bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3743: circPMS1 is a pro-metastatic circular RNA in melanoma
Authors: Mecozzi Nicol , Vera Olga , Karreth Florian
Published: April 2023

Deregulated gene expression is a major driver of melanoma metastasis, a process that results in the majority of melanoma-related deaths. Importantly, these alterations are not limited to protein-coding mRNAs, but also include noncoding RNAs. Despite many recent studies implicating circular RNAs (circRNAs) in cancer development, it is unknown if their deregulation contributes to melanoma metastasis. To identify circRNAs with putative roles in melanomagenesis, we performed RNA sequencing on a panel of melanoma and melanocyte cell lines. 21 differentially expressed circRNAs were validated by qPCR and Sanger sequencing of the backsplice junction. Further analyses of the ratio of the circular and cognate linear RNA transcripts and expression in a larger panel of cell lines identified circPMS1 as a circRNA that is upregulated in melanoma through enhanced backsplicing of the linear PMS1 mRNA. Using transposon-mediated delivery, we examined the effects of stable circPMS1 overexpression on melanoma cells. While proliferation and focus formation were not impacted, migration and invasion were enhanced by circPMS1. Moreover, tail vein injection of circPMS1 overexpressing melanoma cells increased lung metastasis. In addition to this transplant model, we generated the first genetically engineered mouse model of melanoma overexpressing a pro-tumorigenic circRNA. These mice are currently being analyzed. Mechanistically, we find that circularization is required for the pro-migratory effects of the circPMS1 overexpression construct. Additionally, circPMS1 harbors the canonical PMS1 start codon, a predicted ORF spanning over the backsplice junction, and a predicted IRES sequence. Indeed, inserting a flag-tag in the overexpression construct revealed circPMS1 to be translated into a truncated 25kD PMS1 protein as well as a 20kD protein likely arising from an in-frame downstream start codon. Importantly, these truncated circPMS1 proteins are endogenously expressed and increased in melanoma compared to melanocytes while the full-length PMS1 protein is evenly expressed. These findings suggest that circPMS1 promotes melanoma metastasis, at least in part, by coding for truncated circPMS1 proteins. Further work will establish the functions of the circPMS1-encoded proteins and their roles in driving melanoma progression and metastasis.

Citation Format: Nicol Mecozzi, Olga Vera, Florian Karreth. circPMS1 is a pro-metastatic circular RNA in melanoma. [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 3743.
Abstract 3751: Characterizing the role of the y chromosome-expressed long non-coding RNA linc-SPRY3 family in lung cancer radiation resistance
Authors: Westemeier-Rice Emily S.
Published: April 2023

Lung cancer is the leading cause of cancer-related deaths worldwide. A major issue for clinicians and patients alike is inherent resistance to radiation therapy. Recently, certain long non-coding RNAs (lnc-RNAs) have been shown to regulate pathways involved in resistances to lung cancer treatments. Our group recently published that a group of long non-coding RNAs, named the linc-SPRY3 family, play an important role in radiation therapy sensitivity. Originating from the Y-Chromosome, these long non-coding RNAs have been shown in vitro and in vivo to decrease the tumor burden after radiation. Interestingly, certain male Non-Small Cell Lung Cancer (NSCLC) cell lines have been shown to have complete loss of the Y-chromosome (LOY), showing a radiation resistant phenotype. To test this, the DYZ1 region of the Y-chromosome, where the linc-SPRY3 family is expressed, was artificially introduced into radiation resistant male NSCLC cell lines with LOY. Remarkably, by adding the DYZ1 region of the Y-chromosome into these cells, they became more sensitive to radiation and had increased apoptosis. Based on this preliminary data, we hypothesize the linc-SPRY3 family interacts with important pathways in the cell to increase sensitivity to DNA double strand breaks. Through RNA-Sequencing and other preliminary experiments, we found that the linc-SPRY3 family regulates the expression of Cell Division Cycle 6 (CDC6) and Cell Division Cycle 25A (CDC25A) genes. Preliminary data suggests these interactions play a role in influencing NSCLC ability to invade and metastasize. We are currently characterizing this family of linc-RNAs by flow cytometry, in vivo metastasis models, and fluorescent in situ hybridization. We will characterize the mechanism of the linc-SPRY3 family in radiation resistance through genetic modifications and further downstream analysis via the over-expression and knockdown of these linc-RNAs. The characterization of these linc-RNAs will be critical for the development of potential diagnostic or therapeutic therapies.

Citation Format: Emily S. Westemeier-Rice. Characterizing the role of the y chromosome-expressed long non-coding RNA linc-SPRY3 family in lung cancer radiation resistance. [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 3751.
Abstract 3755: Dlx4os: a lncRNA associated with malignant melanocyte transformation
Authors: Ayub Ana Luísa P. , Azevedo Hatylas , Pessoa Diogo , Sejour Leinal , Vlachos Ioannis , Reis Eduardo M. , Slack Frank J. , Jasiulionis Miriam G.
Published: April 2023

