jun family of proteins. Next, we identified peak-gene associations within a ± 500 kb window around the TSS. When we analyzed peaks linked to genes expressed in FP-WT, we found enrichment of the Fos/Jun protein family (p < 1e-34), Ets family, and GGAA-microsatellites. However, the FP-KO were strongly enriched for Fos/Jun protein family (p < 1e-540) with no change in Ets family motif. Knocking out the FP in the A673 cell line significantly shifted the ES lineage. We observed peaks linked to genes positively associated or regulated by the FP were enriched for Fos/Jun family, Ets family, and GGAA-microsatellites. There may be a cooperative interaction between these TFs that affects the genes linked to the FP. The fact that FP loss enriched the Fos/Jun family motifs, but not Ets family or GGAA-microsatellites, suggests that FP loss activates FP-repressed genes and enables chromatin remodeling to open previously inaccessible DNA binding sites. Citation Format: Danh Truong, David McCall, Clement Agyemang, Sandhya Krishnan, Alexander Lazar, Joseph Ludwig. Understanding the molecular drivers that regulate Ewing sarcoma cell fate [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 129.

Abstract 1918: LX-101, a novel, clinical stage, payload-bearing targeted therapy directed to the insulin-like growth factor receptor (IGF-1R), demonstrates potent preclinical anti-tumor activity against multiple cancer types with well-established ties to the IGF-1/IGF-1R pathway

Abstract Objective: LX-101 is a next-generation, targeted therapy directed to the insulin-like growth factor 1 receptor (IGF-1R). LX-101 consists of a proprietary IGF-1 variant coupled to a cytotoxic methotrexate payload. LX-101 was previously evaluated in Phase 1 trials of adult patients with advanced, pretreated cancers, where it was well-tolerated and demonstrated single agent activity. Neither a dose limiting toxicity, nor a maximum tolerated dose was reached, leaving potential room for additional dose escalation and schedule optimization. Multiple aggressive cancers of unmet need have genetic alterations affecting the IGF-1R pathway and/or high IGF-1R expression that correlates with poor outcomes, including certain cancers of the breast, head and neck, Ewing’s sarcoma, osteosarcoma, and others. Prior attempts at inhibiting IGF-1R with non-payload bearing naked monoclonal antibodies or small molecules produced a range of clinical outcomes including some partial and complete responses. None of these agents, however, were ultimately approved, potentially due to suboptimal potency that allowed for escape mechanisms, including redundant signaling pathways, and the lack of enrichment for select tumor types with strong ties to the IGF-1/IGF-1R pathway. Herein, we investigated the anti-tumor activity of the payload-bearing agent LX-101 against IGF-1R-involved cancer cell lines. Methods: Cell lines were incubated with LX-101 (~1.6 - 2500nM). Cell viability was assessed by CellTiter-Glo after 4 days. IC50s were calculated using GraphPad PRISM software. Results: LX-101 displayed potency against head and neck cancer (FaDu, IC50 = 9 nM), triple negative breast cancer (BT-20, IC50 = 17 nM), Ewing’s sarcoma, including EWSR1-FLI1 gene fusion-positive cell lines RD-ES (IC50 = 10 nM), A-673 (IC50 = 14 nM), and SK-ES-1 (IC50 = 29 nM) and EWSR1-ERG gene fusion-positive cell line CADO-ES1 (IC50 = 14 nM). LX-101 was also potent against PAX3-FOXO1 gene fusion positive alveolar rhabdomyosarcoma (SJCRH30, IC50 = 23 nM), osteosarcoma (143B, IC50 = 6 nM; HOS, IC50 = 7 nM; U2OS, IC50 = 32 nM), and neuroblastoma (SK-N-AS, IC50 = 16 nM; IMR-32, IC50 = 20 nM; SH-SY5Y, IC50 = 30 nM). Conclusion: These results demonstrate that LX-101 has potent preclinical anti-tumor activity against multiple cancer cell lines with well-established ties to the IGF-1/IGF-1R pathway, including with different genetic and epigenetic alterations. These data support further clinical development of LX-101 in IGF-1R-involved cancers. New clinical trials are planned. Citation Format: Matt Hoberman, Arkadiusz Z. Dudek. LX-101, a novel, clinical stage, payload-bearing targeted therapy directed to the insulin-like growth factor receptor (IGF-1R), demonstrates potent preclinical anti-tumor activity against multiple cancer types with well-established ties to the IGF-1/IGF-1R pathway [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1918.

Experimental model of primary intraocular lymphoma based on BALB/CaNn strain and A20 cells is optimal for investigational research.

The purpose of this project was to compare the characteristics of two experimental murine models of primary intraocular lymphoma (PIOL) and determine which experimental model is most suitable for further investigational research to elucidate the pathophysiology of PIOL and to find new therapeutical strategies. In both experimental models PIOL was induced in immunocompetent mice with intravitreal injection of syngeneic B-cell lymphoma cell lines. Murine strain C3H/HeN and cell line 38C13 were used in the first model and BALB/CaNn mice and cell line A20 in the second model. During the experiments, thorough clinical evaluation (using photo documentation, ultrasonography, and MRI) and histological evaluation were performed. In both models, the percentage of PIOL development was high, reaching nearly 80%. Disease progression was faster in C3H/HeN with exophthalmos occurring on average on day 10. Vitreous involvement was a predominant sign in the clinical presentation of this group. In BALB/CaNn mice exophthalmos occurred on average on day 22. The predominant clinical sign in the BALB/CaNn group was tumorous infiltration of the retina, optic disc, and tumorous retinal detachment. Slower progression of the disease in BALB/CaNn mice, greater possibility to examine the retina due to mild vitreous involvement, and later occurrence of exophthalmos makes this strain more suitable for further investigational research.

Enhanced Antipediatric Sarcoma Effect of Everolimus with Secukinumab by Targeting IL17A.

