Neuroblastoma Tumor Growth Research Articles

Therapeutic SHPRH-146aa encoded by circ-SHPRH dynamically upregulates P21 to inhibit CDKs in neuroblastoma

Recent research has underscored the significance of circular RNAs (circRNAs) in various cancers, including neuroblastoma (NB). Specifically, circ-SHPRH, a unique circRNA, has been revealed to inhibit tumor growth by sequestering miRNAs or producing the SHPRH-146aa protein. To explore circ-SHPRH's involvement in NB and its potential application in gene therapy, this study examined circ-SHPRH expression in 94 NB tissues and cell lines (SK–N-BE(2), SH-SY5Y) using real-time PCR and fluorescence in situ hybridization (FISH). Functional assays encompassing both overexpression and knockdown experiments in NB cell lines, as well as in vivo investigations, were conducted. RNA-seq analysis revealed a correlation between circ-SHPRH and the pathway of P21 (CDKN1A), a pivotal cell cycle regulator. Validation through PCR and other techniques confirmed that circ-SHPRH upregulated P21 expression. Furthermore, the regulatory role of circ-SHPRH in the P21-CDK pathway was corroborated through SHPRH-146aa expression analysis. Notably, adenovirus-mediated circ-SHPRH overexpression effectively curbed NB tumor growth in NSG mice, while combining circ-SHPRH with everolimus exhibited potential for NB treatment. This study elucidates the remarkable significance of circ-SHPRH in NB and its prospective utility in gene therapy, thereby paving the way for innovative therapeutic approaches.

Development of red blood cell-derived extracellular particles as a biocompatible nanocarrier of microRNA-204 (REP-204) to harness anti-neuroblastoma effect

Neuroblastoma (NB) is the most common extracranial solid tumor in the pediatric population with a high degree of heterogeneity in clinical outcomes. Upregulation of the tumor suppressor miR-204 in neuroblastoma is associated with good prognosis. Although miR-204 has been recognized as a potential therapeutic candidate, its delivery is unavailable. We hypothesized that REP-204, the red blood cell-derived extracellular particles (REP) with miR-204 loading, can suppress neuroblastoma cells in vitro. After miR-204 loading by electroporation, REP-204, but not REP carriers, inhibited the viability, migration, and 3D spheroid growth of neuroblastoma cells regardless of MYCN amplification status. SWATH-proteomics revealed that REP-204 treatment may trigger a negative regulation of mRNA splicing by the spliceosome, suppression of amino acid metabolism and protein production, and prevent SLIT/ROBO signaling-mediated cell migration, to halt neuroblastoma tumor growth and metastasis. The therapeutic efficacy of REP-204 should be further investigated in preclinical models and clinical studies.

Opaganib Downregulates N-Myc Expression and Suppresses In Vitro and In Vivo Growth of Neuroblastoma Cells.

Neuroblastoma (NB), the most common cancer in infants and the most common solid tumor outside the brain in children, grows aggressively and responds poorly to current therapies. We have identified a new drug (opaganib, also known as ABC294640) that modulates sphingolipid metabolism by inhibiting the synthesis of sphingosine 1-phosphate (S1P) by sphingosine kinase-2 and elevating dihydroceramides by inhibition of dihydroceramide desaturase. The present studies sought to determine the potential therapeutic activity of opaganib in cell culture and xenograft models of NB. Cytotoxicity assays demonstrated that NB cells, including cells with amplified MYCN, are effectively killed by opaganib concentrations well below those that accumulate in tumors in vivo. Opaganib was shown to cause dose-dependent decreases in S1P and hexosylceramide levels in Neuro-2a cells, while concurrently elevating levels of dihydroceramides. As with other tumor cells, opaganib reduced c-Myc and Mcl-1 protein levels in Neuro-2a cells, and also reduced the expression of the N-Myc protein. The in vivo growth of xenografts of human SK-N-(BE)2 cells with amplified MYCN was suppressed by oral administration of opaganib at doses that are well tolerated in mice. Combining opaganib with temozolomide plus irinotecan, considered the backbone for therapy of relapsed or refractory NB, resulted in increased antitumor activity in vivo compared with temozolomide plus irinotecan or opaganib alone. Mice did not lose additional weight when opaganib was combined with temozolomide plus irinotecan, indicating that the combination is well tolerated. Opaganib has additive antitumor activity toward Neuro-2a tumors when combined with the checkpoint inhibitor anti-CTLA-4 antibody; however, the combination of opaganib with anti-PD-1 or anti-PD-L1 antibodies did not provide increased antitumor activity over that seen with opaganib alone. Overall, the data demonstrate that opaganib modulates sphingolipid metabolism and intracellular signaling in NB cells and inhibits NB tumor growth alone and in combination with other anticancer drugs. Amplified MYCN does not confer resistance to opaganib, and, in fact, the drug attenuates the expression of both c-Myc and N-Myc. The safety of opaganib has been established in clinical trials with adults with advanced cancer or severe COVID-19, and so opaganib has excellent potential for treating patients with NB, particularly in combination with temozolomide and irinotecan or anti-CTLA-4 antibody.