It is estimated that more than 90% of the human genome is transcribed into non-coding RNAs (ncRNA). It has been demonstrated that ncRNAs have an important role in biological processes such as proliferation, differentiation, and cell migration. Changes in their expression have been associated with several diseases, including cancer. Melanoma is one of the most aggressive types of cancer, with high probability of metastasis and an unfavorable response to therapies. Our lab has developed an in vitro melanoma progression model, which was established by exposing nontumorigenic melanocytes (melan-a) to sustained stress conditions (cycles of adhesion blockage), sequentially generating different cell lines (i.e., 4C, premalignant melanocytes; 4C11-, non-metastatic melanoma cells; and 4C11+, metastatic melanoma cells), in which each cell line represents a distinct stage of tumor progression. Previous RNA sequencing data comparing these 4 cell lines identified a total of 3404 long non-coding transcripts which displayed differential expression (DE) levels when comparing two or more cell lines. We have clustered 531 of them into three distinct signatures: malignancy, epithelial-to-mesenchymal transition (EMT), or metastasis, according to their expression profiles. We also analyzed the neighboring genes of those lncRNAs, as it is known that lncRNAs might regulate gene expression in cis. We identified the lncRNA Dlx4os, highly expressed in undifferentiated and mesenchymal-like 4C and 4C11- cells, and 6 neighboring genes whose expression correlate to the EMT signature and overall survival in human melanoma patients. Among these genes we found Dlx4, which shares the same promoter site with Dlx4os and whose expression correlates to a poor prognosis. We silenced Dlx4os in the 4C11- cells through interference RNA technology. The downregulation of Dlx4os led to expression changes in EMT-related genes, such as Snail1, Sox10 and Mitf, indicating that it might play a role in EMT. Dlx4os downregulation also inhibited cell migration in vitro and tumorigenesis in vivo. These results overall indicate a role of Dlx4os in melanocyte malignant transformation.

Citation Format: Ana Luísa P. Ayub, Hatylas Azevedo, Diogo Pessoa, Leinal Sejour, Ioannis Vlachos, Eduardo M. Reis, Frank J. Slack, Miriam G. Jasiulionis. Dlx4os: a lncRNA associated with malignant melanocyte transformation. [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 3755.
Abstract 3756: miR-145 and its gene targets as therapeutic target and prognostic biomarkers for non-small cell lung cancer
Authors: Cho William C. , Wong Chi F. , Li Leo K. , Fong Alvin H.
Published: April 2023

Introduction: Our previous study found that miR-145 was downregulated in non-small cell lung cancer (NSCLC) tissues and it could inhibit cell proliferation in transfected NSCLC cells. This study aimed to investigate the potential anticancer effect and tumor suppressive function of miR-145, to identify its gene targets, as well as to assess the role of its gene targets as prognostic biomarkers for NSCLC patients.

Materials and Methods: Cell proliferation, transwell migration, and invasion assays were performed on three miR-145 mimics transfected NSCLC cells. The effect of miR-145 in the cell cycle in vitro was assessed by flow cytometry analysis. In vivo transfection of miR-145 was performed to examine the cancer growth in eight BALB/c nude mice. Luciferase reporter assay was used to identify the gene targets. To reveal the role of the identified gene targets in patient survival, the clinical data of 500 lung adenocarcinoma patients from The Cancer Genome Atlas (TCGA) database was used for Kaplan-Meier survival analysis.

Results: Upregulation of miR-145 significantly inhibited proliferation, migration, and invasion in NSCLC cells in vitro. Flow cytometry analysis revealed that the transfection of miR-145 into NSCLC cells did not show any effect in the cell cycle. After 37 days, transfection of miR-145 showed a reduction in tumor size (49% smaller) and tumor weight (57% lighter) than mimic negative in vivo. MicroRNA target prediction database miRTarBase and luciferase reporter assays identified GOLM1 and RTKN as the direct targets of miR-145. TCGA survival analysis found that the high expressions of GOLM1 (p < 0.0001) and RTKN (p < 0.0045) were significantly correlated with poor overall survival in lung adenocarcinoma patients.

Conclusion: Our results revealed that miR-145 inhibited proliferation, migration, and invasion, but did not affect cell cycle regulation in NSCLC. It also caused a reduction in tumor growth and weight. Most importantly, we identified two direct targets (GOLM1 and RTKN) of miR-145, in which RTKN was first identified as a miR-145 target in NSCLC. Moreover, both GOLM1 and RTKN play an imperative role in patients’ overall survival. Taken together, our findings suggest that miR-145 may serve as a molecular therapeutic target for NSCLC, and its gene targets GOLM1 and RTKN may serve as prognostic biomarkers. Their potential clinical applications warrant further investigation.

Citation Format: William C. Cho, Chi F. Wong, Leo K. Li, Alvin H. Fong. miR-145 and its gene targets as therapeutic target and prognostic biomarkers for non-small cell lung cancer. [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 3756.
Abstract 3763: microRNAs associated with metastatic potential in salivary gland mucoepidermoid carcinoma
Authors: Trevizani Maria Eduarda S. , Oliveira Katia K. , Marchi Fabio A. , Bizinelli Daniela , Mariano Fernanda V. , Nagano Cibele P. , Costa Felipe A. , Pinto Clóvis A. , Kowalski Luiz P. , Lourenço Silvia V. , Coutinho-Camillo Cláudia M.
Published: April 2023