In this study, we explored the therapeutic potential of everolimus, an mTOR inhibitor, in a patient-derived xenograft (PDX) of rhabdomyosarcoma, the most prevalent malignant pediatric sarcoma. In addition, rhabdoid tumor cell line A-204 and Ewings sarcoma cell line A-673 were cultured to assess the in vitro effect of everolimus. Furthermore, the cell-derived xenograft (CDX) of A-673 was established and treated with everolimus in vivo. IHC and Western blotting were performed to detect the expressions of pertinent proteins. Results showed that everolimus intervention had limited inhibitory effect on PDX tumor growth compared with cyclophosphamide. Nevertheless, everolimus treatment significantly influenced the phosphorylation levels of S6 kinase beta 1 (S6K1) and eIF4E-binding protein 1 (p-4E-BP1), resulting in the inhibition of angiogenesis in vitro and in vivo. Interestingly, everolimus led to an upregulation in the level of IL17A in sarcoma cells. Notably, when secukinumab, a mAb of IL17A, was combined with everolimus, it synergistically enhanced the inhibitory effect of everolimus on sarcoma cell proliferation in vitro and on the growth of PDX or CDX xenograft tumors in vivo. Importantly, this combination therapy did not affect the mTOR signaling. These results indicate that everolimus exerts an antipediatric sarcoma effect by inhibiting mTOR signal. However, everolimus induces sarcoma cells to produce IL17A, which promotes tumor cell survival and counteracts its antipediatric sarcoma effect. The combination of secukinumab effectively eliminates the effects of IL17A, thereby improving the therapeutic efficacy of everolimus in the context of pediatric sarcomas.

Additive manufacturing of wet-spun chitosan/hyaluronic acid scaffolds for biomedical applications

Additive manufacturing (AM) holds great potential for processing natural polymer hydrogels into 3D scaffolds exploitable for tissue engineering and in vitro tissue modelling. The aim of this research activity was to assess the suitability of computer-aided wet-spinning (CAWS) for AM of hyaluronic acid (HA)/chitosan (Cs) polyelectrolyte complex (PEC) hydrogels. A post-printing treatment based on HA chemical cross-linking via transesterification with poly(methyl vinyl ether-alt-maleic acid) (PMVEMA) was investigated to enhance the structural stability of the developed scaffolds in physiological conditions. PEC formation and the esterification reaction were investigated by infrared spectroscopy, thermogravimetric analysis, evolved gas analysis-mass spectrometry, and differential scanning calorimetry measurements. In addition, variation of PMVEMA concentration in the cross-linking medium was demonstrated to strongly influence scaffold water uptake and its stability in phosphate buffer saline at 37 °C. The in vitro cytocompatibility of the developed hydrogels was demonstrated by employing the murine embryo fibroblast Balb/3T3 clone A31 cell line, highlighting that PMVEMA cross-linking improved scaffold cell colonization. The results achieved demonstrated that the developed hydrogels represent suitable 3D scaffolds for long term cell culture experiments.

Withania Somnifera Extract Mitigates Experimental Acute Graft versus Host Disease Without Abrogating Graft Versus Leukemia Effect.

Acute graft versus host disease (aGvHD) is the major contributor of nonrelapse mortality in alloHSCT. It is associated with an inflammatory immune response manifesting as cytokine storm with ensuing damage to target organs such as liver, gut, and skin. Prevention of aGvHD while retaining the beneficial graft versus leukemia (GvL) effect remains a major challenge. Withania somnifera extract (WSE) is known for its anti-inflammatory, immune-modulatory, and anticancer properties, which are appealing in the context of aGvHD. Herein, we demonstrated that prophylactic and therapeutic use of WSE in experimental model of alloHSCT mitigates aGvHD-associated morbidity and mortality. In the prophylaxis study, a dose of 75 mg/kg of WSE offered greatest protection against death due to aGvHD (hazard ratio [HR] = 0.15 [0.03-0.68], P ≤ .01), whereas 250 mg/kg was most effective for the treatment of aGvHD (HR = 0.16 [0.05-0.5], P ≤ .01). WSE treatment protected liver, gut, and skin from damage by inhibiting cytokine storm and lymphocytic infiltration to aGvHD target organs. In addition, WSE did not compromise the GvL effect, as alloHSCT with or without WSE did not allow the leukemic A20 cells to grow. In fact, WSE showed marginal antileukemic effect in vivo. WSE is currently under clinical investigation for the prevention and treatment of aGvHD.

Low Dose Total Body Irradiation Improves CD19 CAR-T Expansion, Persistence, and Trafficking, Leading to Enhanced Efficacy in Large B-Cell Lymphoma

