Decrease In Viability Research Articles

Abstract 3394: Participation of molecular iodine and PPARG in prostate cancer progression and neuroendocrine differentiation

Abstract Introduction: Advanced prostate cancer (PCa) therapy is based on androgen deprivation; however, cancerous cells develop resistance, and can develop a highly aggressive neuroendocrine (NE) phenotype. Molecular iodine (I2) has antitumoral effects in breast cancer models, in which PPARG receptors represent the main mechanism of action described. In PCa though, PPARG is considered pro-tumoral and I2 effects have not been fully studied. Hence, the aim of this study was to analyze I2 individual and combined effects with androgen deprivation by using both androgen dependent (AD) and independent (AI), in vitro and in vivo PCa models. Methods: In vitro. I2 effects in cell viability and invasion were analyzed in AD (LNCaP) and AI (DU145, PC3) PCa cells. LNCaP cells were also treated with androgen receptor (AR) antagonists. PPARG participation in I2 effects was assessed with GW9962 (antagonist) and rosiglitazone (agonist). NE phenotype was induced in LNCaP by prolonged culture under androgen-depleted conditions and was assessed by measuring the growth and length of neurite-like projections. RT-qPCR was employed to assess the expression of NE markers (SYP, ENO2), and lipogenic genes (FASN, SREBF1). In vivo. Eighteen weeks-old TRAMP mice were subjected to sham surgery or castration, and were provided or not with I2 (0.025%, drinking water) for four weeks. Prostates were subjected to histopathological analysis (H&E and Masson's trichrome) and IHC (AR, SYP, PPARG). Scoring systems were used to grade the severity of high proliferative intraepithelial lesions (HPIN), and desmoplasia. Results: I2 decreased the cell viability and invasion capacity of all PCa cells tested, and when supplied with AR antagonists further enhanced their antiproliferative effects in LNCaP cells. I2 failed to induce the expression of PPARG target genes, and GW9662 did not prevent I2-induced decrease in cell viability. Like androgen deprivation, I2 also caused NE-like morphology in LNCaP cells, but unlike androgen deprived LNCaP cells, I2 treated cells had decreased expression of NE markers. In TRAMP mice, I2 increased the presence of HPIN, reduced nuclear AR protein detection, and had no effect on PPARG. As expected, castration reduced HPIN, caused epithelial atrophy and desmoplasia; decreased total AR levels and PPARG nuclear levels. However, the number of NE (SYP-positive) cells did not change. The addition of I2 to castration did not modify the score of epithelial lesions but increased the desmoplasia and the presence of NE cells. Conclusions: I2 exhibited in vitro antineoplastic effects over epithelial cells, however, these effects did not translate into the in vivo model. PPARG did not seem to mediate I2 effects. Overall, our data suggest that tumoral microenvironment may interfere with I2 epithelial-specific antitumoral effects. Funding: PAPIIT-UNAM (IN217223, IN202322) and CONAHCYT (774791). Citation Format: Carlos Montes de Oca, Lourdes Álvarez, Carmen Aceves, Brenda Anguiano. Participation of molecular iodine and PPARG in prostate cancer progression and neuroendocrine differentiation [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 3394.

Abstract 1796: Role of FASN in pancreatic neuroendocrine cancer cells survival

Abstract Introduction. Pancreatic neuroendocrine tumors (pNETs) are among the most frequently occurring neuroendocrine neoplasms. Up-regulation of fatty acid synthase (FASN) has been reported in many cancers, but the role of lipid metabolism in pNETs remains unclear. The purpose of this study was to determine whether FASN inhibition can be used as a target for pNET treatment. Methods. Immunohistochemistry and western blot analysis were used to determine protein levels of FASN in pNET tissues and cell lines. Expression of FASN was evaluated in pancreatic neuroendocrine primary and metastatic tumors and scored by a pathologist. Neutral lipid droplet levels in pNET cell lines were determined with BODIPY 493/503 staining. Clonogenic and SRB assays were used to evaluate cytotoxic effects of FASN inhibition by TVB-3664 in QGP-1 and BON pancreatic NET cell lines. Western blot analysis was used to determine levels of cleaved PARP and cyclin D1 after FASN inhibition. FASN knockdown was used to determine the effects of FASN downregulation on proliferation and lipid synthesis. Results. FASN expression was determined at the maximum scores of 6 and 5 in 98% of patient samples. BON, QGP-1 and NT-3 cell lines were stained with BODIPY 493/503; the highest levels of neutral lipid droplets were observed in the BON cell line. BON and QGP-1 cells were treated with TVB-3664 in either complete media with fatty acids (FA+) or complete media prepared with fatty acid depleted FBS (FA-). Treatment with TVB-3664 resulted in significant inhibition of BON and QGP-1 cell proliferation and cyclin D1 expression in both FA+ and FA- conditions. Depletion of extracellular FA enhanced the effect of TVB-3664 therapy on NET cells, especially in the BON cell line. FASN inhibition with TVB-3634 suppressed pNET cell proliferation and colony formation. Conclusions. Our study identified high protein levels of FASN in pNET patient samples and cell lines. Moreover, we demonstrated variability in the response of pNET cells to FASN inhibition. QGP cell viability was significantly decreased with FASN inhibition even in the presence of extracellular lipids. FASN inhibition in BON cells had only a minimal effect on cell viability and required depletion of extracellular lipids to achieve a decrease in cell viability and cyclin D1 expression. These data demonstrate that de novo lipid synthesis is involved in the pathogenesis and progression of pNETs. Importantly, our findings show that some pNETs are highly sensitive to FASN inhibition, suggesting that FASN inhibition may be considered as a treatment option in selected pNETs. Citation Format: Benjamin M. Evers, Zeta Chow, Yekaterina Zaytseva, Eun Y. Lee, Courtney M. Townsend, Tianyan Gao, B. Mark Evers, Piotr G. Rychahou. Role of FASN in pancreatic neuroendocrine cancer cells survival [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 1796.