Mucoepidermoid carcinoma (MEC) is the most common malignant tumor of the salivary glands. Metastatic spread occurs in up to 80% of high-grade tumors and it is a strong predictor of poor outcome, however, the mechanisms underlying this process are largely unknown. Large-scale microRNA expression profiling studies of human cancers have demonstrated that dysregulation of miRNA is frequently associated with many cancer types. The aim of this study was to investigate the microRNA profile in metastatic versus non-metastatic MECs. Using Real Time RT-PCR (qPCR) we have analyzed the expression of 384 miRNAs and controls in 4 non-metastatic MECs, 3 MECs with lymph node metastasis, 3 MECs with distant metastasis, and 2 non-neoplastic human salivary gland samples. The microRNA profile was able to discriminate between non-neoplastic and tumor samples and between metastatic and non-metastatic tumors. Considering non-neoplastic samples and non-metastatic MECs, 12 microRNAs were differentially expressed. The non-neoplastic versus lymph node metastasis MEC analysis demonstrated that 10 microRNAs were differentially expressed. Considering non-neoplastic versus distant metastasis MECs, 3 microRNAs were differentially expressed: 1 down-regulated and 2 up-regulated. Comparing non-metastatic MECs versus lymph node metastasis MECs we observed 17 up-regulated microRNAs. Considering non-metastatic MECs versus distant metastasis MECs, 2 microRNAs were up-regulated. One microRNA is differentially expressed between lymph node metastasis MECs and distant metastasis MECs. Our results suggest that microRNA profiles could discriminate the metastatic potential of salivary MECs and might be helpful in diagnostic and prognostic evaluation of patients.

Citation Format: Maria Eduarda S. Trevizani, Katia K. Oliveira, Fabio A. Marchi, Daniela Bizinelli, Fernanda V. Mariano, Cibele P. Nagano, Felipe A. Costa, Clóvis A. Pinto, Luiz P. Kowalski, Silvia V. Lourenço, Cláudia M. Coutinho-Camillo. microRNAs associated with metastatic potential in salivary gland mucoepidermoid carcinoma. [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 3763.
Abstract 3770: Hypoxia-induced circular RNAcircSFMBT2 inhibits tumor malignancy in breast cancer cells
Authors: Liu Chia-Ming , Ke Yi-Chun , Lai Liang-Chuan
Published: April 2023

Breast cancer is the most prevalent cancer among women, and the second major cause of cancer-related deaths in women worldwide. The hypoxic microenvironment of solid tumors is formed in the progress of carcinogenesis, which leads to more malignant and treatment resistance. Recently, circular RNAs (circRNA), a kind of noncoding RNA, were discovered to influence the progress of tumorigenesis, and were induced under hypoxia. However, the regulation and functions of hypoxia-induced circRNA in breast cancer remain unclear. Therefore, the purpose of this study is to identify the regulatory mechanism and its function of a hypoxia-induced circRNAs, circSFMBT2, in breast cancer cells. Firstly, hypoxia-responsive circRNA candidates in breast cancer MCF-7 cells under different oxygen concentrations were identified by RNA-sequencing. Among the differentially expressed circRNAs, circSFMBT2 was chosen for further experiments. The up-regulation of circSFMBT2 under hypoxia was validated by quantitative RT-PCR. The functional assays showed that overexpression of circSFMBT2 inhibited cell proliferation, migration, invasion, and epithelial-mesenchymal transition. Next, in order to investigate the regulatory mechanisms of circSFMBT2, nuclear and cytoplasm fractionation was performed. The results showed circSFMBT2 predominantly localized in the cytoplasm, indicating that the regulatory mechanism of circSFMBT2 might be via interaction with microRNA or RNA-binding proteins (RBP). In order to identify the RBPs, bioinformatic tools, RBPDB, ATtRACT, and RBPmap, were first used to predict the candidate RBPs and validated by RNA pulldown assays. The RNA-binding protein PABPC1 was proved to bind with circSFMBT2. On the other hand, the potential sponging microRNAs were predicted by ENCORI and miRDB, followed by qRT-PCR. No miRNAs were shown to bind with circSFMBT2. Lastly, the functions of circSFMBT2 were explored by identifying its downstream genes using Affymetrix microarrays. The top 10 differentially expressed genes were chosen for validation by quantitative RT-PCR. However, no genes showed significant changes in cells overexpressing circSFMBT2, implying the regulation of circSFMBT2 might not be at the transcriptional level. In summary, circSFMBT2 was able to inhibit the cellular function of breast cancer and bind to PABPC1. With a deeper understanding of the regulatory mechanisms of circSFMBT2, we hope to develop a novel treatment regime for breast cancer.

Citation Format: Chia-Ming Liu, Yi-Chun Ke, Liang-Chuan Lai. Hypoxia-induced circular RNAcircSFMBT2 inhibits tumor malignancy in breast cancer cells. [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 3770.
Abstract 3814: In CLL the U1 snRNA driver mutation alters splicing in multiple genes and pathways
Authors: Senff-Ribeiro Andrea , Almodaresi Fatemeh , Trinh Quang , Shuai Shimin , Spaner David , Puente Xose S. , Campo Elias , Stein Lincoln D.
Published: April 2023