Anti-CD19 chimeric antigen receptor T-cell (CART19) is associated with high response rates in large B-cell lymphoma (LBCL). However, a significant subset of patients relapses due to several mechanisms that include poor expansion, lack of CART19 persistence, and immunosuppressive tumor microenvironment. We hypothesized that low dose total body irradiation (LDTBI) may interfere with these mechanisms and thus enhance the efficacy of CART19 cells in LBCL. To test this hypothesis, we utilized a syngeneic mouse model of LBCL, as they are immunocompetent, to best emulate patients treated in clinic. This model involves transplanting BALB/c mice with GFP/Luciferase transduced mouse derived B cell lymphoma (A20) cells and generating murine CART19 (mCART19). We tested mCART19 cytotoxicity in vitro against A20 cells with or without radiation. We found that exposing A20 cells to 1 Gy radiation prior to co-culturing them with mCART19 led to a significant improvement of the mCART19 cytotoxicity (E:T ratio 2.5:1; p=0.029). This is consistent with our in vitro findings for human LBCL cell lines, where weobserved that exposing LBCL cell lines to 1 Gy radiation significantly improved human CART19 cytotoxicity (E:T ratio 2.5:1: OCI-Ly10 cells: p=0.009; SU-DHL-4 cells: p=0.048). Next, we assessed the activity of mCART19 cells in vivo. Animals were lymphodepleted using cyclophosphamide (Cy) administered 24 hours before mCART19 treatment. LDTBI at 1 Gy was delivered 4 hours before mCART19 administration. In addition to evaluating the benefit of single LDTBI prior to mCART19, we also delivered a second dose of LDTBI 9 days after mCART19 treatment. Thus, we evaluated 6 cohorts as follows: (1) Untreated n=4, (2) Cy/LDTBI n=5 (3) Cy/LDTBI/LDTBI n=3, (4) Cy/mCART19 n=5, (5) Cy/LDTBI/mCART19 n=5, (6) Cy/LDTBI/mCART19/LDTBI n=5. Adding LDTBI to Cy provided a minor improvement compared to untreated controls (median overall survival [OS]= 21 days vs 16.5 days; p=0.009). As expected, Cy/mCART19 improved median OS compared to controls (median OS= 35 day; p=0.003). Strikingly, 8/10 of the mice treated with Cy/LDTBI/mCART19 remained in complete remission (CR) at 6 months post-treatment ( p<0.001), Figure 1. Since one dose of LDTBI prior to mCART19 was very effective, adding the second dose of LDTBI did not result in additional survival benefit. Peripheral blood (PB) was monitored regularly, we observed a 2.8-fold increase of mCART19 expansion in the Cy/LDTBI/mCART19 cohort when compared with the Cy/mCART19 cohort ( p<0.001) 9 days post treatment. Importantly, bone marrow aspirates were performed 6 months post treatment in animals that remained alive, demonstrating sustained B-cell aplasia in 7/8 mice (Cy/LDTBI/mCART19 cohorts) which strongly suggests mCART19 persistence. One animal showed B-cell recovery but remained in CR with no relapse suggesting potential cure. Mice that received Cy/mCART19 with no LDTBI showed evidence of B-cell reappearance 7 days prior to overt relapse. Next, we assessed mCART19 trafficking into the tumor tissues by establishing new cohorts of (1) Cy/mCART19 n=4, (2) Cy/LDTBI/ mCART19 n=4 and harvesting tumor-infiltrated spleen and liver 3 days post treatment. Flow cytometry revealed that LDTBI cohort had significant increase in the amount of mCART19 in the spleen and the liver (3.15-fold; p=0.041, and 2-fold; p=0.012, respectively) compared to Cy/mCART19 cohort, suggesting that LDTBI improves mCART19 tumor-trafficking ability. We have previously reported that exposing human LBCL cells to 1Gy radiation in vitro increases expression of FAS when compared to non-irradiated controls and that FAS blockade impairs CART19 cytotoxicity. Hence, we evaluated the expression of FAS in murine LBCL A20 cells harvested from the syngeneic mouse model 3 days post LDTBI. Consistently, we observed 3.1-fold increase in FAS expression, ( p=0.033). In conclusion, our results show a novel and efficacious approach to harnessing LDTBI with CART19. Based on our findings, LDTBI enhances the efficacy of CART19 leading to improved survival in a syngeneic mouse model. Mechanistically, radiation improves CART19 efficacy by boosting CART19 expansion, persistence, trafficking into tumor sites, and upregulation of FAS in tumor cells, pointing to a clinically translatable benefit.

An Idiotype Vaccine Strategy Utilizing Targeted NGS and Cell Free Protein Synthesis System Ensures Identification and Production of Human Idiotype Antigens, and Shows Promising Results in Preclinical Models of B-Cell Lymphoma with a Novel TLR7/8 Agonist-Based Adjuvant and Anti-PD-L1 Antibody

Background: Non-Hodgkin lymphoma (NHL) is one of the most common hematological malignancies in the world. Despite advances in treatment options, such as chimeric antigen receptor T cells (CAR-T) or bispecific antibodies, many patients still recur after complete remissions (CR) or do not achieve CR. Since B cell malignancies arise from clonal expansion, the B cell receptor (BCR) expressed on the cell surface, idiotype, has been studied as a tumor-specific antigen. Several trials have evaluated idiotype vaccines in clinical setting, but failed to demonstrate efficacy. Limitations have arisen from the uncertainty of BCR sequence confirmation, long turnaround time in manufacturing idiotype antigen, and the absence of strong immune modulators. Here, we utilized targeted next generation sequencing (NGS) for BCR, cell-free protein synthesis (CFPS) system for idiotype antigen manufacturing, and a novel liposomal TLR7/8 agonist-based adjuvant to optimize idiotype vaccine. Methods: To generate the idiotype antigens as single-chain variable fragment (scFv) from A20 lymphoma cell line and biopsy samples from NHL patients, variable regions of BCR heavy and light chain were sequenced by targeted NGS. Total RNA was isolated from A20 cells and tumor tissues from patients, and reverse transcription-polymerase chain reaction (RT-PCR) was performed using constant region-specific primers, resulting in the 1 st strand cDNA. Subsequently, the 2 nd strand cDNA was synthesized using V-gene specific primers and sequenced using an Illumina MiSeq sequencer. We applied targeted NGS to patients' tumor tissues from Asan Medical Center and Seoul National University Hospital to understand whether this method works in human tissues as well. The identified sequence was cloned into a linear DNA template and expressed by CFPS system. We optimized the CFPS system and purification process of idiotype antigen scFv from A20 cell line and human tissues. The liposomal adjuvant ProLNG-001 (TLR7/8 agonist 140 μg) and ProLNG-002 (TLR7/8 agonist 70 μg + TLR4 agonist 5 ug) were synthesized by microfluidics method. Idiotype antigen A20-scFv 50 μg was given alone or mixed with ProLNG-001 or -002. To investigate the efficacy of the idiotype vaccine, BALB/c mice were subcutaneously injected with 5×10 5 of A20 cells. Mice were subcutaneously vaccinated 4 times at 1-week intervals starting from day 2 after tumor inoculation. Atezolizumab 10 mg/kg was administered intraperitoneally 3 times a week for 3 weeks in the idiotype vaccine + immune checkpoint inhibitor combination group (Figure 1a). Results: Targeted NGS has confirmed BCR sequences from A20 cell line and dominant malignant B cell clone from patients' tumor with a success rate of over 90% when sufficient volume (more than 500 mm 3) of biopsy sample was provided for RNA extraction. Also, we were able to express the idiotype scFv antigens determined from 18 NHL patients' tumor samples using CFPS system with a 100% success rate (Figure 1b). The untreated mice (Group 1) showed a tumor growth pattern similar to that of Group 2 (vaccination with antigen only). Vaccination with idiotype antigen mixed with liposomal adjuvants, ProLNG-001 (Group 3) and ProLNG-002 (Group 4), significantly inhibited tumor growth. The combination of anti-PD-L1 antibody and idiotype antigen mixed with ProLNG-002 (Group 5) generated even better overall anti-tumor responses with 14 out of 15 mice showing no residual tumor at day 34 after tumor inoculation. Conclusion: Taken together, the idiotype vaccine strategy using targeted NGS for BCR, CFPS system for idiotype antigen manufacturing, and TLR7/8-agonist-based liposomal adjuvants showed highly effective results in the preclinical syngeneic A20 tumor model. Furthermore, the usage of immune checkpoint inhibitors combined with idiotype vaccine in B cell lymphoma may exert synergistic effects. The CFPS system can be applied to swift, robust and personalized manufacturing of idiotype antigen for individuals with different idiotype sequence in a GMP setting, which was not possible in prior studies. These results support further development of the idiotypic vaccine as a treatment option for non-Hodgkin's B cell lymphomas.

