Limiting Dilution Assay Research Articles

Single-cell landscape identified SERPINB9 as a key player contributing to stemness and metastasis in non-seminomas.

Embryonal carcinoma (EC), characterized by a high degree of stemness similar to that of embryonic stem cells, is the most malignant subtype within non-seminomatous testicular germ cell tumors (TGCTs). However, the mechanisms underlying its malignancy remain unknown. In this study, we employed single-cell RNA sequencing to analyze four non-seminoma samples. Our differential expression analysis revealed high expression of SERPINB9 in metastatic EC cells. We conducted in vitro experiments to further investigate SERPINB9's role in the progression of EC. Functionally, the knockdown of SERPINB9 in NCCIT and NTERA-2 leads to a diminished migratory capability and decreased cis-platin resistance, as demonstrated by Transwell migration assay and drug sensitivity assay. Moreover, embryoid bodies showed reduced size and lower OCT4 expression, alongside heightened expression of differentiation markers AFP, ACTA2, and CD57 in shSERPINB9 cells. In vivo, the role of SERPINB9 in maintaining cancer stemness was validated by the limiting dilution assay. Mechanistically, Bulk RNA-seq further showed downregulation of ERK1/2 signaling and WNT signaling pathways with concomitant upregulation of differentiation pathways subsequent to SERPINB9 knockdown. Additionally, the analysis indicated increased levels of cytokines linked to tertiary lymphoid structures (TLS), such as IL6, IL11, IL15, CCL2, CCL5, and CXCL13 in shSERPINB9 cells, which were further validated by ELISA. Our research indicates that SERPINB9 plays a key role in driving tumor progression by enhancing tumor stemness and suppressing TLS. This study stands as the first to elucidate the molecular signature of non-seminomas at a single-cell level, presenting a wealth of promising targets with substantial potential for informing the development of future therapeutic interventions.

STEM-07. UNVEILING THE ROLE OF HYPOXIA AND TAM-SECRETED TGF-Β1 IN PROMOTING GLIOMA STEM CELL MAINTENANCE VIA YAP/TAZ ACTIVATION IN GLIOBLASTOMA PERI-NECROTIC NICHE

Abstract Glioblastoma (GBM) is a highly malignant brain tumor with rapid growth and poor outcomes. In the tumor microenvironment (TME), necrosis triggers significant reshaping and rapid progression, marked by tumor-associated macrophages (TAMs) and glioma stem cells (GSCs) accumulation in peri-necrotic zones. Analysis of Ivy-GAP and single-cell RNA-Seq data revealed consistent Hippo pathway activation in the hypoxic peri-necrotic zone, despite canonical stem-cell transcription factors such as SOX2, OLIG2, and FOXM1 not being upregulated. We hypothesize that Hippo transcription activation may promote GSC stemness via direct effects of hypoxia and/or TAM-secreted cytokines. Exposing patient-derived GBM neurospheres to 1% hypoxia increased nuclear YAP1 and TAZ expression, along with CYR61, a Hippo pathway readout, compared to normoxia at 48 hours. Single-cell RNA-Seq identified potential hypoxia-regulated genes upstream of YAP/TAZ, including Zyxin, upregulated in 1% hypoxia-treated cultures, possibly facilitating nuclear YAP/TAZ transport. Culturing GBM cells with primary macrophage-conditioned media activated Hippo transcription, with TGF-β1 identified as a hypoxia-specific driver of malignant cell gene expression with single-cell RNA-Seq analysis using NichenetR. Treating GBM cells with TGF-β1 increased CYR61 expression, signifying Hippo transcription activation. Furthermore, we showed that hypoxia and TGF-β1 synergistically maintained GSCs with extreme limiting dilution assays. In the immunocompetent RCAS/tv-a mouse model, Verteporfin, a YAP/TAZ inhibitor, significantly reduced tumor size and prolonged mouse lifespan. TAZ knockdown similarly extended lifespan and reduced tumor growth, with flow cytometry showing a decrease in CD133/CD44 positive GSC populations in knockdown mice. These findings suggest the conclusion that hypoxia and TAM-secreted TGF-β1 directly stimulate YAP/TAZ activation and potentially promote GSC stemness in the GBM peri-necrotic niche.

Effect of interleukin 1b inhibition with canakinumab on inflammation and viral persistence in people with human immunodeficiency virus

