Rh30 Cells Research Articles

Targeting AGAT gene expression – a drug screening approach for the treatment of GAMT deficiency

ABSTRACT Background Targeting the enzyme L-Arginine:glycine amidinotransferase (AGAT) to reduce the formation of guanidinoacetate (GAA) in patients with guanidinoacetate methyltransferase (GAMT) deficiency, we attempted to identify drugs for repurposing that reduce the expression of AGAT via transcriptional inhibition. Research Design and Methods The authors applied a HeLa cell line stably expressing AGAT promoter and firefly luciferase reporter for high-content screening and secondary screening. For further assessment, the authors integrated Nanoluc luciferase as a reporter into the endogenous AGAT gene in HAP1 cell lines and used the human immortalized cell line RH30 as model of GAMT deficiency. Results Screening 6,000 drugs and drug-like compounds, the authors identified 43 and 34 high-score candidates as inhibitors and inducers of AGAT promoter-reporter expression, respectively. After further deselection considering dose response, drug toxicity, topical formulations, price, and accessibility, the authors assessed seven candidates and found none of them demonstrating efficacy in HAP1 and RH30 cells and warranting further assessment. Conclusion The selection of the test models is crucial for screening of gene repressor drugs. Almost all drugs with an impact on gene expression had off-target effects. It is unlikely to find drugs that are selective inhibitors of AGAT expression, rendering pharmacological AGAT gene repression a risky approach for the treatment of GAMT deficiency.

A comparative in vivo study of hyperthermic intraperitoneal chemotherapy with cisplatin versus doxorubicin versus cisplatin plus doxorubicin for the treatment of intra-abdominally disseminated alveolar rhabdomyosarcoma in mice.

Treatment options for advanced intra-abdominal pediatric rhabdomyosarcoma (RMS) with peritoneal sarcomatosis (PS) include cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). However, optimal dosages and combination regimens of drugs used for HIPEC are underexplored. We aimed to evaluate the efficacy of HIPEC with cisplatin, doxorubicin, and their combination in vivo. We established PS/RMS mouse model by intraperitoneally injecting RH30 cells into NOD/LtSz-scid IL2Rγ-null mice. Two weeks post xenotransplantation, mice underwent a single HIPEC procedure at 42°C for 60minutes. Treatment groups received cisplatin (50, 100, and 150mg/m2) and doxorubicin (30, 45, and 60mg/m2), administered alone or combined. The control group underwent an intraperitoneal lavage with isotonic saline. Peritoneal cancer index (PCI) was used to quantify the extent of peritoneal tumor spread. Tissue samples were evaluated regarding proliferation (Ki-67) and apoptosis (caspase 3). Mice treated with cisplatin at 100mg/m2 (PCI of 3.875, p=.007) and 150mg/m2 (PCI of 4.556, p=.026), and doxorubicin at 30mg/m2 (PCI of 2.875, p<.001) and 45mg/m2 (PCI of 4.143, p=.021) showed reduced PCI, with the combination of cisplatin 50mg/m2 and doxorubicin 30mg/m2 showing the most prominent effect (PCI of 3.333, p<.001) compared to the control group (PCI of 8.615). Histologically, there was no difference in Ki-67 or caspase 3 expression among the groups. The cisplatin- and doxorubicin-based HIPEC significantly reduces peritoneal tumor dissemination in vivo. Further investigations are needed to explore the underlying molecular responses to optimize therapeutic strategies.

Development of a novel oncolytic adenovirus controlled by CDX2 promoter for esophageal adenocarcinoma therapy.

Prognosis of esophageal adenocarcinoma (EAC) is still poor. Therefore, the development of novel therapeutic modalities is necessary to improve therapeutic outcomes in EAC. Here, we report a novel promoter-controlled oncolytic adenovirus targeting CDX2 (Ad5/3-pCDX2) and its specific anticancer effect for EAC. We used OE19, OE33, HT29, MKN28, RH30, and HEL299 cell lines. To establish CDX2 overexpressing OE19 cells, pCMV-GLI1 plasmid was transfected to OE19 (OE19 + GLI1). The virus replication and cytocidal effect of replication competent Ad5/3-pCDX2 were analyzed in vitro. Antitumor effect of Ad5/3-pCDX2 was assessed in xenograft mouse models by intratumoral injection of the viruses. Finally, efficacy of combination therapy with Ad5/3-pCDX2 and 5FU was evaluated. EAC cells and HT29 showed high mRNA levels of CDX2, but not MKN28, RH30, and HEL299. We confirmed that deoxycholic acid (DCA) exposure enhanced CDX2 expression in EAC cells and OE19 + GLI1 had persistent CDX2 overexpression without DCA. Ad5/3-pCDX2 showed stronger cytocidal effect in OE19 + GLI1 than OE19, whereas Ad5/3-pCDX2 did not kill CDX2-negative cells. Ad5/3-pCDX2 was significantly replicated in EAC cells and the virus replication was higher in OE19 + GLI1 and OE19 with DCA compared to OE19 without DCA exposure. In vivo, Ad5/3-pCDX2 significantly suppressed OE19 tumor growth and the antitumor effect was enhanced in OE19 + GLI1 tumor. In contrast, Ad5/3-pCDX2 did not show significant antitumor effect in MKN28 tumor. Moreover, Ad5/3-pCDX2 significantly increased the efficacy of 5FU in vitro and in vivo. Ad5/3-pCDX2 showed specific anticancer effect for EAC, which was enhanced by bile acid exposure. Ad5/3-pCDX2 has promising potential for EAC therapy in the clinical setting.