The Immune Checkpoint Siglec-15 in Promoting Immune Dysregulation in Non-Hodgkin's Lymphomas

Outcomes for patients with relapsed or refractory (r/r) hematologic malignancies remain dismal despite therapeutic advances over the last several years. This is particularly striking in patients with r/r aggressive mature B-cell lymphomas such as Diffuse Large B-cell Lymphoma (DLBCL) and Burkitt's Lymphoma (BL). With use of intensive multiagent chemo-immunotherapy, DLBCL, which accounts for approximately 10-20% of pediatric Non-Hodgkin's Lymphoma (NHL) cases and is the most common subtype in adults, is highly curable with 5-year event free survival >75%. However, approximately 30-40% of adults diagnosed with DLBCL will become refractory to or relapse after first-line treatment. Yet even with intensive salvage regimens, r/r DLBCL remains a clinical challenge. There remains an unmet need to identify specific defects in the immune response, among other pathways, that drive lymphomagenesis that will in turn inform the development of novel therapeutics to improve survival in this population of patients. Siglec-15 (Sig-15), a member of the sialic acid binding immunoglobulin-like lectin family of proteins, has recently been identified as a critical immune suppressor that is highly expressed in human cancers and intra-tumoral myeloid cells. Importantly, recent reports have demonstrated inhibition of Sig-15, has a restorative effect on local anti-tumor immune responses and abrogated tumor progression. While reported in solid malignancies and acute leukemias, a role for Siglec-15 in promoting disease progression in aggressive B-cell lymphomas has not yet been described. We have found higher expression of SIGLEC15 in DLBCL cell lines compared to normal B cells at the RNA level, through analyses of the publicly available ONCOMINE database. This was confirmed at the protein level by western blot demonstrating higher Sig-15 expression in various lymphoma cell lines (RAJI, SUDHL6, OCI-LY10, HBL-1) compared to healthy donor PBMCs. Further, immunohistochemistry evaluating Sig-15 expression was performed on a tumor microarray consisting of 139 cases of DLBCL from adult patients along with validation samples from 15 primary pediatric lymphoma cases (DLBCL, BL and Anaplastic Large Cell Lymphoma (ALCL)). Sig-15 was found to be highly expressed, with histologic scores ranging from weak (1+) to strong (3+) in 124 out of the 139 adult cases (Figure 1A) and in all pediatric lymphoma samples but with distinct staining patterns observed in the aggressive B-cell lymphomas. Specifically, Sig-15 appears to be highly expressed and associated with the cell membrane in most DLBCL and Burkitt's lymphoma. Expression is more variable in ALCL, primarily elevated in the cytoplasm at low levels and/or in macrophages (Figure 1A). There is heterogeneity in Sig-15 expression with preliminary analysis of RNA-seq data from a cohort of 693 DLBCL patient samples demonstrates higher levels in the germinal center B-cell (GCB) versus activated B-cell molecular (ABC) subtype of DLBCL when compared to healthy controls (Kruskal-Wallis, *p = 0.041). Finally, using a well-established murine lymphoma model, A20 cells were stably transduced with control, non-silencing shRNA (shNS) or shRNA against Sig-15 and injected into un-irradiated immune competent (wild type, WT) or immune deficient ( Rag1 -/-) BALB/C mice and monitored for signs of lymphoma development. Knockdown of Sig-15 in A20 cells abrogates disease progression in WT but not immune deficient mice (Log-rank, **p <0.0001, n=10 mice/group), consistent with a role for Sig-15 in immune evasion in lymphoma (Figure 1B). Together, these data implicate Sig-15 as an immune checkpoint that may be inhibited therapeutically to promote an immune response to lymphoma cells.