Abstract Introduction Effectively treated people with HIV (PWH) have elevated risk of cardiovascular disease (CVD). Inflammatory markers are predictive of CVD and mortality among PWH. The role of the myeloid system in driving inflammation and atherosclerosis has been recently recognized. Canakinumab (a monoclonal antibody to IL-1β) reduces inflammation and CVD events among people with elevated hsCRP after myocardial infarction without HIV. We evaluated the impact of IL-1β inhibition using canakinumab on HIV parameters, inflammation, HIV persistence markers, arterial inflammation, and hematopoietic activity in PWH. Methods PWH on effective ART who had CVD or were at risk for CVD were randomized 2:1 to receive 150 mg of canakinumab subcutaneously or matched placebo at weeks 0 and 12. Arterial inflammation and bone marrow metabolic activity were assessed using F18 FDG PET/CT at baseline and week 18. T cell and monocyte activation were evaluated using multiparameter spectral cytometry. The viral reservoir was quantified using Tat/rev Induced Limiting Dilution Assay (TILDA), HIV DNA and cell-associated RNA. The primary endpoints were change in CD4 and CD8 count. We assessed group effects over time using linear mixed effects models. Results 33 individuals were randomized (median age of 60 years old (IQR 57.5, 64.5) and 94% male); 25 received canakinumab and 8 received placebo. There were no significant differences at baseline. As expected, IL-1β increased among those treated with canakinumab at weeks 4- 24 (p<0.01 for each) and returned to baseline at week 36. There was one death from sepsis in an individual aged 84 with CVD and poorly controlled diabetes who received canakinumab. There were no significant changes in CD4 count, CD8 count, platelet count, creatinine, AST, or ALT by group. Treatment was not associated with a greater reduction in arterial inflammation in the most diseased segment compared to placebo (-6.9%; 95% CI -30.4 to 24.5%; p=0.62). However, bone marrow metabolic activity decreased in the canakinumab group versus placebo (-14.4%, 95% CI -26.5% to -0.2%; p=0.047). Treatment was associated with increased CD163 expression on monocytes at weeks 24 (p=0.03) and 36 (p<0.001), and lower caspase activity at week 36 (p=0.02). There was expansion of anti-inflammatory CD16+ patrolling monocytes that co-express CD163+CX3CR1+ and a decrease in pro-inflammatory CD16+ patrolling monocytes that are Caspase1+CCR2+. Treatment was not associated with differences in hsCRP, IL-6, IL-16, IL-18, MCP-1, sCD14, sCD163, T cell subsets, NK cell subsets, or viral persistence markers. Conclusion Among treated PWH, targeted inhibition of IL-1β using canakinumab results in decreased bone marrow metabolic activity and a shift toward anti-inflammatory monocyte populations. These findings shed light on mechanisms underlying how targeted IL-1β inhibition using canakinumab prevents CVD events by altering monocyte populations and reducing systemic inflammation.

Type XVII Collagen–Specific CD4+ T Cells Induce Bullous Pemphigoid by Producing IL-5

Bullous pemphigoid (BP) is an autoimmune subepidermal blistering disease caused by anti-type XVII collagen (COL17) antibodies. BP has some immunological features such as eosinophilic infiltration and the deposition of IgE autoantibodies in the skin; however, the mechanism behind such features remains largely unclear. We focused on the autoantigen-specific CD4+ T cells, which are considered to regulate immune response. We established COL17-specific CD4+ T cell lines in vitro. Wild-type mice were immunized with synthesized peptides that include a pathogenic epitope of COL17, and lymphocytes were subjected to a limiting dilution assay. We established 5 T cell lines and examined the pathogenicity by transferring them with COL17-primed B cells into Rag-2-/-/COL17-humanized mice that express human COL17 but not mouse COL17 in the skin. Notably, 3 lines induced BP-like skin changes associated with subepidermal separation and eosinophilic infiltration histologically, and the production of anti-COL17 antibodies. The other 2 lines did not induce such phenotypes. RNA-sequencing analysis revealed that Th2 cytokines, particularly IL-5, were highly expressed in the pathogenic T cell lines. Anti-IL-5 antibody administration significantly reduced the skin changes and attenuated the production of autoantibodies. Thus, the production of IL-5 is critical for COL17-specific CD4+ T cells to induce BP phenotypes in vivo.

1α,25-hydroxyvitamin D/VDR suppresses stem-like properties of ovarian cancer cells by restraining nuclear translocation of β-catenin

Several studies have indicated that 1α,25-hydroxyvitamin D [1α,25(OH)2D3] inhibits the proliferation and metastasis of cancer cells through suppressing epithelial-mesenchymal transition. However, its influence on the translocation of β-catenin remains unclear. In the present study, ovarian cancer stem-like cells (CSCs), including side population (SP) and CD44+/CD117+, were isolated from mouse ovarian surface epithelial (MOSE) cells with malignant transformation. The findings revealed that 1α,25(OH)2D3 obviously reduced the sphere-forming ability, as well as Notch1 and Klf levels. Moreover, the limiting dilution assay demonstrated that 1α,25(OH)2D3 effectively hindered the tumorigenesis of ovarian CSCs in vitro. Notably, treatment with 1α,25(OH)2D3 led to a substantial increase in the cell population of CD44+/CD117+ forming one tumor from ≤ 100 to 445 in orthotopic transplanted model, indicating a pronounced suppression of stemness of ovarian CSCs. Additionally, 1α,25(OH)2D3 robustly promoted the translocation of β-catenin from the nuclear to the cytoplasm through directly binding to VDR, which resulted in decreased levels of c-Myc and CyclinD1 within late MOSE cells. Taken together, these results strongly supported the role of 1α,25(OH)2D3 in inhibiting stem-like properties in ovarian cancer cells by restraining nuclear translocation of β-catenin, thereby offering a promising target for cancer therapeutics.