Small-molecule inhibition of the METTL3/METTL14 complex suppresses neuroblastoma tumor growth and promotes differentiation

The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors invivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.

Abstract 5988: The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma

Abstract Dysregulation of the Ubiquitin Proteasome System has been linked to many human diseases, including cancer. Expression of UBE4B ubiquitin ligase is associated with neuroblastoma patient outcomes and its functional roles in neuroblastoma pathogenesis are not known. We have recently identified a ubiquitin complex ITCH/UBE4B which mediates ubiquitination of Ku70 and c-FLIPL proteins for proteasomal degradation allowing HDAC inhibitor-mediated caspase-8 dependent apoptosis. We also confirmed that the deubiquitinase USP7, critical player in tumor suppression and DNA repair, can be a potential therapeutic target for neuroblastoma. Highly selective USP7 inhibitors have demonstrated significant antitumor activity in preclinical models of adult cancer, and we reported that these inhibitors alone can be more effective against neuroblastoma tumor growth. Our hypothesis is that USP7 inhibition will be more effective against neuroblastoma tumors through destabilization of ITCH/UBE4B complex protein targets allowing a stronger induction of cell death in response to treatment for the children. To evaluate efficacy of USP7 inhibitors in combination with HDAC inhibitors or chemotherapy against neuroblastoma tumor growth, neuroblastoma cell lines depleted for UBE4B or USP7 were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and apoptosis by caspase cleavage and PARP cleavage detection by western blot. We have evaluated also, whether ubiquitination/deubiquitination and degradation rates of p53, Ku70 and c-FLIPL proteins could modulate induction of apoptosis and necroptosis. USP7 inhibition resulted in decreases in cell viability in p53 wild-type neuroblastoma cell lines. USP7 inhibition induced apoptosis and necroptosis by increasing phosphorylation of MLKL, RIPK3 and RIPK1. In UBE4B or USP7 depleted cells, a huge induction of necroptosis and a small rate of apoptosis were observed. We also identified USP7 as a deubiquitinase of Ku70, stabilizing Ku70 at the basal level, leading to stabilization of Ku70/c-FLIPL/Bax complex. USP7 inhibition destabilizes ku70 and c-FLIPL for protein degradation leading to induction of apoptosis as well as necroptosis. UBE4B depletion in neuroblastoma inhibits HDAC inhibitors mediated caspase-8 mediated apoptosis but enhances necroptosis, due to the inhibition of Ku70 and c-FLIPL proteins degradation leading to the blockade of caspase-8 activation and the activation of the necroptosome. Our data suggests that USP7 and ITCH/UBE4B can control the switch between apoptosis and necroptosis in response to USP7 and HDAC inhibitors. USP7 inhibition and ITCH/UBE4B activation by HDAC inhibitors could be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, CARLA SAMPAIO, PETER E. ZAGE. The role of the deubiquitinase USP7 and the E3/E4 ubiquitin ligase complex ITCH/UBE4B in the regulation of cell death in neuroblastoma [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 5988.

Abstract 5120: Direct targeting of the COMPASS complex inhibits neuroblastoma growth by inhibiting cancer stem cells