Combinatorial cellular therapy in pediatric solid tumors with natural killer (NK) and genetically engineered myeloid cells (GEMys).

2561 Background: Early human studies with Natural Killer (NK) cells have shown promise, harnessing their ability to infiltrate into tumors and be tumoricidal. However, these effects can be limited by adenosine, reactive oxygen species, and TGF-β in the highly immune suppressive tumor microenvironment (TME). TGF-β and downstream SMAD3 signaling in Natural Killer (NK) cells have been shown to cause decreased cytokine production, downregulation of activating cell surface receptors, and attenuated cytotoxic function against solid tumors. Exposure to TGF-β during cell culture generates expanded NK cells with increased resistance to TGF-β signaling, dubbed TGF-β imprinted NK cells. In vitrostudies have shown that these cells have increased cytokine production and cytotoxic function in the presence of TGF-β. However, their in vivo trafficking, modulation of the TME, and therapeutic efficacy remain understudied. Our lab has previously shown that when given to tumor-bearing mice, myeloid cells modified to produce IL-12 (IL-12-Genetically Engineered Myeloid cells, aka IL-12 GEMys) are effective at homing to tumor and metastatic sites and, via IL-12 secretion and TME modulation, increase NK cell presence and activation in these sites. They hold great promise as combination therapy, enhancing the effectiveness of other cellular therapies such as NK cell therapy. A current clinical trial exploring TGF-β Imprinted, Ex Vivo Expanded, Universal Donor NK Cell Infusions in relapsed sarcomas is underway (NCT05634369). Methods: In vitro experiments were conducted with the xCELLigence real-time cell analysis system. The human osteosarcoma cell line 143B and human rhabdomyosarcoma cell line RH-30 were evaluated for cell death after co-culture with healthy donor NK cells or TGF-β imprinted NK cells (E:T ratio of 5:1) in isolation and in combination with IL-12 GEMys (E:T ratio of 2:1). Results: TGF-β imprinted NK cells are significantly more effective at tumor cell killing of both 143B and RH30 cell lines in-vitro compared to regular expanded NK cells (WT NK cells), with &gt;90% decrease in cell index compared to 50-60%, respectively, after 48 hours of co-culture. IL-12 GEMys also significantly enhance the cytotoxic effects of WT NK cells, increasing cell killing to 70-80% compared to co-culture with untransduced GEMy controls. Conclusions: These initial in-vitro experiments provide insight into the promising effectiveness of NK-myeloid cell combination therapies in the treatment of pediatric sarcomas. Ongoing in vitroexperiments include cytokine quantification by ELISA and flow cytometric analysis of NK cell activation and proliferation markers. Our preliminary results provide rationale to continue studying the in vivo efficacy of TGF-β imprinted NK cell monotherapy and in combination with IL-12 GEMys in the treatment of tumor-bearing NSG mice.

Abstract 4015: Improvement of CAR T cell potency against low-density targets like L1CAM and FGFR4 in rhabdomyosarcoma