5-10% of patients with the IGHV wild type form of chronic lymphocytic leukemia (CLL) carry a g.3A>C driver mutation in the U1 small nuclear RNA (snRNA). We investigatedthe patterns of mis-splicing and their pathway consequences in three U1-mutant CLL cellline models using long read sequencing in order to better understand the mechanisms ofoncogenicity in tumors carrying this mutation. CLL cell lines (HG3, JVM3 and MEC1) expressing the U1 mutation and their wild-type counterparts were submitted to Oxford Nanopore sequencing to generate full-length RNA transcripts. This transcriptomic datawas evaluated, together with previously obtained short-read data. The g.3A>C mutation occurs at a specific location of the U1 snRNA, a core component of the eukaryotic spliceosome, and acts by altering the 5' splice site (5'SS) recognition sequence to cause consistent patterns of mis-splicing. Long read analysis identified multiple instances of intron retention and suppression of exon skipping, and in silico translation of these mis-splicing events predicted stop-gain and other loss of function mutations in several expressed genes, as well as widespread changes in gene expression levels. We performed a pathway overrepresentation analysis of the altered gene expression patterns in the mutant cells using the Reactome knowledgebase and identified an enrichment in the processes of translation, non-sense mediated decay (NMD) and immune signaling, including interferon signaling. In particular, there was a marked down-regulation of genes related to ribosomal assembly and the translational machinery. A more comprehensiveanalysis of the U1 mutation phenotype in CLL may accelerate the development ofbetter therapeutic and diagnostic approaches in patients.

Citation Format: Andrea Senff-Ribeiro, Fatemeh Almodaresi, Quang Trinh, Shimin Shuai, David Spaner, Xose S. Puente, Elias Campo, Lincoln D. Stein. In CLL the U1 snRNA driver mutation alters splicing in multiple genes and pathways. [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 3814.
Abstract 3819: Naringin nanoparticles inhibit metastasis of lung cancer through TGF-β1-smad2/3 signaling pathway
Authors: Chen Wen
Published: April 2023

Lung cancer causes high mortality due to its high metastasis. Naringin is a naturally occurring flavonoid that is widely found in the various citrus fruits. Naringin is known to have several therapeutic effects including anticancer, anti-pulmonary fibrosis, antimicrobial, anti-inflammatory. However, low bioavailability and solubility of naringin limit its application. This project designed a thermosensitive naringin nanoparticle to investigate whether it might inhibit metastasis of lung cancer by inhibiting TGF-β1-smad2/3 signaling pathway. Cells were treated with various concentrations of naringin nanoparticles and unwrapped naringin for 48 hours. Naringin nanoparticles of 100µmol/L showed obvious inhibitory effect on the larger wound surface of cultured human cancer cells. Westerm blot was used to detect the expression of P-Smad2, Smad2,and α-SMA in A549 cells. The results showed that naringin significantly reduced the expression of EMT-related protein α-SMA in A549 cells in a concentration-dependent manner. Naringin nanoparticles can reverse the EMT process. naringin nanoparticles can inhibit the expression of P-Smad2 protein in A549 cells, which may play a role in inhibiting lung cancer through TGF-β1/Smad signaling pathway.The animal experiment was performed. The mice were given various concentrations of naringin nanoparticles and unwrapped naringin by IV. the protein expression levels of α-SMA, TGF-β1 and P-Smad3 in mice lung tissue were detected by immunohistochemistry. The results showed that naringin nanoparticles 50um/ml,0.2mL/day/time had significant effect on the inhibition of lung cancer tissue migration, and the different doses of naringin nanoparticles showed a certain dose-dependent trend. The expression of TGF-β1 and P-Smad3 in mice treated with naringin nanoparticles was significantly decreased, suggesting that naringin nanoparticles could inhibit the migration of lung cancer cells by inhibiting TGF-β1/Smad pathway.

Citation Format: Wen Chen. Naringin nanoparticles inhibit metastasis of lung cancer through TGF-β1-smad2/3 signaling pathway. [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 3819.
Abstract 3820: Changes in the transcriptome of a triple negative breast cancer cell line following treatment with a water-soluble extract from the leaves of the indigenous Southern African plant Tulbaghia violacea
Authors: Dlamini Zodwa , Hull Rodney , Alouna Mohammed , Penny Clement
Published: April 2023

Triple negative breast cancer (TNBC) accounts for approximately 20% of all breast cancer cases. And is defined by the absence of oestrogen, progesterone, and human epidermal growth factor 2 receptors. This makes it difficult to treat and leads to a low five-year survival rate. In South Africa a large percentage of the population still relies on traditional medicine based on the use of medicinal plants. One of these plants, African garlic (Tulbaghia violacea), that has been used to treat symptoms similar to that observed in cancer. However, the usefulness of these treatments has yet to be clinically proven. Previous studies have revealed that the water-soluble extract from the leaves of T. violacea had anticancer activity against a TNBC cell line. In order to identify the signalling pathways involved in this anti-cancer activity whole transcriptome sequencing was performed using RNA extracted from both TNBC and normal breast cells before and after treatment with the water-soluble T. violacea extract. Paired end sequencing was performed at a depth of 10 million bases for 300 cycles. Data analysis was performed using the Galaxy platform/Sequencing results led us to perform an antibody array assay for proteins involved in the receptor tyrosine kinase pathway. In line with the increased levels of apoptosis previously observed following treatment of the TNBC cell line with the extract, there was an increase in the transcription of genes associated with apoptosis. Decreases were observed in the transcription of genes involved in growth receptor signalling, angiogenesis, and cancer related pathways such as the Wnt, Notch and the PI3K pathway. This work highlights the pathways affected by compounds present within a water-soluble extract of the leaves of T, violacea. Combined with the results of our [previous work, this indicates that the compounds isolated from T, violacea could serve as lead compounds for the development of future therapeutic treatments for TNBC.