Abstract Epigenetic regulator COMPASS Complex, a histone H3K4 methyltransferase has recently been shown to be involved in multiple cancers and attaining focus as a novel therapeutic target. High-risk neuroblastoma (NB) is a heterogeneous pediatric cancer with high mortality and relapse rates. The relapse of refractory cancer is shown to be driven by cancer stem cells (CSCs), which are maintained by epigenetic malfunctions. In the present study, we determined the effects of inhibiting the COMPASS complex on NB CSCs and overall NB growth. A high-throughput shRNA screening of methyltransferases and demethylases reveals the role of COMPASS complex partners MLL1 and Menin in NB proliferation. We found that both MLL1 and Menin are overexpressed in our previously identified CD114+ NB CSCs. Further, MEN1 (gene coding Menin) expression showed an inverse correlation with overall NB patient survival and cancer progression in a comprehensive analysis of 1235 primary NB patient data. Our ChIP results show that the CSF3R gene which codes for CD114 and maintains CD114+ NB CSCs, is regulated by the presence of high H3K4me3 activating marks. Further, we used MI-503 to inhibit MLL1-Menin interaction and also developed MEN1 stable knockdown and found that both strategies significantly inhibit H3K4me3 activity in NB, particularly at the CSF3R gene promoter. This led to decreased CSF3R expression and NB CSC levels. MI-503 significantly inhibits proliferation in different NB cell lines and primary patient-derived xenograft (PDX) lines, with minimal effect on non-cancerous fibroblasts. MI-503 significantly inhibits NB cell cycle progression at the S phase, induces apoptosis, and inhibits NB 3D spheroid growth and development. Interestingly, we overserved differential selectivity of MI-503 in inducing apoptosis and in blocking the cell cycle in NB CSCs in contrast to non-CSCs. MEN1 knockdown also inhibits NB proliferation and 3D spheroid growth. Furthermore, MI-503 in a dose-dependent manner synergistically sensitizes NB to chemotherapy doxorubicin and significantly inhibits NB proliferation and spheroid growth compared to either agent alone. Further, by using the NB xenograft in vivo tumor model, we established that MI-503 significantly inhibits NB tumor growth and tumor metastasis by directly inhibiting tumor NB CSCs, without any observed toxicities. Similar results of significantly reduced tumor growth, metastasis, and NB CSCs were shown by the MEN1 knockdown in vivo. Overall, our data show that the MLL1-Menin of the COMPASS complex directly activates the CSF3R gene to maintain NB CSCs and drive NB pathogenesis. Our data show that MI-503 or MEN1 knockdown leads to significant inhibition of NB growth by directly inhibiting NB CSCs in both in vitro and in vivo tumor models. Further developing these epigenetic-based therapeutic strategies and combining them with current therapies will pave the way for effectively managing NB. Citation Format: Rameswari Chilamakuri, Saurabh Agarwal. Direct targeting of the COMPASS complex inhibits neuroblastoma growth by inhibiting cancer stem 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 5120.

Transmembrane serine protease 6, a novel target for inhibition of neuronal tumor growth

Transmembrane serine protease 6 (Tmprss6) has been correlated with the occurrence and progression of tumors, but any specific molecular mechanism linking the enzyme to oncogenesis has remained elusive thus far. In the present study, we found that Tmprss6 markedly inhibited mouse neuroblastoma N2a (neuro-2a) cell proliferation and tumor growth in nude mice. Tmprss6 inhibits Smad1/5/8 phosphorylation by cleaving the bone morphogenetic protein (BMP) co-receptor, hemojuvelin (HJV). Ordinarily, phosphorylated Smad1/5/8 binds to Smad4 for nuclear translocation, which stimulates the expression of hepcidin, ultimately decreasing the export of iron through ferroportin 1 (FPN1). The decrease in cellular iron levels in neuro-2a cells with elevated Tmprss6 expression limited the availability of the metal forribo nucleotide reductase activity, thereby arresting the cell cycle prior to S phase. Interestingly, Smad4 promoted nuclear translocation of activating transcription factor 3 (ATF3) to activate the p38 mitogen-activated protein kinases signaling pathway by binding to ATF3, inducing apoptosis of neuro-2a cells and inhibiting tumor growth. Disruption of ATF3 expression significantly decreased apoptosis in Tmprss6 overexpressed neuro-2a cells. Our study describes a mechanism whereby Tmprss6 regulates the cell cycle and apoptosis. Thus, we propose Tmprss6 as a candidate target for inhibiting neuronal tumor growth.

LncRNA ZNF674-AS1 drives cell growth and inhibits cisplatin-induced pyroptosis via up-regulating CA9 in neuroblastoma

Neuroblastoma (NB) is a challenging pediatric extracranial solid tumor characterized by a poor prognosis and resistance to chemotherapy. Identifying targets to enhance chemotherapy sensitivity in NB is of utmost importance. Increasing evidence implicates long noncoding RNAs (lncRNAs) play important roles in cancer, but their functional roles remain largely unexplored. Here, we analyzed our RNA sequencing data and identified the upregulated lncRNA ZNF674-AS1 in chemotherapy non-responsive NB patients. Elevated ZNF674-AS1 expression is associated with poor prognosis and high-risk NB. Importantly, targeting ZNF674-AS1 expression in NB cells suppressed tumor growth in vivo. Further functional studies have revealed that ZNF674-AS1 constrains cisplatin sensitivity by suppressing pyroptosis and promoting cell proliferation. Moreover, ZNF674-AS1 primarily relies on CA9 to fulfill its functions on cisplatin resistance. High CA9 levels were associated with high-risk NB and predicted poor patient outcomes. Mechanistically, ZNF674-AS1 directly interacted with the RNA binding protein IGF2BP3 to enhance the stability of CA9 mRNA by binding with CA9 transcript, leading to elevated CA9 expression. As a novel regulator of CA9, IGF2BP3 positively upregulated CA9 expression. Together, these results expand our understanding of the cancer-associated function of lncRNAs, highlighting the ZNF674-AS1/IGF2BP3/CA9 axis as a constituting regulatory mode in NB tumor growth and cisplatin resistance. These insights reveal the pivotal role of ZNF674-AS1 inhibition in recovering cisplatin sensitivity, thus providing potential therapeutic targets for NB treatment.