Abstract Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. Overall survival rates are low for metastatic RMS, emphasizing the urgent need of improved therapies. Chimeric Antigen Receptor (CAR) Ts have shown remarkable results against hematological malignancies, but CAR T cell therapy remains challenging in solid tumors, mainly due to a lack of ideal tumor specific targets, and of the immunosuppressive tumor microenvironment (TME). To maximize antitumoral activity against RMS and to address optimal CAR T persistence in vivo, novel CAR constructs were investigated against upregulated RMS targets. By surfaceome profiling, we recently identified L1CAM as promising candidate for RMS CAR T cell therapy, and confirmed CD276 and FGFR4 expression. CD276- (CD276.CD28HD.28TM.28CSD.3z) and Dual-CAR Ts targeting both CD276 and FGFR4 (F8-FR4.CD28HD.28TM.28CSD.3z) showed tumor eradication in an orthotopic RMS model, whereas FGFR4-CAR Ts exhibited only a partial response in vivo. Moreover, CD276-CAR Ts showed a limited activity against RMS tumors expressing lower amounts of CD276, eradicating the tumor in 1/5 mice, underlying the need of improved CAR T cell potency against low-density target-expressing tumors. First, to improve FGFR4-CAR Ts activity, we optimized the recognition of FGFR4 epitope by modifying the CAR moiety “hinge”. We replaced the CD28-derived hinge with various hinges of different length: no hinge, IgG4 (12aa), IgG4CH3 (119aa), and IgG4CH2CH3 (229aa). Luciferase assays showed effective in vitro killing capacity of FGFR4-CAR Ts expressing an IgG4-derived hinge, with a tumor cell survival less than 10% at Effector to Target (E:T) ratio of 5:1. They also released the double amount of IFN-γ (50pg/ml) compared to the other constructs, when co-incubated with Rh4 cells. Next, to verify the potential of L1CAM as CAR T target for RMS, we generated a panel of L1CAM-CARs expressing either a long (IgG4CH2CH3, 229aa) or a short (CD28, 35aa) Hinge, and a CD28- or 41BB-derived costimulatory domain (CSD). L1CAM.IgG4CH2CH3Hinge.28TM.28CSD.3z CAR Ts outperformed the other constructs, killing ~60% of Rh30 at the E:T ratio of 1:1 and more than 90% at the E:T ratio of 5:1. They also exhibited a 3-fold higher IFN-γ release (120 pg/ml) compared to the other constructs. However, although our examination of patient-derived TMA revealed a strong immunoreactivity for L1CAM, more expressed in aggressive RMS subtypes, the selected L1CAM-CAR Ts against Rh30 cells, exhibiting ~10’000 L1CAM copies, showed limited activity in an in vivo pilot study. Based on these results and on the RMS TME investigation, which revealed consistent collagen (COL4A1, COL4A2 and COL18A1) deposition in RMS-derived mice tumor xenografts, we hypothesize that co-expressing a collagenase, able to partially degrade the dense extracellular matrix surrounding tumors, will lead to an improved CAR T cell infiltration and activity in vivo. Citation Format: Caroline Piccand, Andrea Timpanaro, Stenija Anton-Joseph, Michele Bernasconi, Jochen Karl Rössler. Improvement of CAR T cell potency against low-density targets like L1CAM and FGFR4 in rhabdomyosarcoma [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 4015.

Abstract 2863: CDKN1A prevents IR-induced apoptosis in rhabdomyosarcoma tumor via repressing the pro-apoptotic factor NOXA in a novel apoptotic regulatory axis

Abstract Rhabdomyosarcoma (RMS) is a tumor of the muscle and is the most common soft tissue cancer in children and teens, with approximately 400 to 500 new cases every year in the United States. While we have a good understanding of the interplay between pro- and anti-apoptotic regulators, much remains to be learned about how the decision to trigger apoptosis or not is controlled at the transcriptional level in RMS tumors. We strive to elucidate how pathways that drive apoptosis avoidance are epi-genetically pre-programmed in cells. We have discovered that SNAI2, a transcriptional repressor, acts as a master switch to either activate or dampen apoptosis in response to ionizing radiation (IR). While elevated SNAI2 results in transcriptional repression of the pro-apoptotic factor BIM and protection from apoptosis post-IR, a small number of SNAI2 null cells survive radiation to reproliferate in vivo, revealing the existence of hitherto unappreciated mechanisms that support the survival of irradiated cells. Hence, we hypothesized that there are additional genes or pathways in cells lacking SNAI2 that are co-opted to drive relapse disease. We have identified that CDKN1A or p21 expression is robustly increased in SNAI2 null cells. CDKN1A is thought to be a tumor suppressor. However, our preliminary data indicate that CDKN1A is an oncogene, and in RMS patients, high tumor expression is associated with poor outcomes. We found that CDKN1A expression in combination with SNAI2 can predict resistance to radiation across RMS cell lines. We next developed control RD, Rh30, and Rh18 cells, either CDKN1A or both CDKN1A and SNAI2 ablated using CRISPR/Cas9 reagents with control cells expressing control gRNAs. Ablation of CDKN1A, while not affecting proliferation, led to a significant increase in apoptosis post-radiation, and this effect was increased in SNAI2/CDKN1A double knockout cells. Further, in colony-forming assays, we find that double-ablated cells form significantly fewer colonies than control and single knockout cells. Loss of CDKN1A, SNAI2, or CDKN1A/SNAI2 led to a G2/M cell cycle block, indicating that the cell cycle effects of p21 might not be responsible for the phenotypes observed. Ongoing xenograft experiments have determined the effect we observe in vitro will also translate in vivo. Given that SNAI2 and CDKN1A are heterogeneously expressed, respectively, we will interrogate CDKN1A for its role in protecting differentiated cells from apoptosis. We will determine if CDKN1A prevents apoptosis by repressing the pro-apoptotic factor NOXA in a novel apoptotic regulatory axis specific to differentiated cells. Our study will show that critical primary and alternative apoptosis resistance mechanisms are epi-genetically pre-programmed in cells by SNAI2. Citation Format: Paulomi S. Modi, Myron Ignatius, Long Wang, Prethish Sreenivas. CDKN1A prevents IR-induced apoptosis in rhabdomyosarcoma tumor via repressing the pro-apoptotic factor NOXA in a novel apoptotic regulatory axis [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 2863.