Restriction of YWHAB-mediated YAP cytoplasmic retention as a novel mechanism underlying stemness maintenance and chemo-resistance in peritoneal metastasis of ovarian cancer: A comparative proteomic study.

122 Background: Ovarian cancer (OC) peritoneal metastasis (OCPM) is a significant cause of high mortality of OC. To investigate the mechanisms underlying OCPM stemness maintenance and resistance, we characterized proteomic alterations in residual OCPM tissues after neoadjuvant chemotherapy (NACT), and verified restriction of YWHAB-mediated YAP cytoplasmic retention as a novel important mechanism. Methods: Tumor specimens from HGSOC patients underwent proteomic analysis using TMT and REACTOME for pathway and GO analysis. The OVCAR3 cell line, derived from malignant ascites, and formalin-fixed samples were used for Immunohistochemistry. Ovarian cancer stem-like cells, including cisplatin-resistant cells, were cultured, and ALHD activity was measured by qPCR and ALDEFLUOR Kit. Western blot and Co-IP were used for protein analysis. Sphere formation and limiting dilution assays were performed in vitro and in vivo using BALB/c nude mice, and FACS quantified OCSCs. YWHAB-knockdown cells were created via plasmids, lentivirus, and transfection. Statistical tests included paired and unpaired t-tests, one-way ANOVA, and ELDA for limiting dilution. Data are shown as mean ± SD, with *p< 0.05, **p< 0.01, ***p< 0.001 indicating significance. Results: TMT-based proteomics identified 324 differentially expressed proteins in post-NACT OCPM tissues, with 179 upregulated and 145 downregulated. Bioinformatics revealed novel targets in key pathways, including TUBB, VCP in Hedgehog, YWHAB in Hippo, TLA1/2, SPTA1 in MAPK, and FASN in NOTCH. YWHAB downregulation was confirmed in pPR OCPM tissues and cells, serving as a marker to differentiate pNR from pCR/pPR OCPM (AUC = 0.7673). YWHAB inhibition in pCR OCPM cells enhanced stemness, as seen in sphere formation, OCSC percentages, CD133 expression, ALDH activity, and tumorigenicity. It also induced cisplatin resistance. YWHAB inhibition reduced cytoplasmic YAP retention and increased nuclear YAP, enhancing transcriptional activity. YAP5SA expression negated the effects of YWHAB depletion on stemness and resistance in pCR OCPM cells. Conclusions: In summary, this study explored the potential mechanisms underlying OCPM stemness maintenance and resistance by employing proteomic analysis, and revealed a novel YWHAB-mediated mechanism. This finding indicates that YAP would an important target for eradicating YWHAB-restricted OCSCs in OCPM.

Development of a humanized anti-FABP4 monoclonal antibody for potential treatment of breast cancer

BackgroundBreast cancer is the most common cancer in women diagnosed in the U.S. and worldwide. Obesity increases breast cancer risk without clear underlying molecular mechanisms. Our studies demonstrate that circulating adipose fatty acid binding protein (A-FABP, or FABP4) links obesity-induced dysregulated lipid metabolism and breast cancer risk, thus potentially offering a new target for breast cancer treatment.MethodsWe immunized FABP4 knockout mice with recombinant human FABP4 and screened hybridoma clones with specific binding to FABP4. The potential effects of antibodies on breast cancer cells in vitro were evaluated using migration, invasion, and limiting dilution assays. Tumor progression in vivo was evaluated in various types of tumorigenesis models including C57BL/6 mice, Balb/c mice, and SCID mice. The phenotype and function of immune cells in tumor microenvironment were characterized with multi-color flow cytometry. Tumor stemness was detected by ALDH assays. To characterize antigen-antibody binding capacity, we determined the dissociation constant of selected anti-FABP4 antibodies via surface plasmon resonance. Further analyses in tumor tissue were performed using 10X Genomics Visium spatial single cell technology.ResultsHerein, we report the generation of humanized monoclonal antibodies blocking FABP4 activity for breast cancer treatment in mouse models. One clone, named 12G2, which significantly reduced circulating levels of FABP4 and inhibited mammary tumor growth, was selected for further characterization. After confirming the therapeutic efficacy of the chimeric 12G2 monoclonal antibody consisting of mouse variable regions and human IgG1 constant regions, 16 humanized 12G2 monoclonal antibody variants were generated by grafting its complementary determining regions to selected human germline sequences. Humanized V9 monoclonal antibody showed consistent results in inhibiting mammary tumor growth and metastasis by affecting tumor cell mitochondrial metabolism.ConclusionsOur current evidence suggests that targeting FABP4 with humanized monoclonal antibodies may represent a novel strategy for the treatment of breast cancer and possibly other obesity- associated diseases.