Abstract A105: Pharmacological inhibition of nicotinamide adenine dinucleotide (NAD+) production enzyme nicotinamide phosphoribosyltransferase (NAMPT) impairs cellular survival, energy metabolism, and tumor growth in neuroblastoma (NB) models

Abstract Background: Neuroblastoma is the most common extracranial solid tumor in children. High-risk patients have a poor prognosis with an overall survival rate of <50%, thus novel therapeutic strategies are critically needed. To address this need, we performed a high-throughput drug screen and identified nicotinamide phosphoribosyltransferase inhibitors (NAMPTis) as highly active against neuroblastoma cells compared to other cancer cell types. NAMPT is the rate-limiting enzyme in the nicotinamide adenine dinucleotide (NAD+) salvage pathway, which cancer cells preferentially utilize to generate NAD+, a key co-factor that plays essential roles in energy metabolism. In this study, we investigated the translational potential and mechanistic effects of NAMPT inhibition in preclinical neuroblastoma models. Methods/Results: Using 3 NAMPTis on a panel of 10 neuroblastoma cell lines, we confirmed that a majority of these models were highly responsive to NAMPTis by Incucyte Live-Cell proliferation analysis. Notably, we observed complete growth inhibition in most models at doses <3.2nM of OT-82, a clinically relevant NAMPTi with a more favorable toxicity profile. On-target activity was confirmed by dose-dependent depletion of intracellular NAD+ in cells treated with OT-82. As NAD+ is necessary for energy metabolism, particularly ATP production, we performed a Cell-Titer Glo assay to indirectly assess ATP levels and observed decreases in all models with OT-82 treatment. Glycolysis and oxidative phosphorylation (OXPHOS) are two processes involved in ATP production that utilize NAD+, thus we measured activity of both processes via biochemical and extracellular flux analyses. With OT-82 treatment, we observed decreases in glucose consumption and lactate production in most models, and decreases in OXPHOS in some cell lines. Finally, an in vivo pilot study testing OT-82 in an orthotopic neuroblastoma PDX model demonstrated antitumor activity. Conclusion: Together, these data show that neuroblastoma is susceptible to disruption of the NAMPT pathway and suggest that NAMPTis have translational potential as a novel therapy for neuroblastoma patients. Citation Format: Sophia Varriano, Unsun Lee, Victor Collins, Rosa Nguyen, Sameer Issaq, Ming Sun, Ariana Nelson, Choh Yeung, Craig Thomas, Arnulfo Mendoza, Carol Thiele, Christine Heske. Pharmacological inhibition of nicotinamide adenine dinucleotide (NAD+) production enzyme nicotinamide phosphoribosyltransferase (NAMPT) impairs cellular survival, energy metabolism, and tumor growth in neuroblastoma (NB) models [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A105.

USP7 Inhibition Suppresses Neuroblastoma Growth via Induction of p53-Mediated Apoptosis and EZH2 and N-Myc Downregulation.

Neuroblastoma (NB) is a pediatric malignancy originating from neural crest cells of the sympathetic nervous system that accounts for 15% of all pediatric cancer deaths. Despite advances in treatment, high-risk NB remains difficult to cure, highlighting the need for novel therapeutic approaches. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in tumor suppression and DNA repair, and USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including NB. Therefore, USP7 is a potential therapeutic target for NB. The tumor suppressor p53 is a known target of USP7, and therefore reactivation of the p53 pathway may be an effective therapeutic strategy for NB treatment. We hypothesized that inhibition of USP7 would be effective against NB tumor growth. Using a novel USP7 inhibitor, Almac4, we have demonstrated significant antitumor activity, with significant decreases in both cell proliferation and cell viability in TP53 wild-type NB cell lines. USP7 inhibition in NB cells activated the p53 pathway via USP7 and MDM2 degradation, leading to reduced p53 ubiquitination and increased p53 expression in all sensitive NB cells. In addition, USP7 inhibition led to decreased N-myc protein levels in both MYCN-amplified and -nonamplified NB cell lines, but no correlation was observed between MYCN amplification and treatment response. USP7 inhibition induced apoptosis in all TP53 wild-type NB cell lines. USP7 inhibition also induced EZH2 ubiquitination and degradation. Lastly, the combination of USP7 and MDM2 inhibition showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed NB.