Abstract 138: UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma

Abstract PAX3-FOXO1 positive rhabdomyosarcoma (RMS) is an aggressive pediatric sarcoma with a terrible prognosis. The resultant fusion acts as a chimeric transcription factor, in part remodeling chromatin to inhibit terminal differentiation and maintain cancer cell proliferation. As this oncoprotein is not directly druggable, we have used genetic screens to identify dependencies downstream of PAX3-FOXO1 to improve our understanding of pathogenesis and develop new therapeutic targets. We performed pooled CRISPRi screening in an isogenic system of a patient-derived RMS cell line with or without knockdown of PAX3-FOXO1. Loss of the ubiquitin hydroxylase UCHL5 led to selective depletion of PAX3-FOXO1 positive Rh30 cells compared to PAX3-FOXO1 knockdown cells, suggesting that the fusion creates UCHL5 dependence. We validated this finding in competition growth assays in additional fusion positive (FP) and fusion negative (FN) RMS cell lines, and observed UCHL5 dependency only in the former. By contrast, UCHL5 was dispensable for cell growth and survival in FN RMS cell lines. These data were recapitulated in the large DepMap dataset, which also identified the top genetic co-dependencies of UCHL5 as INO80-complex (INO80c) members. We therefore investigated whether the fusion selective effects of UCHL5 were mediated by the INO80c, of which UCHL5 is an N-terminal member. We observed that knockdown of another N-terminal component of INO80c, NFRKB, phenocopies the fusion-selective effects of UCHL5 loss. Conversely, the catalytic ATPase INO80 was pan-essential, suggesting that only N-terminal subunits of INO80c regulate PAX3-FOXO1 specific functions. INO80c plays an essential role in mitigating DNA replication stress. Indeed, loss of UCHL5 led to DNA damage as measured by γH2AX and slowing of replication forks as measured by fiber assays in FP, but not FN RMS. Unexpectedly, acute degradation of PAX3-FOXO1 also slowed DNA replication fork speed, suggesting a genetic model in which UCHL5 and PAX3-FOXO1 cooperate to resolve replication stress in FP RMS. Finally, in vivo experiments using cell line xenografts confirms that loss of UCHL5 slows the growth of FP RMS, but not FN RMS. These findings suggest that loss of UCHL5 induces replication stress in a fusion-specific manner, and offers both an opportunity to study the mechanisms by which the PAX3-FOXO1 oncoprotein mitigates replication stress, and to develop UCHL5 inhibitors as precision therapies for FP RMS patients. Citation Format: Amit J. Sabnis, Pushpendra K. Sahu, Yue Pan. UCHL5 and PAX3-FOXO1 resolve replication stress in rhabdomyosarcoma [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 138.

CircAFF1 Is a Circular RNA with a Role in Alveolar Rhabdomyosarcoma Cell Migration.

Circular RNAs (circRNAs), covalently closed RNAs that originate from back-splicing events, participate in the control of several processes, including those that occur in the development of pathological conditions such as cancer. Hereby, we describe circAFF1, a circular RNA overexpressed in alveolar rhabdomyosarcoma. Using RH4 and RH30 cell lines, a classical cell line models for alveolar rhabdomyosarcoma, we demonstrated that circAFF1 is a cytoplasmatic circRNA and its depletion impacts cell homeostasis favouring cell migration through the downregulation of genes involved in cell adhesion pathways. The presented data underline the importance of this circular RNA as a new partial suppressor of the alveolar rhabdomyosarcoma tumour progression and as a putative future therapeutic target.

Abstract 1465: A SNAI2/CDKN1A pathway protects rhabdomyosarcoma tumors from radiation

Abstract Rhabdomyosarcoma (RMS) is tumor of the muscle and is the most common soft tissue cancer in children and teens, with approximately 400 to 500 new cases every year in the United States. There are two main subtypes of rhabdomyosarcoma: 1) Alveolar rhabdomyosarcoma (fusion-positive RMS FP-RMS), is classified primarily by the presence of the fusion between PAX3/PAX7 and FOXO1 proteins. 2) Embryonal rhabdomyosarcoma (fusion-negative FN-RMS) is the more common subtype. The survival rate for patients with RMS is 70%, but at relapse it is less than 30%. The standard of care for this disease includes radiation, chemotherapy and surgery. To better understand how RMS tumors survive therapy, we sought to define the role of SNAI2 in the response to radiation treatment. We identified SNAI2 as a critical oncogene that protects RMS tumors from radiation induced apoptosis by repressing pro-apoptotic BIM expression (Cancer Research 2021). SNAI2 additionally prevents FN-RMS tumors and cells from undergoing myogenic differentiation (Nature Comm. 2021). We also showed that MEK maintained SNAI2 post translationally in RAS mutant FN-RMS tumors and combining MEK inhibitor with radiation led to complete tumor regression and delayed event free survival in xenograft and PDX models. However, rare tumor cells grow back after a 2-4 weeks delay (Molecular Cancer Therapeutics, 2022). Hence, we hypothesized that there are additional genes or pathways in cells lacking SNAI2 that are co-opted to drive relapse disease. We have identified that CDKN1A or p21 expression is robustly increased in SNAI2 null cells. CDKN1A is thought to be a tumor suppressor in RMS and other cancers. However, our preliminary data indicate that CDKN1A is an oncogene and in RMS patients, high expression in tumors is associated with poor outcome. We found that p21 expression in combination with SNAI2 can predict resistance to radiation across RMS cell lines. We next developed control RD, Rh30 and Rh18 cells either CDKN1A or both CDKN1A and SNAI2 ablated using CRISPR/Cas9 reagents with control cells expressing control gRNAs. Ablation of CDKN1A while not effecting proliferation led to a significant increase in apoptosis post-radiation and this effect was increased in SNAI2/CDKN1A double knockout cells. Further, in colony forming assays we find that double ablated cells form significantly fewer colonies than control and single knockout cells. Rather unexpectedly, loss of CDKN1A, SNAI2, or CDKN1A/SNAI2 led to a G2/M cell cycle block, indicating that the cell cycle effects of p21 might not be responsible for the phenotypes observed. Ongoing xenograft experiments will determine if the effect we observe in vitro will also translate in vivo. Additionally, we are performing experiments to test how SNAI2 modulates CDKN1A expression. In summary, our study identifies CDKN1A as an oncogene that protects SNAI2 deficient cells from ionizing radiation, thus enabling the survival or rare relapse driving tumor cells. Citation Format: Paulomi S. Modi, Long Wang, Prethish Sreenivas, Myron Ignatius. A SNAI2/CDKN1A pathway protects rhabdomyosarcoma tumors from radiation [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 1465.