Combination Epigenetic Therapy Using YF2, a First-in-Class Histone Acetyltransferase Activator, Restores Immunogenicity in Diffuse Large B-Cell Lymphoma

Diffuse large B-cell lymphoma (DLBCL) is the most common type of non-Hodgkin lymphoma. Molecular studies have identified epigenetic dysregulation as a key driver in germinal center (GC)-derived DLBCL. Mutations in CREBBP/p300 and EZH2 often co-occur and lead to transcriptional repression and decreased immunogenicity as demonstrated by reduced major histocompatibility complex (MHC) expression. Our lab previously reported that YF2, a first-in-class histone acetyltransferase activator discovered at Columbia University, induces cytotoxicity in both cell and murine models of DLBCL. Other epigenetic therapies, such as tazemetostat and belinostat, are approved for use in follicular and T-cell lymphoma, respectively, but have limited single agent activity in DLBCL. We hypothesize that treatment with YF2 in combination with tazemetostat or belinostat increases transcriptional activity and restores tumor immunogenicity in CBP/p300- and EZH2-mutated DLBCL. We utilized several DLBCL cell lines to assess YF2 combination therapy: OCI-Ly7 (CBP/p300 WT, EZH2 WT), SU-DHL-4 (CBP/p300 WT, EZH2 GOF), A20 (CBP/p300 -/-, EZH2 WT), and SU-DHL-6 (CBP/p300 -/-, EZH2 GOF). Cells were treated on day 0 with single agents or with YF2 in combination with either tazemetostat (YF2 + taz) or belinostat (YF2 + bel), retreated on day 3, and collected on day 6 for flow cytometry (n = 4) and western blot (n = 2) analyses. MHC class I/II and β₂ microglobulin (B2M) expression were measured as markers of immunogenicity. To preliminarily assess YF2 combination therapy in vivo, immunocompetent BALB/c mice were engrafted with syngeneic A20 cells, immunized, and segregated (n = 2-4) into treatment groups: control (saline), tazemetostat (300 mg/kg twice daily x 28 days), belinostat (40 mg/kg once daily x 7 days), YF2 (50 mg/kg once daily x 28 days), YF2 + taz, and YF2 + bel. Blood and spleen samples were collected on day 29 to measure T- and B-cell activity via flow cytometry. All fold changes are expressed relative to the corresponding control. Combination therapy generally increased MHC-I, -II, and B2M expression across all cell lines as measured via flow cytometry. In double-mutated SU-DHL-6 cells, MHC-I and -II expression increased 2.0- ( P < 0.01) and 3.1-fold ( P = 0.04), respectively, following YF2 + taz treatment, and increased 1.3- ( P = 0.06) and 4.5-fold ( P < 0.01) after YF2 + bel ( Figure 1). Expression of B2M increased 2.3-fold ( P = 0.01) with YF2 + taz and 1.6-fold ( P = 0.07) with YF2 + bel. Interestingly, YF2 combination therapy was similarly effective in non-mutated OCI-Ly7 cells: MHC-I, -II, and B2M expression increased 1.9- ( P < 0.01), 3.0- ( P < 0.01), and 2.4-fold ( P < 0.01), respectively, following YF2 + taz treatment, and increased 1.5- ( P = 0.01), 3.3- ( P < 0.01), and 2.4-fold ( P < 0.01) with YF2 + bel. Similar expression trends were observed via western blots, but to a lesser degree; MHC-I, -II, and B2M protein increased 1.3-, 1.4-, 1.6-fold with YF2 + taz, and increased 1.1-, 1.6-, and 1.2-fold following YF2 + bel treatment in SU-DHL-6 cells. The combination therapies were tolerated by the mice and decreased tumor size. Splenic MHC-I expression increased 1.2-fold with YF2 + taz and 1.4-fold with YF2 + bel, whereas MHC-II increased 1.3-fold for both combination therapies ( Figure 2). Similarly, circulating B cells displayed increased MHC-I and -II expression with combination therapy. Treatment with YF2 + taz increased the number of circulating CD4 + T cells 1.5-fold and the number of CD8 + T cells 3.5-fold, while YF2 + bel increased the number of central memory and effector memory T cells by 1.6- and 1.9-fold, respectively. To understand the effect of combination therapies on B cell development in the GC, splenic B cells were evaluated and exhibited a 1.9-fold increase of cells in the light zone following YF2 + taz treatment, and a 1.2-fold increase after YF2 + bel exposure. The number of splenic plasma cells increased 1.4- and 1.5-fold following YF2 + taz and YF2 + bel treatments, respectively ( Figure 2). These findings suggest that combination epigenetic therapy may restore homeostatic B cell function and promote B cell cycling through the GC. In summary, these data propose YF2 combination epigenetic therapy as a means to increase immunogenicity in CBP/p300- and EZH2-mutated DLBCL. While our preliminary mouse experiment provided promising initial results, an expanded study is underway to statistically confirm the exciting trends that we observed.

Photoablation with the Aurolase System Reduces T Cell Exhaustion and Synergizes with Immunotherapies in Lymphoma