ALKB homolog 5 (ALKBH5)-induced circPUM1 upregulation facilitated the progression of neuroblastoma via miR-423–5p/PA2G4 axis

BackgroundThe oncogenic role of circPUM1 has been revealed in multiple cancers. Nevertheless, the specific role and molecular mechanism of circPUM1 in neuroblastoma (NB) have never been reported. MethodsThe expression of genes was detected using RT-qPCR and Western Blot assay. The proliferation, migration, and invasion of NB cells were evaluated by CCK-8 and Transwell assays. Besides, mouse model was established to evaluate the effect of circPUM1 on the progression of NB. The interaction among genes was verified through RIP, MeRIP, or Luciferase reporter assay. ResultsThrough our investigation, it was discovered that circPUM1 expression was abnormally elevated in NB tissues and the abundance of circPUM1 was correlated with unfavorable clinical outcomes in NB patients. Besides, the viability and mobility of NB cells as well as NB tumor growth were suppressed by silencing circPUM1. Moreover, bioinformatics prediction and experimental verification demonstrated that circPUM1 was a sponge for miR-423–5p which further targeted proliferation-associated protein 2G4 (PA2G4). The oncogenic effect of circPUM1 on NB was exerted through suppressing miR-423–5p to elevate PA2G4 expression. Finally, we investigated the transcriptional factor causing the upregulation of circPUM1 in NB. The result was that ALKB homolog 5 (ALKBH5), an m6A demethylase, suppressed the m6A modification of circPUM1 and caused the elevation of circPUM1 expression in NB. ConclusionALKBH5 induced the upregulation of circPUM1 to accelerate the development of NB through regulating miR-423–5p/PA2G4 axis.

Abstract 6157: USP7 is a promising thersapeutic target for neuroblastoma

Abstract Background: The ubiquitin-proteasome system (UPS) plays an essential role in post-translational modification of proteins to maintain proteostasis. Ubiquitination of target protein is coordinated by different enzymes, which add and link ubiquitin to each other in specific ways that determine the fate of the protein for to be degraded or to be included into protein complex to induce signaling pathways. Ubiquitination is further regulated by deubiquitinases, which remove ubiquitin and rescue target protein from being degraded or diverted from it signaling role. UPS dysregulation has been linked to many human diseases, including cancer. Ubiquitin-specific protease 7 (USP7) is a deubiquitinase that plays a critical role in immune response, tumor suppression and DNA repair. USP7 overexpression has been associated with tumor aggressiveness in a variety of tumors, including in neuroblastoma (NB). Therefore, USP7 is a potential therapeutic target for NB. Some new specific USP7 inhibitors are highly potent and selective and have demonstrated significant antitumor activity in preclinical models of adult cancer. We hypothesize that inhibition of USP7 alone or in combination with epigenetic drugs like HDAC inhibitors will be more effective against NB tumor growth. To evaluate the efficacy of USP7 inhibition with specific USP7 inhibitors alone or with combination with epigenetic drugs like HDAC inhibitors (Saha) or chemotherapy against NB tumor growth, NB cell lines were treated with increasing concentrations of USP7 inhibitors alone or in combination with chemotherapy or with HDAC inhibitors. Cell proliferation was measured using continuous live cell imaging and cell viability using AlamarBlue assay. Apoptosis was measured by caspase cleavage using Caspase 3/7 apoptosis assay as well as by PARP cleavage by Western Blotting (WB). Changes in various proteins were measured by WB and co-immunoprecipitation were performed to analyze the ubiquitin status of specific protein targets regulated by USP7. Pharmacologic inhibition of USP7 resulted in significant decreases in both cell proliferation and cell viability in vitro only in p53 wild-type NB cell lines. There was no correlation seen between MYCN amplification status and treatment response. USP7 inhibition induced apoptosis as well as necroptosis with dose-dependent increase. Treatment with USP7 inhibitors led to decrease p53 ubiquitination leading increased of p53 protein expression in all sensitive cell lines tested and decreased MYCN protein level in MYCN-amplified cell lines. EZH2, which is a predominant target of USP7 is significantly downregulated with increasing dose of USP7 inhibitors. In addition, the combination of USP7 inhibitors with chemotherapy commonly used in NB or with combination with HDAC inhibitors showed enhanced efficacy. Our data suggests that USP7 inhibition may be a promising therapeutic strategy for children with high-risk and relapsed neuroblastoma. Citation Format: Christophe Le Clorennec, Karen Lee, Peter Zage. USP7 is a promising thersapeutic target for neuroblastoma [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 6157.