UCHL3 induces radiation resistance and acquisition of mesenchymal phenotypes by deubiquitinating POLD4 in glioma stem cells

BackgroundThe high degree of intratumoral genomic heterogeneity is a major obstacle for glioblastoma (GBM) tumors, one of the most lethal human malignancies, and is thought to influence conventional therapeutic outcomes negatively. The proneural-to-mesenchymal transition (PMT) of glioma stem cells (GSCs) confers resistance to radiation therapy in glioblastoma patients. POLD4 is associated with cancer progression, while the mechanisms underlying PMT and tumor radiation resistance have remained elusive.MethodExpression and prognosis of the POLD family were analyzed in TCGA, the Chinese Glioma Genome Atlas (CGGA) and GEO datasets. Tumorsphere formation and in vitro limiting dilution assay were performed to investigate the effect of UCHL3-POLD4 on GSC self-renewal. Apoptosis, TUNEL, cell cycle phase distribution, modification of the Single Cell Gel Electrophoresis (Comet), γ-H2AX immunofluorescence, and colony formation assays were conducted to evaluate the influence of UCHL3-POLD4 on GSC in ionizing radiation. Coimmunoprecipitation and GST pull-down assays were performed to identify POLD4 protein interactors. In vivo, intracranial xenograft mouse models were used to investigate the molecular effect of UCHL3, POLD4 or TCID on GCS.ResultWe determined that POLD4 was considerably upregulated in MES-GSCs and was associated with a meagre prognosis. Ubiquitin carboxyl terminal hydrolase L3 (UCHL3), a DUB enzyme in the UCH protease family, is a bona fide deubiquitinase of POLD4 in GSCs. UCHL3 interacted with, depolyubiquitinated, and stabilized POLD4. Both in vitro and in vivo assays indicated that targeted depletion of the UCHL3-POLD4 axis reduced GSC self-renewal and tumorigenic capacity and resistance to IR treatment by impairing homologous recombination (HR) and nonhomologous end joining (NHEJ). Additionally, we proved that the UCHL3 inhibitor TCID induced POLD4 degradation and can significantly enhance the therapeutic effect of IR in a gsc-derived in situ xenograft model.ConclusionThese findings reveal a new signaling axis for GSC PMT regulation and highlight UCHL3-POLD4 as a potential therapeutic target in GBM. TCID, targeted for reducing the deubiquitinase activity of UCHL3, exhibited significant synergy against MES GSCs in combination with radiation.

Synthetic abnormal mast cell particles successfully mimic neoplastic mast cells by flow cytometry.

Clinical flow cytometry laboratories require quality control materials for assay development, validation, and performance monitoring, including new reagent lot qualification. However, finding suitable controls for populations with uncommonly expressed antigens or for rare populations, such as mast cells, can be difficult. To that end, we evaluated synthetic abnormal mast cell particles (SAMCP), developed together with, and manufactured by, Slingshot Biosciences. The SAMCP's were designed to phenotypically mimic abnormal neoplastic mast cells: they were customized to have the same light scatter and autofluorescence properties of mast cells, along with surface antigen levels of CD45, CD33, CD117, CD2, CD25, and CD30 consistent with that seen in mast cell disease. We evaluated several performance characteristics of these particles using ARUP's high sensitivity clinical mast cell assay, including limit of detection, off-target activity and FMO controls, precision, scatter properties of the particles utilizing several different cytometer platforms, and particle antigen stability. The phenotype of the SAMCP mimicked abnormal mast cells, and they could be distinguished from normal native mast cells. FMO controls demonstrated specificity of each of the markers, and no off-target binding was detected. The limit of detection of the particles spiked into normal bone marrow was found to be ≤0.003% in a limiting dilution assay. The mast cell particles were found to perform similarly on Becton Dickinson Lyric, Cytek Aurora, and Beckman Coulter Navios and CytoFLEX platforms. Within run and between run precision were less than 10% CV. SAMCP were stable up to 13 days with minimal loss of antigen fluorescence intensity. The SAMCP's were able to successfully mimic neoplastic mast cells based on the results of our high sensitivity mast cell flow cytometry panel. These synthetic cell particles represent an exciting and innovative technology, which can fulfill vital needs in clinical flow cytometry such as serving as standardized control materials for assay development and performance monitoring.

Abstract PO1-06-07: EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer

Abstract PO1-06-07: EMSY enhances cancer stem cell self-renewal and tumorigenesis by reshaping methionine metabolism in triple-negative breast cancer

Combination of the Topical Photodynamic Therapy of Chloroaluminum Phthalocyanine Liposomes with Fexinidazole Oral Self-Emulsifying System as a New Strategy for Cutaneous Leishmaniasis Treatment.