Abstract 530: The effect of cardamonin on PD-L1 expression in triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) accounts for about 10% of all breast cancers and does not express estrogen, progesterone, or HER2 receptors. It has an aggressive nature, it is highly metastatic, and because of the lack of therapies, it continues to be a challenge. These cancer cells employ mechanisms to escape the immune system that induce the upregulation of PD-L1 expression, a ligand encoded by the CD274 gene. Because cancer cells tend to produce higher levels of PD-L1 compared to normal cells, therapies that inhibit PD-L1 may be helpful. Evidence also shows that high levels of oxidative stress and inflammation may participate in both the initiation and progression of cancer. Cardamonin, an aromatic enone flavonoid, has displayed an array of pharmacological activities, including modulation of different signaling molecules involved in the development and progression of cancer. This work investigated the cardamonin’s ability to modulate PD-L1 expression and NF-KB in genetically distinct MDA-MB-231 (Caucasian) and MDA-MB-468 (African American) TNBC cell lines. The methodology included cytotoxic assays, ELISA, Real Time-Polymerase Chain Reaction (RT-PCR) assays, and Wes analysis. The results showed that cardamonin treatment caused a dose-dependent decrease in cell viability in both cell lines, ranging from 3.12 µM to 200 µM. The RT-PCR and ELISA data showed that even though IFN-γ stimulated MDA-MB-231 cells showed a higher expression of PD-L1, cardamonin reduced mRNA and protein expression of PD-L1 in both cell lines. As a possible molecular mechanism for PD-L1 inhibition, cardamonin modulated MUC-1, STAT3, and JAK, which are genes that activate the PD1/PD-L1 mechanism. Moreover, data also show that cardamonin modulated NF-KB1 and NF-KB2 genes involved in activating NF-KB signaling. NF-KB is a transcription factor of inflammation and immunity, emerging as a key positive regulator of PD-L1 expression in cancer. Therefore, cardamonin may have the potential utilization as a checkpoint inhibitor that acts by blocking PD-L1 protein, allowing the immune system to recognize and attack the TNBC cancer cells, providing a novel cancer therapy. (Supported by a grant from NIH-NIMHD U54 MD 007582) Citation Format: Patricia Mendonca, Bhonessa Kirpal, Sukhmandeep Kaur, Karam F. A. Soliman. The effect of cardamonin on PD-L1 expression in triple-negative breast cancer cells [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 530.

Protective Effects and Mechanisms of Esculetin against H2O2-Induced Oxidative Stress, Apoptosis, and Pyroptosis in Human Hepatoma HepG2 Cells.

Oxidative stress plays a crucial role in the pathogenesis of many diseases. Esculetin is a natural coumarin compound with good antioxidant and anti-inflammatory properties. However, whether esculetin can protect HepG2 cells through inhibiting H2O2-induced apoptosis and pyroptosis is still ambiguous. Therefore, this study aimed to investigate the protective effects and mechanisms of esculetin against oxidative stress-induced cell damage in HepG2 cells. The results of this study demonstrate that pretreatment with esculetin could significantly improve the decrease in cell viability induced by H2O2 and reduce intracellular ROS levels. Esculetin not only apparently reduced the apoptotic rates and prevented MMP loss, but also markedly decreased cleaved-Caspase-3, cleaved-PARP, pro-apoptotic protein (Bax), and MMP-related protein (Cyt-c) expression, and increased anti-apoptotic protein (Bcl-2) expression in H2O2-induced HepG2 cells. Meanwhile, esculetin also remarkably reduced the level of LDH and decreased the expression of the pyroptosis-related proteins NLRP3, cleaved-Caspase-1, Il-1β, and GSDMD-N. Furthermore, esculetin pretreatment evidently downregulated the protein expression of p-JNK, p-c-Fos, and p-c-Jun. Additionally, anisomycin, a specific activator of JNK, blocked the protection of esculetin against H2O2-induced HepG2 cells apoptosis and pyroptosis. In conclusion, esculetin can protect HepG2 cells against H2O2-induced oxidative stress, apoptosis, and pyroptosis via inhibiting the JNK signaling pathway. These findings indicate that esculetin has the potential to be used as an antioxidant that improves oxidative stress-related diseases.

Abstract 3171: The effect of estrogen and phytoestrogens in Inflammatory breast cancer

Abstract Inflammatory breast cancer is an aggressive and lethal type of breast cancer. Frequently, this cancer is diagnosed in an advanced stage with metastasis. IBC diagnosis is associated with a worse survival rate than other types of breast cancer. The presence of dermal and stromal tumor emboli blocking the lymphatic vessels under the skin is considered a hallmark of IBC and assumed to be responsible for the high metastatic behavior and aggressiveness of IBC disease. To this date, there are no effective targeted therapeutics, especially for those patients that account for approximately 20-40% of IBC cases with triple-negative breast cancer (TNBC) classification. Several studies have shown that estrogen can exert non-genomic effects in IBC and non-IBC TNBC, mediated by the expression of alternate estrogen receptors including estrogen receptor alpha-36, and GPR30. In this context, estrogen can activate non-genomic signaling pathways involved in the acquisition of oncogenic phenotypes such as increased motility and invasion in IBC cells. Phytoestrogens, naturally derived structural analogs to 17β-estradiol (E2), were evaluated as an alternative treatment option using in vitro IBC cell models (SUM149 and SUM190). Given the estrogenic properties of phytoestrogens, the drugs were hypothesized to interact with targets in pathways similar to E2 in non-genomic signaling. Several phytoestrogens including coumestrol (Cou) can induce a cytotoxic effect in TNBC. We hypothesize (1) that estrogen non-genomic signaling has an active role in the aggressive metastatic phenotype of IBC, and (2) that Cou has anticancer activity by inhibiting estrogen non-genomic signaling in IBC. To test these hypotheses, IBC cells were treated with E2 or Cou to determine their effects on the activation of downstream kinases by western blot and phospho-kinase array assays. In addition, transcriptional effects upon treatment with Cou were identified using RNA-seq. A dose-response curve in 2D and 3D models was generated to determine the half-maximal inhibitory concentration (IC50) of Cou. Finally, treatment using the IC50 of Cou caused a decrease in cell viability in the TNBC cell line. Also, in comparison to E2, Cou decreases migration, proliferation, and tumor emboli formation in IBC cell lines. In addition, IBC cells were treated using monotherapy and combination therapy with phytoestrogens including Cou, genistein (GN), and/or 8-prenylnaringenin (8-PN). Observed reductions of cell viability in IBC cell lines treated with the different phytoestrogens and combinations make them worthy of further characterization as anti-cancer therapies in in vitro and in vivo models. In summary, IBC cells are responsive to E2 making this signaling pathway attractive for the development of innovative therapeutic strategies with phytoestrogens as potential candidates for treatment. Citation Format: Esther A. Peterson-Peguero, Keishla Rodriguez-Martir, Madeline Ganshert, Dalissa Negron-Figueroa, Ayeisha N. Colon-Ortiz. The effect of estrogen and phytoestrogens in Inflammatory breast cancer [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 3171.