Citation Format: Zodwa Dlamini, Rodney Hull, Mohammed Alouna, Clement Penny. Changes in the transcriptome of a triple negative breast cancer cell line following treatment with a water-soluble extract from the leaves of the indigenous Southern African plant Tulbaghia violacea. [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 3820.
Abstract 3828: Herbal extracts from lycii radicis corex and achyranthes japonica prevent multiple myeloma progression
Authors: Mehdi Syed Hassan , Lee Sun-OK , Huh Dam , Yoon Donghoon
Published: April 2023

Multiple Myeloma (MM) is a plasma cell malignancy that, despite advances in treatment, remains incurable. In over 90% of MM patients, aberrant bone remodeling occurs and results in osteolytic lesions. Korean traditional medicine has a long-standing interest in healthy foods to enhance the immune system, energy-boost, and Yin-and-Yang balance. Traditional medicine has used herbal extracts (HE) from natural plants with fewer side effects and long-term treatment tolerance. In earlier studies, lycii radices cortex (LRC) and achyranthes japonica (AJ) containing HE have demonstrated the ability to enhance cell growth and mineralization of osteoblast cells while also inhibiting osteoclast differentiation. The current study investigated the effects of LRC+AJ containing HE on murine 5TGM1 MM-bearing mice.This HE significantly increased the mouse survivals of both Treatment before Transplant (TbT) and Treatment after Transplant (TaT) groups with median survivals of undetermined and 52.5 days, respectively, while MM control had a median survival of 42 days. The Mantel-Cox test found that TbT and TaT mice were significantly different from the control group (P=0.0014 and 0.0182, respectively). In histomorphometric analysis, the spines were scanned by micro-CT and revealed that TbT and TaT groups had significantly increased bone volume over total volume (BV/TV) than control. To see if HE affects cell growth of myeloma cells and osteoblast cells, we further investigated the HE on preosteoblast cell line MC3T3-E1, mouse MM cell line 5TGM1-Luc, and human MM cell line U-266. Cells were treated with HE, and viability was assessed at 48 and 96 hours post-treatment. Remarkably, LRC+AJ containing HE increased MC3T3-E1 cell growth while it decreased 5TGM1 and U-266 cell viability. In addition, we found the DAP3G in HE used in this study.Our results demonstrated that LRC+AJ HE prevents MM and promotes bone formation in 5TGM1 engrafted NSG mice. We also found that LRC+AJ HE suppresses myeloma cell growth and enhances osteoblast cell survival. Although it is yet to be defined, a correlation of osteoblast activity and myeloma cell inhibition suggests a possible connection of these events to prevent MM progression.

Citation Format: Syed Hassan Mehdi, Sun-OK Lee, Dam Huh, Donghoon Yoon. Herbal extracts from lycii radicis corex and achyranthes japonica prevent multiple myeloma progression. [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 3828.
Abstract 3832: Apoptotic and cell cycle effects of triterpenes isolated from Phoradendron wattii on a chronic myelogenous leukemia cell line
Authors: Moo-Puc Rosa E. , Sauri Shirley A. Calderón , Villanueva-Toledo Jairo R. , Peraza-Sánchez Sergio R. , Chávez-González Antonieta
Published: April 2023

Introduction: Medicinal plants have been used in traditional medicine practices since ancient times. Mayan traditional medicine uses herbs as treatment to illness that can be related to cancer disease such as Phoradendron wattii. As part of the search for novel cancer treatment two triterpene compounds were isolated from P. wattii: 3-epi-betulinic acid and lupenone with preliminary activity on leukemias. We investigated the cancer treatment abilities of 3-epi-betulinic acid and lupenone on human chronic myelogenous leukemia (K562) cell line and monocytes isolated from peripheral blood using flow cytometry methods.

Results: Lupenone showed low effect on both cells with an IC50 of 177.60 ± 5.07 µg/mL and a selectivity index (SI) of 0.10 times more on K-562 cell line than on monocytes. Nevertheless, 3-epi-betulinic acid showed a moderate anti-CML activity with an IC50 of 38.74 ± 5.07 µg/mL, having a SI of 4.10 on K-562 cells when compared to monocytes. These results indicate that 3-epi-betulinic acid was 41 times more selective for K-562 cell line than lupenone and it also was 7.45 more selective than etoposide as positive control. Additionally, 3-epi-betulinic acid increased cell population in the G0/G1 phase of the cell cycle and apoptotic cells, and it inhibited cell proliferation in a dose-dependent manner. Furthermore, the relative expression of genes associated with apoptosis Bax and caspase 3 increased compared to control.

Conclusion: Combined, these results suggest that 3-epi-betulinic acid plays an important regulatory role of cell cycle and apoptosis. Finally, the 3-epi-betulinic acid investigated in this work is potentially a cheap, safe, and effective alternative to chemotherapy in cancer treatment.