Cutaneous leishmaniasis (CL) is a neglected tropical disease. The treatment is restricted to drugs, such as meglumine antimoniate and amphotericin B, that exhibit toxic effects, high cost, long-term treatment, and limited efficacy. The development of new alternative therapies, including the identification of effective drugs for the topical and oral treatment of CL, is of great interest. In this sense, a combination of topical photodynamic therapy (PDT) with chloroaluminum phthalocyanine liposomes (Lip-ClAlPc) and the oral administration of a self-emulsifying drug delivery system containing fexinidazole (SEDDS-FEX) emerges as a new strategy. The aim of the present study was to prepare, characterize, and evaluate the efficacy of combined therapy with Lip-ClAlPc and SEDDS-FEX in the experimental treatment of Leishmania (Leishmania) major. Lip-ClAlPc and SEDDS-FEX were prepared, and the antileishmanial efficacy study was conducted with the following groups: 1. Lip-ClAlPc (0.05 mL); 2. SEDDS-FEX (50 mg/kg/day); 3. Lip-ClAlPc (0.05 mL)+SEDDS-FEX (50 mg/kg/day) combination; 4. FEX suspension (50 mg/kg/day); and 5. control (untreated). BALB/c mice received 10 sessions of topical Lip-ClAlPc on alternate days and 20 consecutive days of SEDDS-FEX or FEX oral suspension. Therapeutical efficacy was evaluated via the parasite burden (limiting-dilution assay), lesion size (mm), healing of the lesion, and histological analyses. Lip-ClAlPc and SEDDS-FEX presented physicochemical characteristics that are compatible with the administration routes used in the treatments. Lip-ClAlPc+SEDDS-FEX led to a significant reduction in the parasitic burden in the lesion and spleen when compared to the control group (p < 0.05) and the complete healing of the lesion in 43% of animals. The Lip-ClAlPc+SEDDS-FEX combination may be promising for the treatment of CL caused by L. major.

Abstract 3288: Identification of Annexin A1 as a novel driver of lenvatinib resistance in hepatocellular carcinoma

Abstract Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related deaths worldwide. Lenvatinib has recently been approved by the FDA for front-line treatment of HCC patients at advanced stages. However, the survival benefit of lenvatinib is modest due to the development of acquired drug resistance. In this study, we aimed to investigate the mechanisms underlying lenvatinib resistance in HCC and to identify potential therapeutic targets to combat lenvatinib resistance. For this purpose, we established a lenvatinib-resistant (LenR) HCC patient-derived tumor xenograft (PDTX) by administering four rounds of lenvatinib treatment, and their genetic profiles were analyzed using single-cell RNA sequencing (scRNA-seq). Based on single-cell regulatory network inference and clustering (SCENIC) analysis, Annexin A1 (ANXA1), the top gene that was enriched in the HCC cell-like cluster of LenR tumors, was identified and was consistently observed in LenR HCC cell lines and LenR tumors derived from Tp53KO/C-MycOE and RIL175 HCC mouse models. Using an in vitro differentiation model, limiting dilution assay, apoptosis assay and correlation analysis, we demonstrated the role of ANXA1 in the regulation of cancer stemness-driven lenvatinib resistance. Mechanistically, ANXA1 regulates cancer stemness and drug resistance by driving the STAT3/S100A6 signaling cascade, and ANXA1 expression is regulated by SOX2 via promoter activation, as demonstrated by a chromatin immunoprecipitation (ChIP) assay. Using the same set of scRNA-seq data, we showed that LenR HCC tumors reshaped the tumor microenvironment (TME) by inducing M2 macrophage polarization, as evidenced by a higher M2/M1 ratio of tumor-associated macrophages (TAM) in LenR HCC tumors than in their mock counterparts. Based on this observation, we further demonstrated the in vitro role of secretory ANXA1 in LenR HCC cells in inducing macrophage polarization towards the M2 phenotype. In conclusion, ANXA1 may regulate lenvatinib resistance intrinsically via the promotion of cancer stemness and extrinsically via the induction of an immunosuppressive TME. Citation Format: Catherine Yujia GU, Carmen Oi Ning LEUNG, Terence Kin Wah LEE. Identification of Annexin A1 as a novel driver of lenvatinib resistance in hepatocellular carcinoma [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 3288.

Abstract 1433: FOXA1 alterations distinctively drive prostate tumorigenesis or therapy resistance in mice