Abstract 6721: β3-adrenergic receptor correlates with tumor growth and progression in neuroblastoma

Abstract Neuroblastoma (NB) is a heterogeneous, often aggressive, extracranial tumor and the most common malignancy to be diagnosed in the first year of life. It has been already demonstrated that the specific antagonism of the β3-adrenergic receptor (β3-AR) on NB tumor cells affects tumor growth. To confirm the results obtained with the pharmacological approach on the crucial role played by the β3-AR receptor in influencing different pro-tumoral signaling pathways, here we investigated the effect of CRISPR/Cas9-mediated knock-out of β3-AR gene (ADRB3) on human NB cell line BE(2)-C in vitro and in vivo. Inoculation of BE(2)-C β3-AR−/- cells in athymic nude mice (NU(N-Cr)-Foxn1nu) showed a strong reduction of NB tumor growth compared to wild type cells, and in some cases β3-AR knock-out tumor cells failed in producing a tumor mass. RNA Sequencing on these tumor masses showed alterations of signaling pathways related to various tumor mechanisms, such as proliferation, cell motility and cell differentiation. Furthermore, we evaluated by flow cytometry the expression of β3-AR in circulating tumor cells (CTCs) from NB patients with different degrees of disease. Following depletion with anti-CD45 magnetic beads, tumor cells were identified through the expression of specific tumor markers, CD56 and GD2. Results showed that a high expression of β3-AR correlates with poor prognosis, characterized by metastatic or recurrent tumors, compared to patients with a favorable clinical outcome that instead show a low expression of β3-AR. Overall, our results demonstrate the β3-AR involvement in supporting NB tumor growth and suggest a possible role in regulating the metastatic potential of this tumor. Citation Format: Alessia Boaretto, Gennaro Bruno, Nicoletta Nastasi, Claudio Favre, Maura Calvani. β3-adrenergic receptor correlates with tumor growth and progression in neuroblastoma. [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 6721.

Abstract 3246: Targeting Syk reprograms tumor-associated macrophages and enhances responses to immune checkpoint blockade and radiation therapy in high-risk neuroblastoma

Abstract Neuroblastoma (NB) is the most common extracranial solid malignancy in children. As leading regulators of inflammation and tumor progression, tumor associated macrophages (TAMs) have gained major interest as immunotherapeutic targets in NB. Hence targeting or “re-educating” tumorigenic macrophages towards an immunostimulatory phenotype might improve responses to immunotherapy in this childhood cancer. Spleen tyrosine kinase (Syk) has recently been identified by our group as a novel immune-oncology target. We found that Syk is abundantly present in TAMs infiltrated in MYCN and non-MYCN amplified neuroblastoma tumors. Furthermore, genetic deletion or pharmacological inhibition of Syk markedly impaired neuroblastoma tumor growth. This effect was facilitated by macrophages that became immunogenic in the absence of Syk, skewing the suppressive tumor microenvironment towards immunostimulation and activating antitumor immune responses. Moreover, combining Syk inhibitor, R788 along with anti-PDL1 mAb and radiation provided a synergistic effect leading to complete tumor regression and durable anti-tumor immunity in aggressive MYCN-and non-MYCN-driven murine neuroblastoma tumor models. Collectively, our findings demonstrate the importance of Syk inhibitor, R788, in enhancing the anti-tumor immune responses in NB and support the clinical evaluation of R788 either alone or together with radiation and immune check point inhibitors as a potential treatment strategy for NB. Citation Format: Deepak Rohila, In Hwan Park, Timothy Pham, Riley Jones, Pablo Tamayo, Alice Yu, Andrew Sharabi, Shweta Joshi. Targeting Syk reprograms tumor-associated macrophages and enhances responses to immune checkpoint blockade and radiation therapy in high-risk neuroblastoma [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 3246.