Abstract 4759: Targeting slow-cycling persisters in EGFR mutant non-small cell lung cancer

Abstract Activating mutations in the epidermal growth factor receptor (EGFR) gene in non-small cell lung cancer (NSCLC) patients are associated with clinical benefit in the metastatic and adjuvant settings when treated with EGFR inhibitors, such as osimertinib. Despite its dramatic efficacy, most patients are partial responders and refractive disease leave limited treatment options. The objective was to dissect the mechanism of osimertinib resistance in EGFR mutant NSCLC models to identify a means of re-sensitization. Imaging mass cytometric analysis of 76 NSCLC patients demonstrated EGFR expression was inversely correlated to CD105 membrane expression. CD105 is a transforming growth factor beta (TGF-β) family co-receptor that promotes bone morphogenic protein (BMP) signaling and inhibits TGF-β signaling. Significant elevation in cell surface CD105 by osimertinib was furthered by EGFR knockdown in two NSCLC lines with EGFR activating mutations. These lines were subjected to a program of increasing osimertinib concentrations to generate isogenic resistant lines. Heterogeneous drug-tolerant persister cells had significantly higher mutational load, increased CD105 cell surface expression as well as greater expression of bypass signaling factors such as ERK, PI3K, and BMP ligands compared to their parental counterparts. Single cell RNA-seq analysis of parental NSCLC revealed a small population of cells that shared elevated endothelial and pyrimidine metabolism, with a slow-cycling signature that was enriched in the osimertinib-resistant cells. Combining osimertinib with a CD105 neutralizing antibody, carotuximab, reduced the slow-cycling population and restored osimertinib sensitivity in resistant cell lines. The knockdown of CD105 had a more pronounced decrease in cell viability when combined with osimertinib. The osimertinib-resistant lines demonstrated more condensed chromatin and glycolytic state compared to the respective parental lines by ATAC-seq and Single Cell ENergetIc metabolism profiling, respectively. The addition of carotuximab reversed the chromatin and metabolic reprograming of osimertinib resistance. Carotuximab was discovered to initiate CD105 interaction with EGFR and its dominant active variant, EGFRv3. Ultimately, combination therapy of carotuximab and osimertinib resulted in significantly reduced tumor expansion compared to mice treated with either drug alone. Paradoxically, the small tumors from combination therapy had greater mitosis compared to the larger tumors of the osimertinib single agent treated mice. Inhibition of CD105 with carotuximab can overcome resistance to osimertinib and inhibit NSCLC tumor progression. This is a first-in-class pre-clinical foundation for a novel synthetic lethality treatment strategy resulting from the observation of global changes in EGFR-antagonist resistance as opposed to individual mutations identified in tumor subpopulations. Citation Format: Manish Thiruvalluvan, Sandrine Billet, Zhenqiu Liu, Joseph Lownik, Gabrielle Gonzales, Hyoyoung Kim, Anton L. Villamejor, Larry Milshteyn, Kamya Sankar, Edwin M. Posadas, Jean Lopategui, Sungyong You, Karen Reckamp, Neil A. Bhowmick. Targeting slow-cycling persisters in EGFR mutant non-small cell lung cancer [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 4759.

Abstract 6951: The role of estrogen-dependent activation of p38 MAPK signaling pathway in inflammatory breast cancer

Abstract Inflammatory breast cancer (IBC) is a rare and aggressive type of breast cancer in which the lymphatic vessels become blocked by cancer cells in the breast tissue. The five-year survival rate for this type of cancer is around 40%, one of the worst among breast cancer. Most IBCs are estrogen receptor (ER) and progesterone receptor (PR) negative and HER2 positive, with some presenting an amplification of HER2 while others are ER, PR, and HER2 negative, formally known as the triple-negative (TNBC) subtype. However, it has been reported that treatment with 17β-Estradiol (E2) of ER-negative IBC and non-IBC cells can exert rapid non-genomic signaling mediated by ERα-36, GPR30, and/or estrogen receptor beta, activating MAPK pathways involved in oncogenic phenotypes. Previous data generated in our lab showed that when cells were treated with estradiol it led to increased phosphorylation levels of p38. However, the specific role p38 MAPK signaling takes on in IBC is unknown. Thus, this study centers on examining the role of estradiol and p38 regulation in the triple-negative IBC cell line model, SUM149PT. We hypothesize that upon estradiol treatment p38 signaling pathway is activated resulting in the over-expression of pro-inflammatory cytokines which regulate pro-tumorigenic phenotypes including the growth and survival of IBC cells. Inflammatory cytokines can promote pro-oncogenic features by promoting cell proliferation, angiogenesis, and immune suppression. We treated SUM149 IBC cells with 10nM of estradiol and measured the levels of human cytokines using a Human Cytokine Antibody Array. In addition, treatment using SB202190, a potent p38 inhibitor, caused a significant decrease in cell viability of several breast cancer cell lines in a dose-dependent manner. Ongoing studies include assessing the effects of p38 inhibition proliferation, invasion, and migration. In summary, a better characterization of the effect of E2 and p38 activation in ER-negative cell lines may provide great insight into novel targeted therapies for IBC. Citation Format: Keyla V. Ramirez, Adrian D. Bonilla-González, Jeisac Guzman-Rivera, Esther A. Peterson-Peguero. The role of estrogen-dependent activation of p38 MAPK signaling pathway in inflammatory breast cancer [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 6951.