The modulation of iron metabolism affects the Rhabdomyosarcoma tumor growth in vitro and in vivo

Rhabdomyosarcoma (RMS) is an aggressive rare neoplasm that derives from mesenchymal cells, which frequently develops resistance to the current therapies and the formation of metastases. Thus, new therapies are needed. The alteration of iron metabolism in cancer cells was effective in reducing the progression of many tumors but not yet investigated in RMS. Here we investigated the effect of iron modulation in RMS both in vitro and in vivo. We first characterized the most used RMS cell lines representing the most common subtypes, embryonal (ERMS, RD cells) and alveolar (ARMS, RH30 cells), for their iron metabolism, in basal condition and in response to its modulation. Then we investigated the effects of both iron overload and chelation strategies in vitro and in vivo. RMS cell lines expressed iron-related proteins, even if at lower levels compared to hepatic cell lines and they are correctly modulated in response to iron increase and deprivation. Interestingly, the treatment with different doses of ferric ammonium citrate (FAC, as iron source) and with deferiprone (DFP, as iron chelator), significantly affected the cell viability of RD and RH30. Moreover, iron supplementation (in the form of iron dextran) or iron chelation (in the form of DFP) were also effective in vivo in inhibiting the tumor mass growth both derived from RD and RH30 with iron chelation treatment the most effective one. All the data suggest that the iron modulation could be a promising approach to overcome the RMS tumor growth. The mechanism of action seems to involve the apoptotic cell death for both iron supplementation and chelation with the concomitant induction of ferroptosis in the case of iron supplementation.

Redundant Signaling as the Predominant Mechanism for Resistance to Antibodies Targeting the Type-I Insulin-Like Growth Factor Receptor in Cells Derived from Childhood Sarcoma.

Antibodies targeting insulin-like growth factor 1 receptor (IGF-1R) induce objective responses in only 5% to 15% of children with sarcoma. Understanding the mechanisms of resistance may identify combination therapies that optimize efficacy of IGF-1R-targeted antibodies. Sensitivity to the IGF-1R-targeting antibody TZ-1 was determined in rhabdomyosarcoma and Ewing sarcoma cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. The BRD4 inhibitor, JQ1, was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of receptor tyrosine kinases (RTK) was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1-resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (∼5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other phosphorylated RTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared with other IGF-1R-targeted antibodies tested against this model.

Anticancer bioactivity of zerumbone on pediatric rhabdomyosarcoma cells

PurposeNatural products are generally regarded as safe and have been shown to mediate anticancer activities against a variety of cell types. Zerumbone is a natural cyclic sesquiterpene derived from the rhizome of Zingiber zerumbet, which has attracted extensive attention in the recent decade for anticancer activities. The present study investigates the in vitro effect of zerumbone on rhabdomyosarcoma cells.MethodsTwo rhabdomyosarcoma cell lines (RD and RH30) were used as the model system. The growth inhibition of zerumbone was measured by MTT-assay, apoptosis via flow cytometry, gene expression by real-time PCR, the migration by transwell assay, and intracellular signaling by Western blotting.ResultsZerumbone shows anticancer effects on RD and RH30 cells in a dose-dependent manner via cell growth inhibition and induction of apoptosis. Exposure of RD and RH30 cells on zerumbone also resulted in a decrease of migration and downregulation of the hedgehog pathway.ConclusionsTaken together, our study provided the first evidence that zerumbone imparted strong inhibitory and apoptotic effects on pediatric rhabdomyosarcoma cell lines and merit further investigation as a promising candidate for the anticancer therapy.