Abstract Androgen receptor (AR) signaling is critical for survival of prostate cancer (PCa) cells, thus making androgen deprivation therapy (ADT; e.g., castration) as the mainstay for treatment. Notably, oncogenic functions of AR rely on chromatin-binding regulatory proteins, which includes a pioneer transcription factor called FOXA1. FOXA1 de-compacts chromatin to enable AR’s binding to the DNA and expression of it's target genes. Recently, our lab found FOXA1 alterations to recur within three distinct structural classes in over 35% of metastatic castration resistant PCa (mCRPC) in Caucasian men. Subsequent studies found FOXA1 mutations to be prevalent in over 40% of primary PCa in Chinese men, positioning FOXA1 as a principal oncogene in this disease. Yet, hitherto, no studies have defined the pathobiology of FOXA1 alterations in mouse models. Here, we have developed the first-in-field transgenic mice with conditional overexpression of FOXA1 mutants in the prostate luminal epithelia. We found truncal FOXA1 Class1 mutants to initiate luminal hyperplasia in a monogenic model, or hyperproliferative prostate adenocarcinoma in a compound Trp53-deficient genetic background. Mechanistically, Class1 mutants concurrently upregulate the AR and mTORC1/2 pathways to drive transformation, with this being the first report of FOXA1-driven PCa formation in mice. In contrast, FOXA1 Class2 mutants—which are acquired in mCRPC—do not drive prostate luminal transformation. Instead, single-cell multi-omics (RNA+ATAC) profiling of Class2-mutant mouse prostate tissue uncovered extensive transcriptional remodeling of epithelial cells into a luminal stem-like cell fate, leading to a dramatic 15-20-fold expansion of the progenitor population vs control tissue. These luminal stem cells are similar to the Club/Hillock cells detected in the human prostate that have been implicated in driving resistance to ADT. Consistently, we found Class2-mutant prostates to show minimal atrophy upon castration, with immunohistological assessment uncovering a higher density of Ki67+ luminal epithelial cells relative to wild type and Class1 tissues. Class2-mutant organoids also showed higher subcutaneous grafting ability in mice in limiting dilution assays. Mechanistically, we found the cistromically-dominant Class2 mutants to pioneer over 40,000 neo-enhancer elements harboring motifs of stemness-associated transcription factors, which in turn instruct the ADT-resistant luminal progenitor gene program. Altogether, findings from our mouse models uncover FOXA1’s versatility as a driver oncogene that, depending on the mutation type, either activates enhancer-wired luminal tumorigenesis (Class1-initiating event) or therapy resistance-associated stemness (Class2-promoting alteration) gene programs in the mouse prostate tissue. Citation Format: Sanjana Eyunni, Abhijit Parolia, Eleanor Young, James Matthew George, Rahul Mannan, Sandra E. Carson, Yuping Zhang, Jean Tien, Mustapha Jaber, Jie Luo, Matthew Pang, Rohit Mehra, Xuhong Cao, Fengyun Su, Rui Wang, Marcin Cieslik, Dong Kee Lee, Jianming Xu, Arul M. Chinnaiyan. FOXA1 alterations distinctively drive prostate tumorigenesis or therapy resistance in mice [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 1433.

Abstract 5111: Rational synergistic radiosensitization strategies for group 3 medulloblastoma

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is comprised of 4 groups, termed Wnt, Shh, Group 3 and Group 4, each characterized by distinct molecular and clinical profiles. MYC-amplified Group 3 MB is associated imminent fatal metastatic relapse following standard treatment approaches, which currently include surgery, craniospinal irradiation and chemotherapy, highlighting an urgent need for identification of therapeutic vulnerabilities for these tumours. As such, using 4 novel patient-derived models of Group 3 MB that faithfully recapitulate the molecular and histological tumor characteristics, we characterised response to radiation of Group 3 MB in vitro to show that these tumors do not possess intrinsic radiation resistance. Furthermore, cumulative radiation experiments do not increase bulk radiation resistance in MB cells, highlighting the contribution of dynamic cell states to radiation response. In contrast, exposure to radiation leads to changes in cell clonogenic capacity as measured by limited dilution assays. Furthermore, RNA-seq data uncovered altered transcriptomic programs in response to cumulative radiation treatment. In particular, we observe differential regulation of genes encoding potassium ion channels and their regulators as well as genes encoding ECM and cell adhesion proteins. As part of our goal to evaluate the current multimodal treatment strategy of MB, we also show that a set of standard chemotherapeutic agents (etoposide, thiotepa and lomustine) currently used in the clinic do not produce a potent response at optimal doses in vitro, potentially accounting for abysmal survival outcomes seen in chemotherapy treated MB patients. As such, we conducted a screen of 144 FDA approved cancer drugs and identified therapeutics that selectively target Group 3 MB cells in contrast to normal tissue controls (foetal neural stem cells) in the low nanomolar dose range. In particular, topoisomerase inhibitor topotecan produces a potent response specific to MYC-amplified Group 3 MB, and sensitizes 2 patient-derived MB models to gamma irradiation. Altogether, our data characterizes Group 3 MB molecular and cellular responses to current treatment modalities and provides rationale for new clinical strategies to sensitize patients to radiation that will potentially allow to reduce the required radiation doses to treat these aggressive tumors, reduce the incidence of radiation treatment-related morbidity as well as improve cure rates. Citation Format: Julija Povilaikaite, Kaitlin Stitz, Nikhita Austin, Vijay Ramaswamy. Rational synergistic radiosensitization strategies for group 3 medulloblastoma [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 5111.