Abstract 4231: A biobank of bladder cancer organoids as a platform for screening new therapeutic approaches

Abstract In patients with bladder cancer (BC) immune therapies have recently demonstrated an improvement for overall survival rates compared to chemotherapy alone. However, several challenges persist and it remains an unmet need to develop novel therapeutic strategies. Organoids have emerged as a promising animal-free tool revealing patients’ heterogeneity and allowing large preclinical therapeutic screening. Urosphere developed a BC organoids biobank with phenotypic, pharmacological and molecular characterization. The aim of this study was to demonstrate the relevance of our organoids models and their use for validation of therapeutic approaches such as targeted or CAR-T cell therapies using EGFR as target. Molecular characterization of the organoids was performed by exome and whole transcriptome sequencing. Cell architecture and differentiation were assessed by labeling cytokeratins (CK) 5, 17 and 20, and uroplakin 3 (UPK3). Proliferation and tumor status were validated with Ki67 and GATA3, respectively. To evaluate targeted-therapy, organoids were treated with erlotinib for 5 days and cell viability was measured with CellTiter-Glo3D® (CTG) assay. To validate CAR-T cell approach, organoids were co-cultured with anti-EGFR or control anti-CD19 CAR-T cells at 4 effector:target ratios. Cell viability was measured with CTG and Caspase-Glo® 3/7 3D assays. Pro-inflammatory and granzyme B release were analyzed by Luminex® assay. Phenotypically, organoids showed proliferating structures whose tumor status was confirmed by GATA3 expression. Cellular heterogeneity present in original tumors was also found, with differential expression of CKs showing the presence of basal (CK5/CD17) and/or differentiated superficial (CK20/UPK3) cells. Organoids from 2 models with high transcriptomic EGFR expression were selected and protein expression was confirmed by immunofluorescence. For targeted therapy, erlotinib presented significant efficacy in both models with a better activity in the model presenting the highest EGFR expression. After 48h of co-culture with anti-EGFR CAR-T cells, a decrease of cell viability associated with an increase of apoptosis was observed. After 72h of co-culture, an increase in the secretion of pro-inflammatory cytokines (IFN-γ and TNF-α) and granzyme B by anti-EGFR CAR-T cells was also observed. In co-cultures with organoids higher expressing EGFR, CAR-T cells activation appeared higher compared to those co-cultured with organoids lower expressing EGFR. Organoids reproduce characteristics of the tumor from which they are derived, making them a predictive preclinical model. In an EGFR targeting approach, the use of organoids to evaluate both targeted- and CAR-T cell therapies was validated with concordant results. This robust translational model is fully transposable to other therapeutic approaches, whether in a traditional culture model or in a co-culture system. Citation Format: Emilie Decaup, Claire Béraud, Anne Sophie Bajeot, Xavier Gamé, Nicolas Monjotin, Pascal Rischmann, Philippe Lluel, Mathieu Roumiguié. A biobank of bladder cancer organoids as a platform for screening new therapeutic approaches [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 4231.

Resveratrol-Loaded Polydimethylsiloxane-Silica Hybrid Materials: Synthesis, Characterization, and Antitumoral Activity.

In this work, hybrid materials within the polydimethylsiloxane-silica (PDMS-SiO2) system, synthesized via the sol-gel method, were developed and characterized for their potential to incorporate and release the bioactive compound resveratrol (RES). RES was incorporated into the materials with a high loading efficiency (>75%) using the rotary evaporator technique. This incorporation induced the amorphization of RES, resulting in enhanced solubility and in vitro release when compared to the free polyphenolic compound. The release profiles displayed pH dependence, exhibiting notably faster release at pH 5.2 compared to pH 7.4. The gradual release of RES over time demonstrated an initial time lag of approximately 4 h, being well described by the Weibull model. In vitro cytotoxicity studies were conducted on human osteosarcoma cells (MG-63), revealing a concentration-dependent decrease in cell viability for RES-loaded samples (for concentrations >50 µg mL-1).

Abstract 1723: Targeting SMARCA1 in rhabdomyosarcoma: Potential therapeutic implications

Abstract Background: Rhabdomyosarcoma (RMS) is the most common soft tissue in pediatric sarcoma, and studies demonstrate that RMS arises from skeletal muscle precursor cells. RMS, genetically divided into two subtypes: PAX-FOXO1 fusion positive RMS (FP-RMS), which is driven by chromosomal translocation involving PAX3 or PAX7 genes with FOXO1 and PAX-FOXO1 fusion negative RMS (FN-RMS), which is marked by mutations in RAS isoforms and some other genes. Chromatin was one of the earliest identified targets for cancer therapy. Several chromatin remodeling proteins are associated with cancer progression processes such as proliferation, differentiation, apoptosis, and tumorigenesis. Components of chromatin remodeling complexes have been classified as both oncogenes and tumor suppressors. The ISWI family protein, SMARCA1 has been implicated in tumorigenesis for several cancer types. ISWI complexes regulate cell differentiation and proliferation in other cell systems, but their impact on myogenesis is not well understood. In this study, we will characterize the function of SMARCA1 in RMS cells and skeletal muscle. We hypothesize that SMARCA1 acts to modulate chromatin accessibility and drive RMS tumorigenic growth. Methods: We employed RNA-seq, ATAC-seq, and CUT&RUN to study the impact of SMARCA1 in RMS cells. Furthermore, phenotypic experiments were performed to determine the influence of SMARCA1 on differentiation and proliferation. Results: SMARCA1 is expressed at a high level in RMS tissues but not in muscle tissues. Our findings show that SMARCA1 is modestly upregulated during differentiation in normal myogenesis. Upon treatment with trametinib, FP-RMS cell lines with SMARCA1 knockout exhibited a dramatic decrease in cell confluency and viability. Intriguingly, the control FP-RMS cell lines demonstrated increased SMARCA1 protein levels in response to trametinib, which could suggest a resistance mechanism to the treatment. Trametinib also affected SMARCA1 mRNA expression in FN-RMS cells. Additionally, it was discovered that the elimination of SMARCA1 not only resulted in a reduction in cell migration but also led to a failure of these cells to develop spheres, unlike the control cells, where the migration gap healed in the usual manner and the spheres were observed. These findings may align with our hypothesis that SMARCA1 regulates chromatin accessibility, potentially impacting adhesion properties and signaling pathways. Analysis of gene clusters in RMS transcriptomic data indicated that SMARCA1 co-expresses with a RAS effector, RALA. SMARCA1 is also clustered with HDAC2. In addition, SMARCA1 interacted with HDAC2 in RMS cells, as determined by co-immunoprecipitation experiments. Conclusions: Our findings suggest that SMARCA1 may influence RMS tumor growth. This study might contribute to a deeper understanding of SMARCA1's role in RMS and support its credentialing as a novel therapeutic avenue. Citation Format: Ashwaq Aljabri, Katie Hebron, Yuliya Kriga, Juan Manuel Caravaca, Aiysha Althobaiti, Stacey Stauffer, Angela Kim, Lauren Stoak, Morgan Porter, Jyoti Shetty, Bao Tran, Matthew Geisler, Judith Davie, Marielle Yohe. Targeting SMARCA1 in rhabdomyosarcoma: Potential therapeutic implications [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 1723.