Neuroblastoma Differentiation: The Untapped Potential of Mitochondrial Uncouplers

While the goal of most anticancer treatments is to kill cancer cells, some therapies halt cancer progression by inducing cancer cell differentiation. For example, retinoic acid induces neuroblastoma cell differentiation in vitro and is used as maintenance therapy for children with high-risk neuroblastoma. A new study by Jiang and colleagues has revealed the mitochondrial uncoupler niclosamide ethanolamine (NEN) induces neuroblastoma cell differentiation in vitro and slows neuroblastoma tumor growth in vivo. Mitochondrial uncoupler molecules alter cell metabolism by forcing cells to "burn" more nutrients, resulting in a switch from anabolic to catabolic metabolism. NEN-induced neuroblastoma cell differentiation was associated with disruption of Warburg metabolism, epigenetic remodeling, and downregulation of key oncogenic drivers of neuroblastoma development, including MYCN. NEN is currently used as an antiparasitic worm treatment and is safe to use in children but has poor pharmacokinetic properties. However, derivatives of NEN and structurally distinct uncouplers that have improved pharmacokinetic properties are in development. Results of this study ignite the idea that mitochondrial uncouplers could be used as differentiating agents and expand the pharmacotherapy toolkit to treat cancer, including neuroblastoma. See related article by Jiang et al., p. 181.

Calreticulin Expression Controls Cellular Redox, Stemness, and Radiosensitivity to Function as a Novel Adjuvant for Radiotherapy in Neuroblastoma.

Radiotherapy (RT) is currently only used in children with high-risk neuroblastoma (NB) due to concerns of long-term side effects as well as lack of effective adjuvant. Calreticulin (CALR) has served distinct physiological roles in cancer malignancies; nonetheless, impact of radiation on chaperones and molecular roles they play remains largely unknown. In present study, we systemically analyzed correlation between CALR and NB cells of different malignancies to investigate potential role of CALR in mediating radioresistance of NB. Our data revealed that more malignant NB cells are correlated to lower CALR expression, greater radioresistance, and elevated stemness as indicated by colony- and neurospheroid-forming abilities and vice versa. Of note, manipulating CALR expression in NB cells of varying endogenous CALR expression manifested changes in not only stemness but also radioresistant properties of those NB cells. Further, CALR overexpression resulted in greatly enhanced ROS and led to increased secretion of proinflammatory cytokines. Importantly, growth of NB tumors was significantly hampered by CALR overexpression and was synergistically ablated when RT was also administered. Collectively, our current study unraveled a new notion of utilizing CALR expression in malignant NB to diminish cancer stemness and mitigate radioresistance to achieve favorable therapeutic outcome for NB.

ERK Inhibitor Ulixertinib Inhibits High-Risk Neuroblastoma Growth In Vitro and In Vivo.

Neuroblastoma (NB) is a pediatric tumor of the peripheral nervous system. Approximately 80% of relapsed NB show RAS-MAPK pathway mutations that activate ERK, resulting in the promotion of cell proliferation and drug resistance. Ulixertinib, a first-in-class ERK-specific inhibitor, has shown promising antitumor activity in phase 1 clinical trials for advanced solid tumors. Here, we show that ulixertinib significantly and dose-dependently inhibits cell proliferation and colony formation in different NB cell lines, including PDX cells. Transcriptomic analysis revealed that ulixertinib extensively inhibits different oncogenic and neuronal developmental pathways, including EGFR, VEGF, WNT, MAPK, NGF, and NTRK1. The proteomic analysis further revealed that ulixertinib inhibits the cell cycle and promotes apoptosis in NB cells. Additionally, ulixertinib treatment significantly sensitized NB cells to the conventional chemotherapeutic agent doxorubicin. Furthermore, ulixertinib potently inhibited NB tumor growth and prolonged the overall survival of the treated mice in two different NB mice models. Our preclinical study demonstrates that ulixertinib, either as a single agent or in combination with current therapies, is a novel and practical therapeutic approach for NB.

Interplay: The Essential Role between INSM1 and N-Myc in Aggressive Neuroblastoma.