Combination therapy of doxorubicin and Sildenafil inhibits the growth of pediatric rhabdomyosarcoma

BackgroundOverexpression of phosphodiesterase type 5 (PDE5) has been detected in many types of malignant tumors. Sildenafil, a potent and selective inhibitor of a cGMP-specific PDE5, has been found to enhance the cytotoxic activity of different chemotherapeutic agents including doxorubicin. The combined therapy of doxorubicin with Sildenafil might reduce the possible side effects of chemotherapy while maintaining synergistic anticancer activity. The present study explored for the first time the effects of Sildenafil, alone and in combination with doxorubicin, on pediatric RMS cells.MethodsHuman RMS cells lines RH30 and RD were used. Western blotting and RT-PCR were performed to analyze PDE5 expression in RMS cells. Cell viability was determined using MTT assay. Cell migration was analyzed via transwell chambers, clonal growth and mitotic cell death were analyzed using the clonogenic assay. FACS analysis was performed to evaluate reactive oxygen species (ROS) and apoptosis.ResultsSildenafil significantly decreased cell viability and migration of RD and RH30 cells. The exposure of RMS cells to doxorubicin resulted in a dose-dependent decrease in their viability. Simultaneous administration of Sildenafil enhanced this effect. The incubation of the RMS cells with Sildenafil in the presence of doxorubicin significantly increased the proportion of apoptotic cells and ROS production compared to the treatment with Sildenafil alone.ConclusionsThe results of our study indicate a link between PDE5 inhibition by Sildenafil and decreased calcium signalling leading to significantly impaired viability, migration, and colony forming of RMS cells. Increased ROS production and apoptosis are mechanisms relevantly contributing to this observation.

Abstract 2020: Investigation of CYP450 enzymes as therapeutic targets in rhabdomyosarcoma

Abstract Introduction: Rhabdomyosarcoma (RMS) is a rare soft tissue sarcoma (STS) that predominantly affects children and teenagers. It is the most frequent STS in children and accounts for around 5-8% of total childhood cancers. The improvement of chemotherapeutic regimens has raised RMS survival chances over the last decades. However, the mortality rate remains exceptionally high in high-risk RMS patients. Duocarmycins have arisen as an opportunity for targeted therapy in cancer. These ultrapotent anticancer agents have been reengineered to generate bioprecursors that are selectively activated bycertain cytochrome P450 (CYP) isoforms (e.g. CYP1A1, CYP1B1, CYP2W1). The overexpression of these isoforms in tumors provides the possibility to locoregionally bioactivate duocarmycin prodrugs only in the tumor site. Previous studies have shown that duocarmycins can be chemically modified and deactivated by removal of a hydroxyl group in the pharmacophore, which can be reinstalled by CYPsto generate a potent DNA-targeted cytotoxic metabolite. In the past, a consistent CYP2W1overexpression in patient-derived RMS biopsies as compared to matched, non-transformed tissue was revealed. Results: Ongoing studies are focused on measuring CYP1A1, CYP1B1 and CYP2W1 expression levels in RMS cell lines and in three-dimensional spheroids as well as evaluating the antiproliferative activity of duocarmycin bioprecursors in these models. With the exception of RH30 cells (IC50=0.2µM forICT2700) the IC50 for CYP1A1- and CYP2W1-bioactivated duocarmycins (ICT2700 and ICT2706,respectively) lie in the low micromolar range in RMS monolayer cultures, suggesting that CYP1A1 andCYP2W1 might not be a suitable target in these 2D cancer models. Consistently, only RH30 cells displayed a high CYP1A1 expression in monolayer cultures among the RMS cell line panel, and noCYP2W1 expression could be identified. However, preliminary data suggests that CYP1A1 and CYP2W1enzymes might be restored when spheroids are generated from RMS cell lines. A functional 7-ethoxyresorufin-O-deethylase (EROD) assay has been validated to analyze CYP1A1- and CYP1B1-specific enzymatic activity in RMS cell lines. Vorinostat (SAHA) is an epigenetic drug currently used in the clinic for cancer treatment, and it has been associated with modulatory effects on CYP1expression. Our data suggests cell line-dependent synergistic or antagonistic effects of SAHA when combined with ICT2700, and these correlate with SAHA-induced CYP1A1 upregulation and downregulation respectively at both gene and protein levels. Conclusion: Evaluation of CYP target expression in RMS is being established to assess if duocarmycin bioprecursorscan be progressed as a novel type of therapy in RMS. Interestingly, CYP450 expression in RMS cell lines seems to vary depending on the complexity of the in vitro model used. Citation Format: Enric Arasanz Picher, Klaus Pors. Investigation of CYP450 enzymes as therapeutic targets in rhabdomyosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2020.