Abstract 1637: EPHB4 is a key regulator of G1/S transition in hepatocellular carcinoma

Abstract Effective therapeutic regimens against advanced hepatocellular carcinoma (HCCs) have recently improved but it remains very limited. Targeting receptor tyrosine kinases (RTKs) has proven to one of the main ways of achieving better survival outcome in HCC patients, yet the precise mechanism for this success remains to be elusive. We have systematically investigated the expression profiles of RTKs transcriptomic datasets i.e. The Cancer Genome Atlas - Liver hepatocellular carcinoma (TCGA-LIHC) (n=371) and our in-house cohort of paired i.e. HKU-QMH (n=41), and identified EPHB4 as one of the most abundantly expressed and frequently mutated RTKs in human HCCs. EPHB4 expression was found to be significantly correlated with Alpha Fetoprotein (AFP) expression, a factor commonly used in the diagnosis of HCCs. Furthermore, EphB4 has been demonstrated to play a regulatory role in VEGF/VEGFR-mediated angiogenesis, critical targets for current successful drugs. We confirmed abundant membrane EphB4 expression in HCC clinical specimens and human HCC cell line models using immunohistochemistry. Clinicopathological correlation analysis of EPHB4 mRNA expression revealed that its overexpression is associated with aggressive tumor features including presence of tumor microsatellite, liver invasion, venous invasion, and absence of tumor encapsulation. Functionally, stably knocking down EPHB4 (shEPHB4) resulted in a strong reduction of proliferation and self-renewal rates of multiple HCC cell lines in vitro. It significantly reduced tumor growth rates in subcutaneous and liver orthotopic models in vivo. Furthermore, limiting dilution assay showed EPHB4 plays a role in tumor initiation property of HCC cells. Mechanistically, we performed Gene Set Enrichment Analysis (GSEA) of transcriptome profiles for shEPHB4 with respective controls and found that knocking down EPHB4 causes HCC cells undergo drastic changes in several cell cycle checkpoints including G1/S transition. Using thymidine blockade, we further confirmed that knockdown of significantly hinders the transition of HCC cells from G1 to S phase of cell cycle. Our data suggests a strong link between EphB4 and cell cycle. Conclusion: We found EphB4 plays a functional role in regulating G1/S transition in human HCCs and understanding regulatory role of EphB4 on cell cycle progression may provide mechanistic insights into the how to effectively target HCC in the clinic. Citation Format: Abdullah Husain, Elley Y. Chiu, Hoi-Tang Ma, Daniel W. Ho, Karen M. Sze, Lo-Kong Chan, Irene O. Ng. EPHB4 is a key regulator of G1/S transition in hepatocellular carcinoma [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 1637.

Long non-coding RNA lung cancer-associated transcript-1 promotes glioblastoma progression by enhancing Hypoxia-inducible factor 1 alpha activity.

Hypoxia is associated with poor prognosis in many cancers including glioblastoma (GBM). Glioma stem-like cells (GSCs) often reside in hypoxic regions and serve as reservoirs for disease progression. Long non-coding RNAs (lncRNAs) have been implicated in GBM. However, the lncRNAs that modulate GSC adaptations to hypoxia are poorly understood. Identification of these lncRNAs may provide new therapeutic strategies to target GSCs under hypoxia. lncRNAs induced by hypoxia in GSCs were identified by RNA-seq. Lung cancer-associated transcript-1 (LUCAT1) expression was assessed by qPCR, RNA-seq, Northern blot, single molecule FISH in GSCs, and interrogated in IvyGAP, The Cancer Genome Atlas, and CGGA databases. LUCAT1 was depleted by shRNA, CRISPR/Cas9, and CRISPR/Cas13d. RNA-seq, Western blot, immunohistochemistry, co-IP, ChIP, ChIP-seq, RNA immunoprecipitation, and proximity ligation assay were performed to investigate mechanisms of action of LUCAT1. GSC viability, limiting dilution assay, and tumorigenic potential in orthotopic GBM xenograft models were performed to assess the functional consequences of depleting LUCAT1. A new isoform of Lucat1 is induced by Hypoxia inducible factor 1 alpha (HIF1α) and Nuclear factor erythroid 2-related factor 2 (NRF2) in GSCs under hypoxia. LUCAT1 is highly expressed in hypoxic regions in GBM. Mechanistically, LUCAT1 formed a complex with HIF1α and its co-activator CBP to regulate HIF1α target gene expression and GSC adaptation to hypoxia. Depletion of LUCAT1 impaired GSC self-renewal. Silencing LUCAT1 decreased tumor growth and prolonged mouse survival in GBM xenograft models. A HIF1α-LUCAT1 axis forms a positive feedback loop to amplify HIF1α signaling in GSCs under hypoxia. LUCAT1 promotes GSC self-renewal and GBM tumor growth. LUCAT1 is a potential therapeutic target in GBM.