Citation Format: Rosa E. Moo-Puc, Shirley A. Calderón Sauri, Jairo R. Villanueva-Toledo, Sergio R. Peraza-Sánchez, Antonieta Chávez-González. Apoptotic and cell cycle effects of triterpenes isolated from Phoradendron wattii on a chronic myelogenous leukemia cell line. [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 3832.
Abstract 3842: Berberine recognition of the dGMP-bound PDGFR-β promoter G-quadruplex: Structural insight into specific drug targeting
Authors: Wang Kai-Bo , Dickerhoff Jonathan , Han Yichen , Yang Danzhou
Published: April 2023

PDGFR-β (Platelet-Derived Growth Factor Receptor beta) is a receptor tyrosine kinase whose overexpression drives the progression and metastasis of many cancers. It is an established anticancer target and PDGFR-β inhibition via kinase inhibitors has been actively sought-after. However, PDGFR-β kinase inhibitors often lack specificity. The PDGFR-β gene promoter forms DNA G-quadruplexes that are transcription silencer whose stabilization by small molecules represses PDGFR-β expression. Therefore, targeting PDGFR-β promoter G-quadruplexes for transcriptional silencing is a promising alternative strategy for cancer therapy. G-quadruplex is an exciting family of non-canonical DNA secondary structures. DNA G-quadruplex within the PDGFR-β gene promoter forms novel vacancy pockets (vG4) that can be filled-in by guanine metabolites such as dGMP and cGMP. This presents an attractive target for small molecule targeting. Herein, we present the binding of the natural product berberine with the unique PDGFR-β dGMP-vG4 binary complex to form a novel ternary complex, the first case observed in DNA G-quadruplexes. Using circular dichroism, fluorescence, and nuclear magnetic resonance (NMR) spectroscopy, we demonstrated that berberine binds with high affinity and strongly stabilizes the dGMP-fill-in PDGFR-β vG4. Our determined high-resolution NMR structure of this ternary complex in K+-containing solution reveals a 2:1 binding stoichiometry of berberine to the dGMP-fill-in PDGFR-β vG4. Significantly, one berberine directly stacks upon and extensively covers the dGMP. This direct binding mode provides structural basis for targeting the unique PDGFR-β promoter G-quadruplex by small molecules and suggests an intriguing strategy of designing drug-like, guanine-conjugated molecules to selectively silence the PDGFR-β gene. Because vG4 formation and dGMP fill-in is a unique property of the PDGFR-β promoter G-quadruplex, our study provides a structural basis for rationally designing small molecule drugs to specifically target the PDGFR-β promoter G-quadruplex to inhibit oncogenic PDGFR-β for cancer therapeutics.

Citation Format: Kai-Bo Wang, Jonathan Dickerhoff, Yichen Han, Danzhou Yang. Berberine recognition of the dGMP-bound PDGFR-β promoter G-quadruplex: Structural insight into specific drug targeting. [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 3842.
Abstract 3843: Potent human ClpP protease agonists with anticancer properties bind the enzyme with improved structural complementarity and alter the mitochondrial N-terminome
Authors: Mabanglo Mark F. , Wong Keith S. , Barghash Marim M. , Graves Lee M. , Iwanowicz Edwin J. , Houry Walid A.
Published: April 2023

The mitochondrial ClpP protease is responsible for mitochondrial protein quality control through specific degradation of proteins involved in several metabolic processes. ClpP overexpression is also required in many cancer cells to eliminate ROS-damaged proteins and to sustain oncogenesis. Targeting ClpP to dysregulate its function using small molecule agonists is a novel strategy in cancer therapy. Here, we synthesized novel imipridone-derived compounds and related chemicals, which we characterized using biochemical, biophysical, and cellular studies. Using X-ray crystallography, we found that these compounds have enhanced binding affinities due to their greater shape and charge complementarity with the surface hydrophobic pockets of ClpP. N-terminome profiling of cancer cells upon treatment with one of these compounds revealed the global proteomic changes that arise and identified the structural motifs preferred for protein cleavage by compound-activated ClpP. Together, our studies provide the structural and molecular bases by which dysregulated ClpP affects cancer cell viability and proliferation.

Citation Format: Mark F. Mabanglo, Keith S. Wong, Marim M. Barghash, Lee M. Graves, Edwin J. Iwanowicz, Walid A. Houry. Potent human ClpP protease agonists with anticancer properties bind the enzyme with improved structural complementarity and alter the mitochondrial N-terminome. [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 3843.
Abstract 3851: Targeting RET solvent-front mutants with an alkynyl nicotinamide-based inhibitor
Authors: Khatri Ujjwol , Dayal Neetu , Hu Xueqing , Larocque Elizabeth , Naganna Nimishetti , Shen Tao , Liu Xuan , Holtsberg Frederick W. , Aman M. Javad , Sintim Herman O. , Wu Jie
Published: April 2023

Selpercatinib (LOXO-292, LY3527723) and pralsetinib (BLU-667) are first-in-class RET-targeted cancer therapy drugs. However, secondary RET mutations that confer selpercatinib/pralsetinib resistance have been identified, necessitating development of next-generation RET Tyrosine kinase inhibitors (TKIs). While the G810C/R/S/V mutations located at the RET kinase solvent-front site were detected in selpercatinib-treated patients, it was unclear whether all of these and other potential G810 mutants are resistant to selpercatinib and pralsetinib. We profiled selpercatinib and pralsetinib on all six possible G810 mutants derived from single nucleotide substitution. Surprisingly, the G810V mutant found in a clinical study was not resistant to selpercatinib or pralsetinib. Besides G810C/R/S, G810D also conferred selpercatinib/pralsetinib resistance. We found that alkynyl nicotinamide compounds such as HSN608 have better drug-like properties than alkynyl benzamides. HSN608 inhibited RET and RET V804M gatekeeper mutant, and all six G810 mutants with low nanomolar IC50s in the BaF3/KIF5B-RET mutant cell model. In cell derived xenograft (CDX) tumors driven by KIF5B-RET(G810C), HSN608 caused regression of the selpercatinib-resistant tumors. This study clarifies the sensitivities of different RET solvent-front mutants to selpercatinib and pralsetinib, and identifies an alkylnyl nicotinamide-based RET TKI for inhibiting selpercatinib/pralsetinib-resistant G810 mutants.