Abstract 3210: Raptamer-drug conjugates as molecularly targeted cancer therapeutics

Abstract The use of aptamers as a therapeutic targeting moiety holds the promise of building on the success of antibody-drug conjugates (ADCs). While ADCs are an attractive modality, they face limitations, including limited penetration into solid tumors and a complicated production process for large-scale manufacturing. Aptamers have several potential advantages over antibodies as a targeting moiety including: (i) a smaller size that enables better tumor penetration; (ii) chemical synthesis which allows rapid scale-up and ease of incorporating modifications; (iii) a superior safety profile from lack of immunogenicity and (iv) room temperature stability. Therefore, the use of aptamers in conjunction with drugs (similar to ADCs) holds promise for development of targeted therapeutics. We have identified a novel therapeutic target for certain cancers (COF-01) using proteomics data and immunohistochemistry from patient-derived tumors. Both the COF-01 protein and related mRNA expression was shown to be significantly increased in non-small cell lung cancer, head and neck cancer and pancreatic cancer. Here, we present a new therapeutic approach that targets COF-01-positive cancer cells using next generation aptamer conjugates. We performed a cell- and protein-based selection using our proprietary bead-based combinatorial libraries to generate next-generation base-modified ssDNA aptamers (Raptamers) against COF-01. Binding affinities of the COF-01 Raptamer candidates were determined by concentration curves using biolayer interferometry. The dissociation constants for the COF-01 Raptamers ranged from 80 nM - 200 nM. COF-01-specific Raptamer-drug conjugates (Rap-DCs) were developed by conjugating the Raptamers to monomethyl auristatin E via a cleavable linker. In vitro efficacy data showed the COF-01 Rap-DC can selectively induce cytotoxicity in different types of COF-01-positive human cancer cells (A431, CAL 27, BxPC-3). There was a significant decrease in cell viability of COF-01-positive cells when compared to COF-01-negative control cells (IC50 values ranged from 5.19 nM - 14.11 nM). In vivo efficacy testing in a COF-01-postive cell-derived xenograft mouse model showed a significant decrease in tumor volume in animals treated with the COF-01 Rap-DC when compared to saline- and erlotinib-treated controls. This provides proof-of concept for the use of high-affinity Raptamers against novel molecular targets, such as COF-01. This platform can be used to develop Rap-DCs against emergent neo-antigens on cancer cells that have escaped chemotherapy. Citation Format: Stephanie P. Vega, Nancy E. Ward, Uksha Saini, Leniher Castan-Chibas, Garima Kaushik, Daniel Ciznadija, Dev Chatterjee, Michael J. Heffernan, Atul Varadhachary, Michael Ritchie. Raptamer-drug conjugates as molecularly targeted cancer therapeutics [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 3210.

Abstract 7224: Investigation of the efficacy of duocarmycin SA in combination with FDA approved drugs in acute myeloid leukemia cells in vitro

Abstract Acute myeloid leukemia (AML) is a hematological malignancy that is characterized by the arrest of hematopoietic stem cell differentiation, clonal expansion of myeloid hematopoietic precursors and poor patient survival outcomes. Despite therapeutic advancements, the prognosis of AML patients remains poor and improving patient survival outcomes continues to be a challenge. Therefore, the objective of this study was to investigate the efficacy of Duocarmycin SA (DSA- an antitumor antibiotic that induces DNA alkylation) in combination with FDA approved drugs currently used in the treatment of AML. In this study, DSA was evaluated in combination with doxorubicin (an anthracycline that disrupts topoisomerase II mediated DNA repair) or etoposide (a topoisomerase II inhibitor used to treat relapsed/refractory AML) in human AML cells in vitro. We hypothesized that DSA in combination with doxorubicin or etoposide would exhibit synergistic effects by improving the efficacy of both doxorubicin and etoposide in AML cells. The human AML cell lines (Molm-14 and HL60) were used for all studies. The phenotype of the cells was confirmed by flow cytometry and the IC50 of the drugs were determined using the MTT assay. Our results showed that the AML cells were CD45+, CD33+, CD13+ and CD4+. The IC50 of DSA alone for Molm-14 cells and HL60 cells were 11.12 pM and 114.8 pM, respectively. DSA alone induced DNA double-stranded breaks, significantly decreased the production of colonies and significantly increased apoptosis in AML cells in a dose-dependent manner. The IC50 of doxorubicin alone and etoposide alone was 68.6 nM and 129.7 nM, respectively. DSA in combination with the FDA approved AML drugs showed increased cytotoxic efficacy by observable decreases in AML cell viability. In summary, 1) picomolar concentrations of DSA alone was sufficient to significantly reduce AML cell viability; 2) DSA in combination with doxorubicin or etoposide significantly reduced AML cell viability and reduced the IC50 concentration of doxorubicin and etoposide. Thus, highlighting the therapeutic potential of using DSA in combination with doxorubicin or etoposide as a drug combination strategy to improve AML patient survival outcomes by decreasing the concentration of drugs necessary to treat patients and reducing off-target toxicity. Citation Format: William A. Chen, Diego Aguilar, Leena So, Yasmeen Jawhar, Nhi Nguyen, Natalie Drew, Terry G. Williams, Carlos A. Casiano, Sinisa Dovat, Kristopher Boyle, Olivia L. Francis-Boyle. Investigation of the efficacy of duocarmycin SA in combination with FDA approved drugs in acute myeloid leukemia cells in vitro [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 7224.