Role of SOX18 in Promoting Tumorigenesis in Pediatric Cancer Cell Lines

Sarcomas constitute a high percentage (∼13%) of cancer‐related deaths among pediatric patients between 0‐19 years of age, with Rhabdomyosarcoma (RMS) being the most common pediatric soft tissue sarcoma and Ewing Sarcoma being the second most common malignant bone tumor in children. Yet, survival for those who develop such metastatic sarcomas remains below 20‐30%. Interestingly, SOX family proteins are known to be up regulated in various cancer types and play a role in cancer progression (tumorigenesis, metastasis, etc.). More specifically, targeted knockdown of SOX18 has been shown to suppress various tumorigenic properties in cancer cell lines including osteosarcoma cells, hepatocellular carcinoma cells and breast cancer cells. Additionally, prior studies showed inhibition of IGFR leads to compensatory pathway activation via other RTK receptors resulting in continued cell survival. Upregulation of SOX18 was observed in such cases. Thus, it is important to clarify the role of SOX18 in RMS and ES. Ewing Sarcoma (ES8) and Rhabdomyosarcoma (RH41) cell lines were infected with shSOX18 to produce knockdown and selected via puromycin resistance. Western blotting and RT‐qPCR was used to analyze baseline and resultant protein and mRNA levels, respectively after knockdown. Results showed that the shRNA was effective in SOX18 knockdown and lead to reduced cell proliferation. Cell migration, cell proliferation and colony formation assays were used to characterize effects of knockdown. Further evaluation of knockdown effects on angiogenesis was analyzed using chick chorioallantoic membrane (CAM) assays. For ES8 and RH41 cell lines, SOX18 knockdown led to reduced cell migration, cell proliferation and colony formation. We anticipate characterization of the role of SOX18 in RMS and ES may inform future development of novel targeted treatments and increased understanding of role of SOX18 in activation of RTK compensatory pathways during IGFR inhibition in pediatric sarcomas.

Novel non-covalent LSD1 inhibitors endowed with anticancer effects in leukemia and solid tumor cellular models

LSD1 is a histone lysine demethylase proposed as therapeutic target in cancer. Chemical modifications applied at C2, C4 and/or C7 positions of the quinazoline core of the previously reported dual LSD1/G9a inhibitor 1 led to a series of non-covalent, highly active, and selective LSD1 inhibitors (2–4 and 6–30) and to the dual LSD1/G9a inhibitor 5 that was more potent than 1 against LSD1. In THP-1 and MV4-11 leukemic cells, the most potent compounds (7, 8, and 29) showed antiproliferative effects at sub-micromolar level without significant toxicity at 1 μM in non-cancer AHH-1 cells. In MV4-11 cells, the new derivatives increased the levels of the LSD1 histone mark H3K4me2 and induced the re-expression of the CD86 gene silenced by LSD1, thereby confirming the inhibition of LSD1 at cellular level. In breast MDA-MB-231 as well as in rhabdomyosarcoma RD and RH30 cells, taken as examples of solid tumors, the same compounds displayed cell growth arrest in the same IC50 range, highlighting a crucial anticancer role for LSD1 inhibition and suggesting no added value for the simultaneous G9a inhibition in these tumor cell lines.

MET Inhibition Sensitizes Rhabdomyosarcoma Cells to NOTCH Signaling Suppression.

Rhabdomyosarcoma (RMS) is a pediatric myogenic soft tissue sarcoma. The Fusion-Positive (FP) subtype expresses the chimeric protein PAX3-FOXO1 (P3F) while the Fusion-Negative (FN) is devoid of any gene translocation. FP-RMS and metastatic FN-RMS are often unresponsive to conventional therapy. Therefore, novel therapeutic approaches are needed to halt tumor progression. NOTCH signaling has oncogenic functions in RMS and its pharmacologic inhibition through γ-secretase inhibitors blocks tumor growth in vitro and in vivo. Here, we show that NOTCH signaling blockade resulted in the up-regulation and phosphorylation of the MET oncogene in both RH30 (FP-RMS) and RD (FN-RMS) cell lines. Pharmacologic inhibition of either NOTCH or MET signaling slowed proliferation and restrained cell survival compared to control cells partly by increasing Annexin V and CASP3/7 activation. Co-treatment with NOTCH and MET inhibitors significantly amplified these effects and enhanced PARP1 cleavage in both cell lines. Moreover, it severely hampered cell migration, colony formation, and anchorage-independent growth compared to single-agent treatments in both cell lines and significantly prevented the growth of FN-RMS cells grown as spheroids. Collectively, our results unveil the overexpression of the MET oncogene by NOTCH signaling targeting in RMS cells and show that MET pathway blockade sensitizes them to NOTCH inhibition.

The Effect of Direct and Indirect EZH2 Inhibition in Rhabdomyosarcoma Cell Lines.