Citation Format: Ujjwol Khatri, Neetu Dayal, Xueqing Hu, Elizabeth Larocque, Nimishetti Naganna, Tao Shen, Xuan Liu, Frederick W. Holtsberg, M. Javad Aman, Herman O. Sintim, Jie Wu. Targeting RET solvent-front mutants with an alkynyl nicotinamide-based inhibitor. [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 3851.
Abstract 3933: RRM2 inhibition by COH29 is a potential therapeutic strategy for atypical teratoid rhabdoid tumors
Authors: Giang Le Hien , Wong Tai-Tong , Chang Che-Chang
Published: April 2023

Introduction: Atypical teratoid rhabdoid tumors (ATRT) are a rare but aggressive malignancy in central nervous system, commonly occurring in early childhood. ATRT patients face a dismal prognosis despite aggressive therapy. Development of both effective and protective targeted therapy is an emerging necessity. It has been reported that RRM2 is a master driver of aggressiveness and a poor prognosis biomarker in several cancers. However, little is known about RRM2 function in ATRT. The aim of this study is to assess the roles of RRM2 in ATRT and elucidate the therapeutic potential of RRM2 to cure the ATRT.

Methods: Expression of RRM2 was evaluated by molecular profiling analysis and was confirmed by IHC in both ATRT patients and PDX tissues. Follow-up in vitro studies used 3 different cell lines to elucidate the oncogenic role of RRM2 in ATRT cells. The efficacy of COH29, an RRM2 inhibitor, was assessed in vitro. ATRT model of SCID mice orthotopically injected with ATRT cells were used to test the efficiency of COH29 for in vivo. Western blot and RNA-sequencing were used to determine the mechanisms of RRM2 transcriptional activation in ATRT.

Results: Molecular profiling results revealed that RRM2 was relatively highly expressed in ATRT tissues and cell lines. Clinical relevance analysis results showed that the RRM2 expression level was associated with poor prognosis among ATRT patients. Knockdown of RRM2 by shRNAs significantly reduced ATRT cells colonies formation, proliferation and migration. A similar but more pronounced inhibitory effect was observed when COH29 was used to target RMM2. Inhibition of RRM2 by shRNAs or COH29 significantly induced cell death via DNA damage and/or apoptosis pathways. In vivo results demonstrated that COH29 could suppressed ATRT tumor growth and extend mice overall survival.

Conclusion: Our study identified RRM2 as a potential mediator of oncogenic cellular functions in ATRT cell lines. We propose that highly expressed RRM2 is associated with poor patient outcomes, and inhibition of RRM2 by COH29 may present a significant therapeutic value in ATRT.

Citation Format: Le Hien Giang, Tai-Tong Wong, Che-Chang Chang. RRM2 inhibition by COH29 is a potential therapeutic strategy for atypical teratoid rhabdoid tumors. [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 3933.
Abstract 3939: Inhibition of USP7 upregulates USP22 through desuppression of transcriptional activity of Sp1 in human cancer cells
Authors: Zhang Keqiang , Sun Ting , Li Wendong , Wu Jun , Raz Dan
Published: April 2023

Deubiquitinases (DUBs) play important roles in various human cancers and targeting DUBs is considered as a novel anticancer therapeutic strategy. Overexpression of ubiquitin specific protease 7 and 22 (USP7 and USP22) are associated with malignancy, therapy resistance, and poor prognosis in many cancers. Although both DUBs are involved in the regulation of similar genes and signaling pathways, such as histone H2B monoubiquitination (H2Bub1), c-Myc, FOXP3, and P53, the interdependence of USP22 and USP7 expression has never been described. In the study, we found targeting USP7 via either siRNA-mediated knockdown or pharmaceutical inhibitors dramatically upregulates USP22 in cancer cells. Mechanistically, the elevated USP22 occurs through a transcriptional pathway, possibly due to desuppression of the transcriptional activity of SP1 via promoting its degradation upon USP7 inhibition. Importantly, the elevated USP22 leads to significant activation of downstream signal pathways including H2Bub1 and c-Myc, which may potentially enhance cancer malignancy and counteract the anticancer efficacy of USP7 inhibition. Importantly, targeting USP7 further suppresses the in vitro proliferation of USP22-knockout (USP22-Ko) A549 and H1299 lung cancer cells and induces a stronger activation of P53 tumor suppressor signaling pathway. In addition, USP22-Ko cancer cells are more sensitive to a combination of cisplatin and USP7. USP7 inhibitor treatment further suppresses the in vivo angiogenesis and growth and induced more apoptosis in USP22-Ko cancer xenografts. Taken together, our findings demonstrate that USP7 inhibition can dramatically upregulate USP22 in cancer cells; and targeting USP7 and USP22 may represent a more effective approach for targeted cancer therapy, which warrants further study.