Introduction: Immunotherapy is less active in non-Hodgkin lymphoma due to immune evasion from the tumor microenvironment. For diffuse large B cell lymphoma (DLBCL), the overall response rates (ORR) for immune checkpoint inhibitors (ICIs) were low (4-18%). Bispecific antibodies (BiAb), such as FDA-approved Epcoritamab and Glofitamab, had a complete response (CR) rate of 39% for DLBCL. Methods that alter local and systemic immune microenvironments are needed to improve the response to immunotherapy. Unlike radiation, photothermal therapy (PTT) can generate a strong T-cell activation signal. PTT is created by irradiating specially designed gold nanoparticles with near-infrared (NIR) light. The nanoparticles absorb the light and transfer the energy into heat, leading to very localized ablations. The local high heat stimulates a cascade of pro-inflammatory cytokines, including IL-6, IL-1β, TNF-α, G-CSF, GM-CSF, and CCL2. We previously found that PTT with toll-like receptor 9 agonists had a significantly higher CD8 to CD4 ratio, cytotoxic T cell to regulatory T cell ratio, and effector memory T cells in a murine lymphoma model. The AuroLase system, which uses silica gold nanoshells and an FDA-cleared laser, has been undergoing clinical trials for various solid tumors. Here, we explore the systemic immune effects of the PTT device in a lymphoma murine model and evaluate its synergistic effects with ICIs and BiAbs. Methods: The AuroLase system and AuroShells were provided by Nanospectra. The lymphoma model uses A20 cells implanted in Balb/c mice. Single flank tumors were used for immunophenotyping, and a dual tumor model was used to evaluate the systemic anti-lymphoma effects of PTT and combination therapies. PTT (day 0) was performed by injecting 20ul of nanoshells intratumorally and then irradiating the tumor with the laser at 4W for 30sec-1min. Spleens were collected on days (d) 1, 4, and 8 for immunophenotype by flow cytometry. For systemic immune response, we used a dual tumor model and treated the left side with PTT on d0. Anti-PD1 (aPD1) antibodies and the CD20xCD3 BiAbs were injected intraperitoneally on d1, 3, and 5. Results: In a single tumor lymphoma model, PTT did not change the size of the spleen or the total T cell count, including CD4, CD8, and regulatory T cell (Treg), compared to tumor naïve or tumor-bearing mice. However, the treatment reduced splenocyte T cell PD1 levels on d1 & 4 post-ablation, down to the level of tumor naive mice, and returned to elevated levels by d8, similar to untreated mice. CD4 and CD8 T cell PD1 levels dropped from 3.49% to 0.78% and 28% to 0.45%, respectively, post-ablation (p<0.001). Treg PD1 levels also dropped from 11.5% to 2.4%. There was a reduction of the central memory T cells (CD44 hi CD62 hi) population on d1&4 post-ablation but started to increase by d8. The PD1 expression pattern was similar to the prior, with a reduction on d1&4 and recovery by d8. The effector memory T cells (CD44 hi CD62 lo) had an increased population by d8 with the same PD1 reduction pattern. The B cell population didn't change, but there was an increase in B cell maturation (CD80 +) on d1&4. As for the myeloid cells, there is a sharp increase in dendritic cell population on d1 and increased maturation/activation on d1&4. There was an increased population of macrophages on d4. There was a reduction of myeloid-derived suppressor cells on d1. In the dual tumor model, the treated tumor had a dramatic loss of tumor volume due to PTT. The untreated tumor did not have a tumor reduction with PTT alone. However, when combined with anti PD1 (aPD1) therapy with just three doses, the untreated side had significant tumor growth reduction. By day 20, the PTT+aPD1 group had a tumor size of 378 mm 3, while the PTT alone and aPD1 alone groups were 1207 and 985 mm 3, respectively. There was also a survival advantage with PTT+aPD1. Similarly, we found that PTT+BiAb significantly delayed tumor growth compared to PTT only. Survival data is still ongoing. Conclusion: PTT in lymphoma is understudied and can effectively activate an anti-lymphoma response. PTT resulted in a marked reduction in PD1 expression on all T cells and activated several myeloid immune cells. We found that PTT combined with aPD1 therapy or BiAbs had improved tumor suppression in a lymphoma model. This system provides a platform and opportunity to translate PTT into the clinics.

Ferroptosis Inhibition Generates TCF-1 + CAR-T Cells with Enhanced Persistence and Cytotoxicity

[Introduction] Long-term in vivo persistence of chimeric antigen receptor (CAR) T-cells is associated with sustained response after CAR-T cell therapy against hematological malignancies. Ferroptosis is a novel form of programmed cell death that refers to iron-dependent regulated cell death resulting from lipid peroxidation and consequent cytomembrane rupture. The iron, lipid, amino acid, and glutathione metabolisms strictly govern the beginning and execution of ferroptosis. Recent studies demonstrated that ferroptosis limited longevity of virus-specific memory T cells (Wang Y: Nat Immunol 2022). However, the role of ferroptosis in CAR-T cells remains to be clarified. We herein explored the role of ferroptosis in CAR-T cells both in vitro and in vivo. [Methods] For CAR-T cell generation, purified T cells from naïve mice were stimulated with anti-CD3/CD28 antibodies for 48 hrs and then incubated with retroviral vector encoding anti-CD19 CAR (1D3-28Z.1-3; Addgene) for another 48 hrs, in the presence or absence of 32 µM of ferrostatin-1 (Fer-1), an inhibitor of the iron-dependent production of lipid hydroperoxide, or 10 µM of deferoxamine (DFO), an iron chelator. Flow cytometric analyses of phenotype of CAR-T cells, accumulation of reactive oxygen species (ROS), lipid peroxidation, and cytokine production were performed 96 hrs after T-cell stimulation. ROS were quantified using CellROX Green dye, and lipid peroxidation was evaluated using Lipid Peroxidation Assay Kit (abcam). [Results] First, we tested whether CAR-T manufacturing process could induce ferroptosis. We found a substantial accumulation of ROS in CAR-T cells compared to their pre-culture levels, along with a significant increase in lipid peroxidation, indicating that manufacturing process induces ferroptosis of CAR-T cells. Next, we tested whether ferroptosis inhibition by the lipid peroxidation inhibitor Fer-1, could impact the quality of CAR-T cells. When added to the culture, Fer-1 significantly reduced lipid peroxidation in CAR-T cells and significantly increased the proportion of CAR-T cells with a progenitor phenotype, TCF-1 +Tim-3 -, which is known to be associated with longevity of CAR-T cells. Importantly, ferroptosis inhibition significantly enhanced proliferative capacity and cytotoxicity against syngeneic A20 B-cell leukemia in vitro. Next, we tested whether ferroptosis inhibition by the iron chelator DFO could improve CAR-T cell functions. Similar to the effects of Fer-1, DFO mitigated lipid peroxidation of CAR-T cells in association with increased TCF-1 +Tim-3 - population, while it did not affect ROS accumulation. DFO-treated CAR-T cells demonstrated enhanced cytotoxicity against A20 cells compared with control CAR-T cells, confirming that ferroptosis inhibition improves anti-tumor effects of CAR-T cells. Finally, we examined whether ferroptosis inhibition could enhance antitumor effects of CAR-T cells in vivo. Naïve BALB/c mice were intravenously (i.v.) injected with 1.0 x 10 6 luciferase-expressing A20 cells on day 0, followed by i.v. injection of 1.0 x 10 5 Fer-1- or DFO-treated CAR-T cells or control CAR-T cells 7 days later. In mice injected with Fer-1-treated CAR-T cells, the numbers of CAR-T cells in peripheral blood on day 16 was significantly higher than those in mice injected with control CAR-T cells, suggesting that ferroptosis inhibition improved the persistence of CAR-T cells in vivo ( Fig. 1). In vivo bioluminescence imaging showed that both Fer-1-treated and DFO-treated CAR-T cells demonstrated significantly improved anti-tumor effects compared to control CAR-T cells ( Fig. 2). [Conclusions] We for the first time showed that ferroptosis limits the persistence and anti-tumor effects of CAR-T cells, while ferroptosis inhibition induces TCF-1 +Tim-3 - CAR-T cells and improves the therapeutic effect of CAR-T cell therapy. Figure 1. The number of CAR-T cells in the peripheral blood on day 16 after tumor inoculation. Fer-1, ferrostatin-1; *, P < 0.05; P < 0.01. Figure 2. The fold change of the total flux at each timepoint relative to the total flux of the baseline in the mice injected with DFO-treated CAR-T cells, Fer-1-treated CAR-T cells, and control CAR-T cells on day 7 after tumor inoculation. Fer-1, ferrostatin-1; DFO, deferoxamine; *, P < 0.05.