Abstract 691: Preclinical evaluation of valproic acid in combination with radiation in orthotopic models of glioblastoma

Abstract Background: Valproic acid (VPA) is an FDA-approved anti-seizure medication that has been demonstrated to have an anti-tumor and radiosensitization effect on multiple cancer cells including aggressive brain cancer, glioblastoma (GBM). VPA is thought to exert its anti-tumor effects in part through inhibition of histone deacetylases (HDACs), upregulation of apoptosis and autophagy, and disruption of DNA repair mechanisms. Overall survival of patients treated with VPA in combination with TMZ and radiation therapy (RT) was significantly improved compared to historical controls (mOS 29.6 months). Hence, to establish a preclinical benchmark, we investigated the effectiveness of VPA alone and in combination with the radiotherapy in patient-derived xenograft (PDX) models of GBM in vitro and in vivo. Methods: Anti-proliferative effect of VPA in combination with radiation was assessed across multiple GBM cells lines (long-term cultures and PDX lines). Athymic mice were implanted with PDX GBM cells orthotopically and were randomized into four cohorts: vehicle, VPA, IR (6 gy), VPA + IR (6 gy). Animals were treated with 300mg/kg/day of VPA alone or in combination with 6Gy of radiation. Treatments were performed either via intraperitoneal route for 3 consecutive days or via a gastric gavage 5 days on/2 days off for the duration of the study. For pharmacodynamics (PD), animals were sacrificed after 3 days of drug treatment at 2hr, 24hr and 4 days post last dose and processed for immunohistochemical (IHC) analysis for acetylated H3K9/14, KI67, pH2AX, and cleaved caspase 3. Results: VPA treatment demonstrated a dose-dependent decrease in GBM cell viability and enhancement of radiation sensitivity. Pharmacodynamic analysis of tumor-bearing mice demonstrated a significant increase in acetylated H3K9/14 levels upon treatment with combination of VPA and irradiation. Elevation of acetylated H3K9/14 levels was present 2 hours after the administration of the last dose of VPA and it persisted for 4 days. Treatment of mice with irradiation upregulated levels of the apoptosis marker, cleaved caspase 3, which was further enhanced upon VPA and irradiation combination treatment. No reduction in tumor volume or survival benefit was seen in VPA alone or combination cohorts. Conclusion: Consistent with previous reports, treatment of PDX GBM line with VPA reduced cell viability in vitro, increased histone acetylation, and cell death. However, these changes were not sufficient to provide survival benefit in the intracranial model and warrants further investigation into the genetic requirements for radiation sensitization by VPA. Citation Format: Mariya Stavnichuk, Zorana Opachich, Dylan Huttenlocher, James McNamara, Jennifer Molloy, Xu He, Sonam Patel, Nader Sanai, An-Chi Tien, Shwetal Mehta. Preclinical evaluation of valproic acid in combination with radiation in orthotopic models of glioblastoma [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 691.

Encapsulating Epigallocatechin-3-Gallate (EGCG) in Chitosan Nanoparticles Influenced Multiple Genes Controlling Oncostatic Signaling Pathways in HepG2 Cells

AbstractThe primary polyphenol in green tea, epigallocatechin-3-gallate (EGCG), is the primary active polyphenol in green tea that showed a remarkable anticancer effect in a variety of cancer types including liver cancer. However, its anticancer impact on gene expressions related to liver cancer proliferation and apoptosis is limited. The current study investigated the oncostatic influence of chitosan encapsulating EGCG (Chit-nanoEGCG) on human hepatocellular carcinoma HepG2 cells compared with its native form and the conventional anticancer drug cisplatin in vitro. The Chit-nanoEGCG caused a dose-dependent decrease in cell viability and significantly induced apoptosis compared with control. Flow cytometric analysis confirmed the results of RT-qPCR, displaying a significant increase in the expression of transcriptomes (P53, Bax, Caspase-3, Caspase-9, and PARP) with a significant decrease in the antiapoptotic Bcl-2 gene. Also, there was a significant decrease in transcription genes (OCT4 and SOX2) and receptor genes (CD133, NOTCH1, c-MET, and Ezrin) but an increase in CD95 expression. Furthermore, there was a highly significant decrease in oncogenes (mTOR, PI3K, RALA, and BMI) in HepG2 cells after Chit-nanoEGCG treatment when compared with the control group. The current findings indicate that Chit-nanoEGCG had a significant impact on a number of genes involved in controlling signaling pathways to inhibit the proliferation and development of HepG2 cells. In addition, encapsulating EGCG in chitosan nanoparticles increases its antitumor effectiveness compared to its native form, which encourages the use of various polyphenols in nanochemotherapy for cancer treatment. The finding that the nanoparticles (NPs) influenced the expression of genes involved in apoptosis and proliferation enables the development of medical chemotherapeutic agent for the treatment of hepatocellular carcinoma. Graphical Abstract

Abstract 3165: Targeting angiogenesis for improved survival in mantle cell lymphoma