Citation Format: Keqiang Zhang, Ting Sun, Wendong Li, Jun Wu, Dan Raz. Inhibition of USP7 upregulates USP22 through desuppression of transcriptional activity of Sp1 in human cancer cells. [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 3939.
Abstract 3948: DDX5 helicase resolves G-quadruplex and transactivates MYC expression
Authors: Wu Guanhui , Xing Zheng , Chen Luying , Tran Elizabeth J. , Yang Danzhou
Published: April 2023

DDX5 (DEAD-box protein 5) plays an important role in cell proliferation, differentiation, and tumorigenesis. DDX5 is a founding member of the DEAD-box RNA helicase family and known as a non-processive RNA helicase. It acts as a transcriptional co-activator of many cancer-associated genes, such as MYC, however, the underlying molecular mechanism is unknown. MYC is one of the most critical oncogenes and has a DNA G-quadruplex in its proximal promoter region (MycG4) that functions as transcriptional silencer. Guanine-rich DNA and RNA sequences can form G-quadruplex, a non-canonical secondary structure. However, MycG4 is highly stable and its regulatory role in transcription requires active unfolding. Here, we report that DDX5 unfolds MycG4 with extreme efficiency, and thereby transactivates MYC expression. To understand the effects of DDX5 on MycG4, we characterized the unfolding of MycG4-DNA within the DDX5-MycG4 complex using DNA footprinting, FRET and CD spectroscopy. While DDX5 is known as a dsRNA helicase, our results showed that DDX5 is a highly active DNA G4-resolvase. Strikingly, MycG4 unfolding requires neither ATP hydrolysis nor extended loading of single-stranded flanking. Our protein-binding-ELISA experiments revealed specific and high-affinity binding of DDX5 to G4 structures regardless of whether the substrate is DNA or RNA. To elucidate the cellular functions of DDX5, we analyzed publicly available DDX5 ChIP-seq data and identified G-rich sequences in cancer cells as chromatin binding sites of DDX5. Moreover, our ChIP and Western Blot results showed that DDX5 is enriched at the MYC promoter and activates MYC transcription. Importantly, G-quadruplex interactive small molecules can inhibit the DDX5 interaction with the promoter MycG4 and DDX5-mediated transcriptional activation of the MYC gene. In addition, knock-down of DDX5 expression with DDX5-specific siRNA in cancer cells resulted in downregulation of MYC expression and sensitization to G4-interactive small molecules. In summary, we identified resolving DNA and RNA G-quadruplexes as novel function of DDX5. Our results establish that the MYC-transactivation by DDX5 is mediated through unfolding of the MYC promoter quadruplex and thereby establish a new molecular target to suppress MYC for cancer intervention.

Citation Format: Guanhui Wu, Zheng Xing, Luying Chen, Elizabeth J. Tran, Danzhou Yang. DDX5 helicase resolves G-quadruplex and transactivates MYC expression. [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 3948.
Abstract 395: Resistance to tyrosine kinase inhibitor confers an immunosuppressive microenvironment and cross-resistance to immunotherapy through AXL/PDPK1 axis in liver cancer
Authors: Xie Yunong , Huang Bosheng , Yu Huajian , Loh Jia-Jian , Leung Rainbow Wing-Hei , Lee Terence , Ma Stephanie , Tong Man
Published: April 2023

Systemic therapy is the current standard of care for unresectable advanced hepatocellular carcinoma (HCC), the most malignant form of liver cancer. Clinical practice guideline for HCC includes the management of the second-line immunotherapies after patients become unresponsive to first-line targeted therapies using tyrosine kinase inhibitors (TKI), which is expected to generate optimal therapeutic effect due to the distinct mechanism of action between TKIs and immunotherapies. However, evidence from clinical trials suggests that the response to immunotherapy following TKI treatment is often compromised in TKI-resistant HCC as compared with treatment-naïve HCC, indicating a potential cross-resistance to immunotherapy, which impedes the optimal therapeutic effect of the second-line immunotherapies. In the present study, we first explored the therapeutic effect of anti-PD-1 immunotherapy in TKI-resistant HCC and successfully validated the putative cross-resistance to immunotherapy after TKI treatment. We further observed that TKI-resistant HCC exhibits an immune-evasive microenvironment, characterized by decreased CD8+ T cell and dendritic cell infiltration. This is potentially caused by the intratumoral activation of receptor tyrosine kinase AXL and its downstream target PDPK1 which protect TKI-resistant HCC cells from mitochondrial oxidative stress and the cytosolic leakage of mitochondrial DNA, which subsequently suppresses cGAS-STING together with its downstream interferon pathways and immunogenic cell death. Genetic manipulation of AXL and PDPK1 in TKI-resistant HCC cells abrogated the protective effect and thus activated the immunogenic responses. Blockade of AXL with a selective inhibitor BGB324 re-sensitized TKI-resistant HCC to anti-PD-1 treatment. The current findings suggest that targeting AXL/PDPK1 signaling axis might overcome cross-resistance to second-line immunotherapy for HCC patients who progress on TKI treatment.

Citation Format: Yunong Xie, Bosheng Huang, Huajian Yu, Jia-Jian Loh, Rainbow Wing-Hei Leung, Terence Lee, Stephanie Ma, Man Tong. Resistance to tyrosine kinase inhibitor confers an immunosuppressive microenvironment and cross-resistance to immunotherapy through AXL/PDPK1 axis in liver cancer [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 395.