Evaluation of Cytotoxicity of Hyaluronic Acid/Chitosan/Bacterial Cellulose-Based Membrane.

Novel wound dressing materials are required to non-cytotoxic with a viable cell ratio of above 92%. Herein, the cytotoxicity of hyaluronic acid/chitosan/bacterial cellulose-based (BC(CS/HA)) membranes are evaluated and compared to that of alginate/chitosan/bacterial cellulose-based (BC(CS/Alg)) membranes was investigated. Multilayer membranes with up to ten CS/HA or CS/Alg layers were prepared using the layer-by-layer (LBL) method. Scanning electron microscopy showed that the diameters of the fibers in the BC(CS/Alg) and BC(CS/HA) membranes were larger than those in a BC membrane. The cytotoxicity was analyzed using BALB-3T3 clone A31 cells (mouse fibroblasts, 1 × 104 cells/well). The BC(CS/HA)5 and BC(CS/HA)10 membranes exhibited high biocompatibility, with the cell viabilities of 94% and 87% at 5 d, respectively, compared to just 82% for the BC(CS/Alg)5 and BC(CS/Alg)10 membranes with same numbers of layers. These results suggested that BC(CS/HA)5 is a promising material for wound dressings.

CD44 Modulates Cell Migration and Invasion in Ewing Sarcoma Cells.

The chimeric EWSR1::FLI1 transcription factor is the main oncogenic event in Ewing sarcoma. Recently, it has been proposed that EWSR1::FLI1 levels can fluctuate in Ewing sarcoma cells, giving rise to two cell populations. EWSR1::FLI1low cells present a migratory and invasive phenotype, while EWSR1::FLI1high cells are more proliferative. In this work, we described how the CD44 standard isoform (CD44s), a transmembrane protein involved in cell adhesion and migration, is overexpressed in the EWSR1::FLI1low phenotype. The functional characterization of CD44s (proliferation, clonogenicity, migration, and invasion ability) was performed in three doxycycline-inducible Ewing sarcoma cell models (A673, MHH-ES1, and CADO-ES1). As a result, CD44s expression reduced cell proliferation in all the cell lines tested without affecting clonogenicity. Additionally, CD44s increased cell migration in A673 and MHH-ES1, without effects in CADO-ES1. As hyaluronan is the main ligand of CD44s, its effect on migration ability was also assessed, showing that high molecular weight hyaluronic acid (HMW-HA) blocked cell migration while low molecular weight hyaluronic acid (LMW-HA) increased it. Invasion ability was correlated with CD44 expression in A673 and MHH-ES1 cell lines. CD44s, upregulated upon EWSR1::FLI1 knockdown, regulates cell migration and invasion in Ewing sarcoma cells.

Targeting CD47 in lymphoid malignancies with CAR T cells.