Abstract Mantle cell lymphoma (MCL) is a subtype comprising 6% of all NHL cases and is characterized by overexpression of cyclin D1 due to chromosomal translocation t(11;14) in the nodal MCL subtype. One of the best discriminatory genes between these conventional and indolent MCL tumors is SOX11; in addition, SOX11 has been shown to promote angiogenesis in MCL. Further SOX11 protein expression in MCL patients correlated with increased microvascular density and poor outcomes. Bulk transcriptomics analysis was performed on MCL patients (n=24) and CD19-purified B cells from healthy donors (HD,n=3). To understand the role of the angiogenic process in MCL, we queried 141 genes related to angiogenesis (GO:0001525). Our analysis showed that many genes involved in angiogenesis were predominantly overexpressed in MCL compared to HD, including FLT1 and KDR (p-value;0.05; FC>2). SOX11 has been shown to regulate PDGFA in MCL cell lines. Consistent with this observation, we also see upregulated PDGFA levels in MCL patients. However, PDGFA upregulation was not statistically significant in our analysis. Further expression of VEGFR2 is overexpressed in MCL Patients (n=122) compared to naïve or activated-B cells from HD. We validated that VEGFR2 protein levels were higher in MCL primary patients than in healthy donor PBMC. Further, in two independent cohorts, high VEGFR2 expression shows significant adverse effects on 5-year overall survival (OS). To explore the potential of targeting VEGFR, we utilized lucitanib, which potently and selectively inhibits VEGFRs. Treatment with lucitanib resulted in a dose-dependent decrease in cell viability and cellular death as determined by Annexin PI staining; further, MCL cells treated with lucitanib showed inhibition of autophosphorylation of VEGFR2 and demonstrated PARP cleavage associated with cytotoxicity in-vitro. To assess the role of VEGFR2 inhibitor lucitanib as a single agent in MCL growth in-vivo, we utilized two different murine models of MCLs. First, we used cell-line-derived xenograft (CDX) models of MCL where Jeko-1 and HS5 cells were injected together, followed by treatment (lucitanib;15mg/kg QOD; IP) and measured tumor volume until the ERC of any experimental mice was reached. Kaplan-Meier survival graphs were plotted, demonstrating that lucitanib treatment prolongs survival in-vivo. Further, to confirm if the lucitanib can provide an overall survival benefit in the systemic PDX MCL model, we injected the PDX cells via the tail vein in NSG mice and followed human-B2M levels for disease progression, and overall survival was the endpoint. Lucitanib treatment significantly prolonged median overall survival significantly in the systemic MCL model. Our data demonstrates that targeting angiogenesis in MCL can provide therapeutic benefits, as demonstrated in preclinical MCL models. Citation Format: Satishkumar Singh, Anuvrat sircar, Audrey Odier, Narendranath Epperla, Natarajan Muthusamy, Samir Parekh, Lalit Sehgal. Targeting angiogenesis for improved survival in mantle cell lymphoma [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 3165.

Polymeric Nanoparticles Enable mRNA Transfection and Its Translation in Intervertebral Disc and Human Joint Cells, Except for M1 Macrophages.

Chronic lower back pain caused by intervertebral disc degeneration and osteoarthritis (OA) are highly prevalent chronic diseases. Although pain management and surgery can alleviate symptoms, no disease-modifying treatments are available. mRNA delivery could halt inflammation and degeneration and induce regeneration by overexpressing anti-inflammatory cytokines or growth factors involved in cartilage regeneration. Here, we investigated poly(amidoamine)-based polymeric nanoparticles to deliver mRNA to human joint and intervertebral disc cells. Human OA chondrocytes, human nucleus pulposus (NP) cells, human annulus fibrosus (AF) cells, fibroblast-like synoviocytes (FLS) and M1-like macrophages were cultured and transfected with uncoated or PGA-PEG-coated nanoparticles loaded with EGFP-encoding mRNA. Cell viability and transfection efficiency were analyzed for all cell types. Nanoparticle internalization was investigated in FLS and M1-like macrophages. No significant decrease in cell viability was observed in most conditions. Only macrophages showed a dose-dependent reduction of viability. Transfection with either nanoparticle version resulted in EGFP expression in NP cells, AF cells, OA chondrocytes and FLS. Macrophages showed internalization of nanoparticles by particle-cell co-localization, but no detectable expression of EGFP. Taken together, our data show that poly (amidoamine)-based nanoparticles can be used for mRNA delivery into cells of the human joint and intervertebral disc, indicating its potential future use as an mRNA delivery system in OA and IVDD, except for macrophages.

Wireless electrostimulation for cancer treatment: An integrated nanoparticle/coaxial fiber mesh platform

Cancer, namely breast and prostate cancers, is the leading cause of death in many developed countries. Controlled drug delivery systems are key for the development of new cancer treatment strategies, to improve the effectiveness of chemotherapy and tackle off-target effects. In here, we developed a biomaterials-based wireless electrostimulation system with the potential for controlled and on-demand release of anti-cancer drugs. The system is composed of curcumin-loaded poly(3,4-ethylenedioxythiophene) nanoparticles (CUR/PEDOT NPs), encapsulated inside coaxial poly(glycerol sebacate)/poly(caprolactone) (PGS/PCL) electrospun fibers. First, we show that the PGS/PCL nanofibers are biodegradable, which allows the delivery of NPs closer to the tumoral region, and have good mechanical properties, allowing the prolonged storage of the PEDOT NPs before their gradual release. Next, we demonstrate PEDOT/CUR nanoparticles can release CUR on-demand (65 % of release after applying a potential of −1.5 V for 180 s). Finally, a wireless electrostimulation platform using this NP/fiber system was set up to promote in vitro human prostate cancer cell death. We found a decrease of 67 % decrease in cancer cell viability. Overall, our results show the developed NP/fiber system has the potential to effectively deliver CUR in a highly controlled way to breast and prostate cancer in vitro models. We also show the potential of using wireless electrostimulation of drug-loaded NPs for cancer treatment, while using safe voltages for the human body. We believe our work is a stepping stone for the design and development of biomaterial-based future smarter and more effective delivery systems for anti-cancer therapy.

Abstract 4760: Inhibition of FASN postpones development of resistance to BRAF inhibitors in colorectal cancer