METTL16 promotes liver cancer stem cell self-renewal via controlling ribosome biogenesis and mRNA translation

BackgroundWhile liver cancer stem cells (CSCs) play a crucial role in hepatocellular carcinoma (HCC) initiation, progression, recurrence, and treatment resistance, the mechanism underlying liver CSC self-renewal remains elusive. We aim to characterize the role of Methyltransferase 16 (METTL16), a recently identified RNA N6-methyladenosine (m6A) methyltransferase, in HCC development/maintenance, CSC stemness, as well as normal hepatogenesis.MethodsLiver-specific Mettl16 conditional KO (cKO) mice were generated to assess its role in HCC pathogenesis and normal hepatogenesis. Hydrodynamic tail-vein injection (HDTVi)-induced de novo hepatocarcinogenesis and xenograft models were utilized to determine the role of METTL16 in HCC initiation and progression. A limiting dilution assay was utilized to evaluate CSC frequency. Functionally essential targets were revealed via integrative analysis of multi-omics data, including RNA-seq, RNA immunoprecipitation (RIP)-seq, and ribosome profiling.ResultsMETTL16 is highly expressed in liver CSCs and its depletion dramatically decreased CSC frequency in vitro and in vivo. Mettl16 KO significantly attenuated HCC initiation and progression, yet only slightly influenced normal hepatogenesis. Mechanistic studies, including high-throughput sequencing, unveiled METTL16 as a key regulator of ribosomal RNA (rRNA) maturation and mRNA translation and identified eukaryotic translation initiation factor 3 subunit a (eIF3a) transcript as a bona-fide target of METTL16 in HCC. In addition, the functionally essential regions of METTL16 were revealed by CRISPR gene tiling scan, which will pave the way for the development of potential inhibitor(s).ConclusionsOur findings highlight the crucial oncogenic role of METTL16 in promoting HCC pathogenesis and enhancing liver CSC self-renewal through augmenting mRNA translation efficiency.

Cancer associated fibroblasts serve as an ovarian cancer stem cell niche through noncanonical Wnt5a signaling

Frequent relapse and chemoresistance cause poor outcome in ovarian cancer (OC) and cancer stem cells (CSCs) are important contributors. While most studies focus exclusively on CSCs, the role of the microenvironment in providing optimal conditions to maintain their tumor-initiating potential remains poorly understood. Cancer associated fibroblasts (CAFs) are a major constituent of the OC tumor microenvironment and we show that CAFs and CSCs are enriched following chemotherapy in patient tumors. CAFs significantly increase OC cell resistance to carboplatin. Using heterotypic CAF-OC cocultures and in vivo limiting dilution assay, we confirm that the CAFs act by enriching the CSC population. CAFs increase the symmetric division of CSCs as well as the dedifferentiation of bulk OC cells into CSCs. The effect of CAFs is limited to OC cells in their immediate neighborhood, which can be prevented by inhibiting Wnt. Analysis of single cell RNA-seq data from OC patients reveal Wnt5a as the highest expressed Wnt in CAFs and that certain subpopulations of CAFs express higher levels of Wnt5a. Our findings demonstrate that Wnt5a from CAFs activate a noncanonical Wnt signaling pathway involving the ROR2/PKC/CREB1 axis in the neighboring CSCs. While canonical Wnt signaling is found to be predominant in interactions between cancer cells in patients, non-canonical Wnt pathway is activated by the CAF-OC crosstalk. Treatment with a Wnt5a inhibitor sensitizes tumors to carboplatin in vivo. Together, our results demonstrate a novel mechanism of CSC maintenance by signals from the microenvironmental CAFs, which can be targeted to treat OC chemoresistance and relapse.

Studying the number of ammonium-oxidizing bacteria in bottom sediments of Lake Baikal

Using the limiting dilution assay, the number of ammonium-oxidizing bacteria in the bottom sediments of the area of the Baikal Pulp and Paper Mill (BPPM, Southern Baikal) was determined. The maximum number of bacteria under study was determined at the station where treated wastewater from the city of Baikalsk is discharged into the lake.

A Bedaquiline, Pyrazinamide, Levofloxacin, Linezolid, and Clofazimine Second-line Regimen for Tuberculosis Displays Similar Early Bactericidal Activity as the Standard Rifampin-Based First-line Regimen.

In 2018 the World Health Organization recommended a switch to an all oral bedaquiline-based second-line regimen for treatment of drug-resistant tuberculosis (DR-TB). How these new second-line regimens fare in comparison to first-line regimens for treatment of drug-sensitive tuberculosis (DS-TB) is not well known. In this study, we contemporaneously enrolled subjects with DS-TB (n = 31) or DR-TB (n = 23) and assessed their response to therapy with first-line (rifampin, isoniazid, ethambutol, pyrazinamide) or second-line (bedaquiline, pyrazinamide, levofloxacin, linezolid, clofazimine) regimens, respectively. We found that the early bactericidal activity of first- and second-line regimens was similar during the first 2 weeks of therapy as determined by BACTEC MGIT, colony-forming units, and a liquid limiting dilution assay capable of detecting differentially detectable/culturable Mycobacterium tuberculosis. Furthermore, an identical percentage (77.8%) of subjects from the DS-TB and DR-TB cohorts converted to culture negative after 2 months of therapy. Despite presenting with more advanced disease at time of treatment, subjects with DR-TB receiving an all oral bedaquiline-based second-line treatment regimen displayed a similar microbiological response to therapy as subjects with DS-TB receiving a first-line treatment regimen.