2513 Background: Despite major therapeutic advances of chimeric antigen receptor (CAR) T cells directed against CD19 in relapsed and refractory non-Hodgkin lymphoma (NHL), two thirds of patients experience relapse within the first two years. Nearly one third of these relapses are associated with CD19 antigen-loss escape thus leveraging alternate CAR targets may improve outcomes. CD47, an anti-phagocytic “don’t eat me” receptor known to be overexpressed in NHL, is an ideal CAR target as it is potentially indispensable for tumor progression. Methods: CD47-CAR binding domains were engineered either as a camelid single variable heavy chain nanobody (CD47-VHH-CAR) or as the CD47 binding domain of cognate receptor SIRPα (SIRPα-CAR), each combined with a CD28 costimulatory domain and a CD3ζ activation domain with two mutated ITAMs (1XX). In vitro co-culture assays were performed with lymphoid mouse tumor cell lines BM185 and A20. For in vivo experiments, syngeneic BALB/c mice were sublethally irradiated for lymphodepletion then injected intravenously with 1x105 luciferase+ BM185 cells. Two days later, mice were injected with 2x106 CAR T cells, and bioluminescence imaging was performed to monitor tumor progression. For bio-localization experiments, CAR T cells were co-transduced with a separate vector encoding luciferase. Results: CD47-CAR T cell expansion in vitro was hindered due to fratricide, which can be rescued by CD47 CRISPR knockout. Both CD47-VHH-CAR and SIRPa-CAR T cells exhibited potent and specific in vitro lysis of BM185 and A20 cells. CD47-VHH-CAR T cells showed transient anti-leukemia activity, however, survival was not significantly prolonged. T cell bio-localization analysis revealed that, compared to CD19-CAR T cells, CD47-VHH-CAR T cells exhibited prolonged retention in the thorax with minimal bone marrow trafficking, suggesting CD47 antigen-specific pulmonary sequestration, and inferior expansion. SIRPα-CAR T cells engineered to secrete pro-inflammatory cytokine IL-18 significantly improved CAR T cell expansion, resulting in bone marrow trafficking and clearance of BM185 tumor. However, recipients of IL-18-secreting SIRPa-CAR T cells experienced lethal CAR T cell-related toxicity. To improve the safety of CD47-CAR T cells, we engineered an NFAT-responsive vector expressing the CD47-CAR modified with E. coli-derived dihydrofolate reductase drug destabilization domain to limit CD47-CAR expression to T cells exposed to the stabilizing antibiotic trimethoprim and receiving a licensing NFAT signal from a CD19-CAR. Conclusions: In vivo activity of CD47-targeting CAR T cells is limited by thoracic retention and limited expansion, which can be partially overcome by overexpression of IL-18, though enhanced toxicity precludes survival prolongation. Our findings suggest that strategies to spatiotemporally restrict CAR expression or activation will be necessary to achieve safe and efficacious CD47-CAR T cell activity.

Chemical Constituents of Primulina eburnea (Gesneriaceae) and Their Cytotoxic Activities.

Two new ursane-type triterpenes, eburnealactones A and B (1 and 2), one new flavonoid, eburneatin A (6), and one new phenylethanoid glycoside, chiritoside D (7), along with 9 known compounds (3-5, 8-13) were isolated from the whole plant of Primulina eburnea. Their structures were elucidated by comprehensive spectroscopic data analysis (IR, UV, NMR, and HR-ESI-MS). All the compounds were evaluated for their cytotoxic activities. Compound 1 showed significant cytotoxic activities against MKN-45 cell lines and 5637 cell lines with the IC50 values of 9.57 μM and 8.30 μM, respectively. Compound 1 exhibited moderate cytotoxic activities against A549 and PATU8988T cell lines with the IC50 values of 30.70 μM and 38.22 μM, respectively. Compound 6 exhibited moderate cytotoxic activities against MKN-45, HCT116, PATU8988T, 5637 and A-673 cell lines with the IC50 values of 19.69 μM, 16.44 μM, 18.07 μM, 11.51 μM and 18.15 μM, respectively. Compound 5 showed moderate cytotoxic activities against A549 cell lines with the IC50 values of 24.06 μM.

Abstract 3559: Understanding Ewing sarcoma cell fate

Abstract Like several other translocation-positive sarcomas, Ewing sarcoma (ES) exists exclusively in a high-grade, undifferentiated state. We believe that the pathognomonic EWS-FLI1 fusion protein (FP) responsible for ES is intimately linked to cell fate, differentiation, and plasticity. Over the last two decades, an emerging body of research has begun the challenging task of deciphering how the FP - via its action as an aberrant transcription factor- blocks differentiation and promotes reprogramming toward a quasi-stem-like cell with features of neuroectodermal or mesenchymal stem cells. We compared ES transcriptome data to other cancers in an unsupervised comparison between the gene expression profile of cancer cells in the Cancer Cell Line Encyclopedia (CCLE) and tumor expression datasets. While ES is traditionally classified as a bone cancer, it does not cluster near other bone cancer subtypes (e.g., osteosarcomas) or soft-tissue sarcomas. To that end, we sought to better understand how targeting the FP affected ES differentiation and lineage. Using a CRISPR knockout (CRISPR-KO) model of A673, we targeted Exon 4 of the EWSR1 protein in the n-terminus of the FP and generated a clonal pool (EWSR1 CRISPR KO). We used single-cell RNA sequencing (scRNA-seq) to identify the transcriptional changes after FP knockout (FP KO). We identified five cell clusters in the data. We observed the expression of FP target genes (EWS-FLI1 On), PRKCB, and LIPI and saw robust expression in four of the clusters suggesting normal FP expression. Conversely, FP repressed genes (EWS-FLI1 Off), LOX and IGFBP3, were elevated in cluster 2, which contained only EWSR1 CRISPR KO cells. This suggests that the FP was successfully knocked out in cluster 2 (FP EWSR1 KO). Next, we asked how the FP KO changed the lineage/fate of A673 cells. We hypothesized that loss of the FP would induce mesenchymal/fibroblastic fate in ES cells. To answer this, we used SingleR, which compares the gene expression profile of query cells to the CCLE data set, to identify the nearest cell type. Interestingly, all clusters except the FP EWSR1 KO cluster identified closely with ES and related tumors. The FP EWSR1 KO cluster, which expressed EWS-FLI1 ‘off’ genes, did not resemble ES. Using a granular identification with cell lines, we confirmed that the highest score was A673 since these were A673 cell lines. However, the FP EWSR1 KO resulted in a phenotype unrelated to A673 and more closely resembled cell lines with fibroblastic phenotype. Our data suggest that targeting the FP significantly changed ES lineage. While the FP KO did not lead to cell death, our results suggest a novel gene regulatory network acts to sustain ES lineage. Further perturbations in this network may lead to a better understanding of ES treatment, particularly as novel EWS-FLI1-directed therapies emerge. Citation Format: Danh Truong, Sandhya Krishnan, Alexandar Lazar, David McCall, Joseph A. Ludwig. Understanding Ewing sarcoma cell fate. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3559.