Simple SummaryRhabdomyosarcoma is the most common soft tissue tumor in children. Its two major subtypes show epigenetic alterations that are associated with poor prognosis. Therefore, targeting these epigenetic alterations by pharmacological intervention could be a therapeutic approach. We investigated two different types of substances that interfere with the epigenetic process of histone methylation. We performed studies in two cell lines that carry characteristics of the major rhabdomyosarcoma subtypes. The aim of this study was to find out if the substances differ in their effect on tumor-related cellular functions and to find out if the tumor subtypes differ in their response to the substances. These findings may contribute to a better assessment of the feasibility of pharmacological intervention directed against histone methylation in subtypes of rhabdomyosarcoma.Enhancer of Zeste homolog 2 (EZH2) is involved in epigenetic regulation of gene transcription by catalyzing trimethylation of histone 3 at lysine 27. In rhabdomyosarcoma (RMS), increased EZH2 protein levels are associated with poor prognosis and increased metastatic potential, suggesting EZH2 as a therapeutic target. The inhibition of EZH2 can be achieved by direct inhibition which targets only the enzyme activity or by indirect inhibition which also affects activities of other methyltransferases and reduces EZH2 protein abundance. We assessed the direct inhibition of EZH2 by EPZ005687 and the indirect inhibition by 3-deazaneplanocin (DZNep) and adenosine dialdehyde (AdOx) in the embryonal RD and the alveolar RH30 RMS cell line. EPZ005687 was more effective in reducing the cell viability and colony formation, in promoting apoptosis induction, and in arresting cells in the G1 phase of the cell cycle than the indirect inhibitors. DZNep was more effective in decreasing spheroid viability and size in both cell lines than EPZ005687 and AdOx. Both types of inhibitors reduced cell migration of RH30 cells but not of RD cells. The results show that direct and indirect inhibition of EZH2 affect cellular functions differently. The alveolar cell line RH30 is more sensitive to epigenetic intervention than the embryonal cell line RD.

Flavonoids kaempferol and quercetin are nuclear receptor 4A1 (NR4A1, Nur77) ligands and inhibit rhabdomyosarcoma cell and tumor growth

BackgroundFlavonoids exhibit both chemopreventive and chemotherapeutic activity for multiple tumor types, however, their mechanisms of action are not well defined. Based on some of their functional and gene modifying activities as anticancer agents, we hypothesized that kaempferol and quercetin were nuclear receptor 4A1 (NR4A1, Nur77) ligands and confirmed that both compounds directly bound NR4A1 with KD values of 3.1 and 0.93 μM, respectively.MethodsThe activities of kaempferol and quercetin were determined in direct binding to NR4A1 protein and in NR4A1-dependent transactivation assays in Rh30 and Rh41 rhabdomyosarcoma (RMS) cells. Flavonoid-dependent effects as inhibitors of cell growth, survival and invasion were determined in XTT and Boyden chamber assays respectively and changes in protein levels were determined by western blots. Tumor growth inhibition studies were carried out in athymic nude mice bearing Rh30 cells as xenografts.ResultsKaempferol and quercetin bind NR4A1 protein and inhibit NR4A1-dependent transactivation in RMS cells. NR4A1 also regulates RMS cell growth, survival, mTOR signaling and invasion. The pro-oncogenic PAX3-FOXO1 and G9a genes are also regulated by NR4A1 and, these pathways and genes are all inhibited by kaempferol and quercetin. Moreover, at a dose of 50 mg/kg/d kaempferol and quercetin inhibited tumor growth in an athymic nude mouse xenograft model bearing Rh30 cells.ConclusionThese results demonstrate the clinical potential for repurposing kaempferol and quercetin for clinical applications as precision medicine for treating RMS patients that express NR4A1 in order to increase the efficacy and decrease dosages of currently used cytotoxic drugs.

Hybrid amorphous TiO2/polymer nanomaterials trigger apoptosis of pediatric cancer cells upon ultrasound irradiation

Sonodynamic therapy is a cost-effective, minimally invasive and localized anticancer therapy based on the in situ generation of reactive oxygen species by combining low-intensity ultrasound (US), oxygen, and a sonosensitizer and it minimizes systemic toxicity. In this work, we initially study the effect of poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) block copolymers of different architecture, molecular weight, and hydrophilic-lipophilic balance on the size, size distribution and morphology of hybrid amorphous TiO2/polymer sono-responsive nanoparticles (NPs) produced by a sol-gel process that comprises the synthesis and aging of a Ti(IV)-acetone oxo-organo complex, its mixing with the PEO-PPO block copolymer and their nanoprecipitation in water. Regardless of the PEO-PPO block copolymer used in the synthesis, the properties of the hybrid NPs are governed by the age of the oxo-organo complex. At the same time, we show the ability to incorporate a variety of amphiphiles with different hydrophilic-lipophilic balance, and thus, different encapsulation capacities of hydrophobic cargos. Morphological analysis by high-resolution–transmission electron microscopy shows that all the NPs are rounded. Next, we demonstrate that these hybrid NPs induce the formation of reactive oxygen species upon irradiation with the therapeutic US. In addition, they exhibit good compatibility and uptake by the Rh30 cell line, a model of rhabdomyosarcoma (a pediatric tumor of connective tissue) and do not cause significant hemolysis upon exposure of up to 24 h. Finally, we investigate the pathway by which these nanomaterials kill Rh30 cells by using annexin-V/propidium iodide staining and flow cytometry and demonstrate that, upon US induction, they trigger cell apoptosis. To the best of our knowledge, this is the first work reporting on the sono-responsive performance of an amorphous TiO2-based nanomaterial and its potential application in the sonodynamic therapy of cancer.