Abstract Introduction: Mutations in proto-oncogene BRAF occur in about 10-15% of CRC patients and BRAFV600E is the most common. For patients who undergo BRAF-targeted therapy, resistance develops 4-6 months after treatment initiation and results in more aggressive disease. We found that development of resistance to BRAF inhibitors (BRAFi) is associated with an increase in lipid metabolism and expression of fatty acid synthase (FASN). FASN is a key enzyme of lipid synthesis overexpressed in CRC. Therefore, our hypothesis is that inhibition lipid metabolism via FASN will postpone development of resistance to BRAFi. Methods: We established CRC cells resistant to PLX8394, a novel BRAFi. To evaluate differences in parental and resistance cells, CellTiterGlo 2.0, PrestoBlue, CytoSelectTM Invasion Assay, Triglyceride Assay, Seahorse XF, and confocal microscopy were used. Combination of PLX8394 and TVB3664 or C75 (FASN inhibitors) was tested on cell viability, colony formation, and synergy studies in parental and BRAFi resistant cells. Results: The development of resistance to BRAFi promotes cellular proliferation and increases cyclin D and survivin expression in vitro and in vivo. BRAFi resistance is also associated with an increase in invasive properties and loss of E-cadherin expression. Metabolic changes include an increase in lipid metabolism, oxidative phosphorylation, and triglycerides storage. RNAseq and western blot analysis show significant upregulation of FASN in BRAFi resistant cells. Using cell viability and soft agar colony formation assays, we show that combined PLX8394 and TVB3664 treatment leads to a significantly higher decrease in cell viability and colony formation as compared to each drug alone in parental cells but not in BRAFi resistant cells. The calculation of a Bliss synergy score confirms that combination treatment with C75 and PLX8394 has synergetic effect in BRAFV600E cells. To further confirm that FASN contributes to resistance to BRAFi, we show that HT29 FASN shRNA cells are more susceptible to PLX8394 treatment as compared to control cells. Importantly, our data show that the long-term treatment with PLX8394 in combination with TVB3664 postpones development of resistance to BRAFi as compared to cells treated with PLX8394 alone. Conclusion: Our study demonstrates that resistance to BRAFi is associated with a significant increase in proliferation, metastasis, and lipid metabolism. We demonstrate that combination of FASN inhibitors and BRAFi postpones development of resistance in BRAFV600E cells. However, FASN inhibition does not sensitize cells to BRAFi in already resistance cells, suggesting that this approach cannot be used to overcome acquired resistance to BRAFi. In summary our data suggest that an addition of TVB3664 at the beginning of BRAF treatment regimen could be an efficacious treatment strategy for BRAFV600E CRC patients. Citation Format: Mariah Geisen, Josiane weber-Tessmann, Courtney Kelson, Daheng He, Chi Wang, Abu Saleh Mosa Faisal, Jill Kolesar, Yekaterina Zaytseva. Inhibition of FASN postpones development of resistance to BRAF inhibitors in colorectal cancer [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 4760.

Abstract 7593: Photodynamic therapy improves therapeutic management of glioblastoma

Abstract Glioblastoma (GBM) remains a formidable challenge in the realm of oncology, primarily due to the limitations of the standard of care therapies. Photodynamic therapy (PDT), which exploits the light-activated potential of 5-aminolevulinic acid (5-ALA), emerges as a promising avenue to combat GBM. In this study, primary patient-derived cell lines, Ox5 core, and G7, served as critical subjects, exposed to light excitation at wavelengths of 410 nm, 528 nm, and 810 nm, both with and without 5-ALA (protoporphyrin IX). The central objective was to rigorously assess the efficacy of PDT, either with or without 5-ALA, in reducing cell viability and curtailing clonogenic expansion. This evaluation was performed within the context of the existing standard of care for GBM, encompassing Temozolomide (TMZ) and Radiotherapy. Our results unveiled striking insights into the potential of PDT as an innovative therapeutic strategy. Notably, Ox5 cells exhibited a remarkable reduction in viability, exceeding 70%, following exposure to PDT with 410 nm light and 5-ALA at a concentration of 50μM. A similar, albeit slightly less profound, effect was observed in G7 cells, with a viability decrease of more than 53%. These outcomes, meticulously quantified through the MTT viability assay (n=4), were reinforced by statistical significance, as indicated by p-values falling below 0.05 and, lower 0.001. Furthermore, clonogenic assays employing G7 cells provided an additional layer of evidence, demonstrating a substantial inhibition of clonogenic potential following PDT treatment with 410 nm light and 5-ALA. Immunohistochemistry techniques, particularly focusing on the cleaved caspase-3 marker, in both Ox5 core and G7 cells, underscoring the mechanistic basis of the observed cell death. In stark contrast, the conventional standard of care, involving TMZ monotherapy, was tested, and the results across four biological replicates were less significant. The relative viability of Ox5 and G7 cells exhibited no statistical significance, with p-values exceeding 0.05, implying a limited impact of this regimen on cell viability. Moreover, clonogenic assays conducted under the standard of care protocol (TMZ+/-Radiotherapy) unveiled the persistence of colonies, even at higher radiation doses of 4 and 6 Gy.In summary, PDT, specifically when implemented with 410 nm light and 5-ALA, represents a breakthrough strategy that leads to substantial cell death in both Ox5 and G7 cells. This compelling alternative therapy shines a hopeful light on the horizon of GBM treatment, providing a potentially transformative approach that can augment the current standard of care. Citation Format: Nazar Vasyliv, Karin Williams. Photodynamic therapy improves therapeutic management of glioblastoma [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 7593.

Knockdown of nicotinamide N-methyltransferase suppresses proliferation, migration, and chemoresistance of Merkel cell carcinoma cells in vitro.

Merkel cell carcinoma (MCC) is an aggressive skin cancer, with a propensity for early metastasis. Therefore, early diagnosis and the identification of novel targets become fundamental. The enzyme nicotinamide N-methyltransferase (NNMT) catalyzes the reaction of N-methylation of nicotinamide and other analogous compounds. Although NNMT overexpression was reported in many malignancies, the significance of its dysregulation in cancer cell phenotype was partly clarified. Several works demonstrated that NNMT promotes cancer cell proliferation, migration, and chemoresistance. In this study, we investigated the possible involvement of this enzyme in MCC. Preliminary immunohistochemical analyses were performed to evaluate NNMT expression in MCC tissue specimens. To explore the enzyme function in tumor cell metabolism, MCC cell lines have been transfected with plasmids encoding for short hairpin RNAs (shRNAs) targeting NNMT mRNA. Preliminary immunohistochemical analyses showed elevated NNMT expression in MCC tissue specimens. The effect of enzyme downregulation on cell proliferation, migration, and chemosensitivity was then evaluated through MTT, trypan blue, and wound healing assays. Data obtained clearly demonstrated that NNMT knockdown is associated with a decrease of cell proliferation, viability, and migration, as well as with enhanced sensitivity to treatment with chemotherapeutic drugs. Taken together, these results suggest that NNMT could represent an interesting MCC biomarker and a promising target for targeted anti-cancer therapy.