Simple SummaryNeuroblastoma (NB) is a cancer that starts in certain very early forms of nerve cells of the sympathetic nervous system, most often found in an embryo or fetus. Symptoms may include bone pain, an abdominal mass, frequent urination, limping, anemia, spinal cord weakness, or bruising of the eye area. N-Myc is a key driver of high-risk NB. An elevated expression of N-Myc often predicts a poorer prognosis, in both time to tumor progression and overall survival rate. We discovered a transcription factor, insulinoma-associated-1 (INSM1), as the downstream target gene of N-Myc. INSM1 has emerged as a novel NB biomarker that plays a critical role in facilitating NB tumor cell development. Both N-Myc and INSM1 demonstrate high clinical relevance to NB. Therefore, further understanding the association of INSM1 and N-Myc functions in aggressive NB should be beneficial for future NB treatment.An aggressive form of neuroblastoma (NB), a malignant childhood cancer derived from granule neuron precursors and sympathoadrenal lineage, frequently comprises MYCN amplification/elevated N-Myc expression, which contributes to the development of neural crest-derived embryonal malignancy. N-Myc is an oncogenic driver in NB. Persistent N-Myc expression during the maturation of SA precursor cells can cause blockage of the apoptosis and induce abnormal proliferation, resulting in NB development. An insulinoma-associated-1 (INSM1) zinc-finger transcription factor has emerged as an NB biomarker that plays a critical role in facilitating tumor cell growth and transformation. INSM1 plays an essential role in sympathoadrenal cell differentiation. N-Myc activates endogenous INSM1 through an E2-box of the INSM1 proximal promoter, whereas INSM1 enhances N-Myc stability via RAC-α-serine/threonine protein kinase (AKT) phosphorylation in NB. The ectopic expression of INSM1 stimulates NB tumor growth in contrast to the knockdown of INSM1 that inhibits NB cell proliferation. The clinical pathological result and bioinformatics analysis show that INSM1 is a strong diagnostic and a prognostic biomarker for the evaluation of NB progression. The INSM1/N-Myc expression shows high clinical relevance in NB. Therefore, targeting the INSM1/N-Myc-associated signaling axis should be a feasible approach to identifying new drugs for the suppression of NB tumor growth.

Depletion of Foxp3+ regulatory T cells but not the absence of CD19+IL-10+ regulatory B cells hinders tumor growth in a para-orthotopic neuroblastoma mouse model.

Adaptive immune cells with regulatory function reportedly mediate immune escape in a variety of tumors. Little is known regarding the relevance of the most prominent regulatory cell populations, namely Foxp3+ T regulatory cells (Tregs) and CD19+IL-10+ B regulatory cells (Bregs), for neuroblastoma (NB) survival. After establishing a novel immunocompetent syngeneic NB mouse model where orthotopic tumors can be generated after intrarenal injection of NB975A cells, we studied the importance of Tregs and Bregs in Foxp3-DTR mice whose Tregs can be depleted by diphtheria toxin (DT) application as well as in CD19-specific IL-10 deficient mice that lack IL-10+ Bregs (CD19cre+/- × IL-10fl/fl mice). We observed Foxp3 Treg cells in tumors from wild type mice. On the contrary, Bregs or B cells were scarce. Specific depletion of Tregs in Foxp3-DTR mice resulted in an 85% reduction of tumor volume and weight compared to DT-treated wild type mice and untreated Foxp3-DTR mice. In contrast, NB tumor growth was not affected in CD19-specific IL-10 deficient mice. Similarly, mice lacking mature B cells (μMT mice) and CD19 deficient mice (CD19cre mice) showed no change in growth pattern of NB tumors. In Treg-depleted mice, reduced tumor growth was associated with an increased concentration of IFN-gamma, TNF-alpha, IL-4, IL-6, and IL-10 in isolated splenocytes. In summary, transient ablation of Tregs but not absence of Bregs hindered the growth of NB, strongly suggesting the therapeutic potential of targeting Tregs for this aggressive childhood tumor.

DRR1 promotes neuroblastoma cell differentiation by regulating CREB expression.

Neuroblastoma is the most common cancer in infants and the most common extracranial solid tumor in childhood. DRR1 was identified to be downregulated in poorly differentiated ganglion cells from neuroblastoma model mice. However, the roles of DRR1 in neuroblastoma remain largely unclear. The neuroblastoma cells were induced to differentiate, and the expression of DRR1 was detected. The expression of the neuroblastoma cell differentiation markers was analyzed in DRR1 shRNA- or DRR1-expressing vector-treated neuroblastoma cells. The downstream genes of DRR1 were screened with ChIP-seq assay. Finally, TNB1 cells were infected with DRR1 shRNA and CREB expressing vector containing lentivirus, and the expression of the cell differentiation markers, cell cycle distribution and tumor growth were analyzed. The expression of DRR1 was increased in differentiated neuroblastoma cells, and downregulation of DRR1 expression inhibited the differentiation of neuroblastoma cells. Further experiments indicated that CREB is a candidate downstream gene of DRR1, and it mediates neuroblastoma cell differentiation. Moreover, overexpression of CREB rescued the effect of DRR1 shRNA on cell differentiation, cell cycle distribution and tumor growth in neuroblastoma. DRR1-CREB axis modulates the differentiation of neuroblastoma cells and is associated with the outcome of neuroblastoma patients. DRR1 is involved in regulation of the differentiation of neuroblastoma. Binding with actin is essential for DRR1 to regulate neuroblastoma cell differentiation. CREB is a candidate downstream gene of DRR1 in regulating of the differentiation of neuroblastoma.