Neuroblastoma Patient-derived Xenografts Research Articles

Abstract 2609: Vesicular LGALS3BP is a neuroblastoma biomarker and a therapeutic target for combination therapy with antibody-drug conjugate and checkpoint inhibitors

Abstract We have recently shown that targeting circulating LGALS3BP with a maytansinoid-based non-internalizing antibody-drug conjugate (ADC) resulted in tumor eradication of high-risk NMYC-amplified neuroblastoma patient-derived xenografts. LGALS3BP is a high molecular weight, hyperglycosylated and secreted protein which is enriched at the surface of cancer-derived extracellular vesicles (EVs). Here, using a neuroblastoma pseudometastatic preclinical model, we extended our previous work demonstrating that EVs-associated LGALS3BP strictly correlates with disease progression and dissemination. Moreover, to investigate whether LGALS3BP ADC, named 1959sss/DM4, possesses immune-modulating properties, we analyzed DM4 ability to induce immunogenic cell death (ICD). Interestingly, using NXS2 murine neuroblastoma cells, we found that DM4 induced a time-dependent surface expression of three different ICD marker, i.e. calreticulin, HSP70 and HSP90. As it was recently demonstrated that to limit immune surveillance NXS2 cells increase PD-L1 expression in response to IFNγ production in the tumor immune infiltrate, we evaluated potential synergism between LGALS3BP targeting ADC and an anti-PD1 immune check-point inhibitor. To this aim, we genetically manipulated NXS2 cells which resulted nearly negative for murine LGALS3BP expression. Therefore, using a lentivirus-based gene transduction system, we obtained NXS2 cells producing large amounts of vesicular human LGALS3BP. Of note, the vesicular LGALS3BP could be quantified by ELISA and imaged by confocal microscopy using 1959 antibody. Strikingly, while single agent treatment showed a limited antitumor activity in NXS2-LGALS3BP syngeneic model, the LGALS3BP ADC/anti-PD1 combination resulted in a marked inhibition of tumor growth, which was followed by a prolonged survival. In conclusion, our findings establish that vesicular LGALS3BP is a potential biomarker for liquid biopsy and reveals this protein as a suitable target for therapeutic strategy that combines 1959sss/DM4 ADC with immune checkpoint blockade for the treatment of LGALS3BP expressing neuroblastoma. Citation Format: Gianluca Sala, Emily Capone, Ilaria Cela, Giulio Lovato, Alessia Lamolinara, Manuela Iezzi, Cosmo Rossi, Vincenzo De Laurenzi, Rodolfo Ippoliti, Stefano Iacobelli. Vesicular LGALS3BP is a neuroblastoma biomarker and a therapeutic target for combination therapy with antibody-drug conjugate and checkpoint inhibitors [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 2609.

Targeted Degradation of XIAP is Sufficient and Specific to Induce Apoptosis in MYCN-overexpressing High-risk Neuroblastoma.

XIAP degradation is sufficient to kill MYCN-amplified neuroblastoma which overexpresses and relies on XIAP as a brake against cell death, without affecting normal cells.

Macrophage infiltration promotes regrowth in MYCN-amplified neuroblastoma after chemotherapy

ABSTRACT Despite aggressive treatment, the 5-year event-free survival rate for children with high-risk neuroblastoma is <50%. While most high-risk neuroblastoma patients initially respond to treatment, often with complete clinical remission, many eventually relapse with therapy-resistant tumors. Novel therapeutic alternatives that prevent the recurrence of therapy-resistant tumors are urgently needed. To understand the adaptation of neuroblastoma under therapy, we analyzed the transcriptomic landscape in 46 clinical tumor samples collected before (PRE) or after (POST) treatment from 22 neuroblastoma patients. RNA sequencing revealed that many of the top-upregulated biological processes in POST MYCN amplified (MNA+) tumors compared to PRE MNA+ tumors were immune-related, and there was a significant increase in numerous genes associated with macrophages. The infiltration of macrophages was corroborated by immunohistochemistry and spatial digital protein profiling. Moreover, POST MNA+ tumor cells were more immunogenic compared to PRE MNA+ tumor cells. To find support for the macrophage-induced outgrowth of certain subpopulations of immunogenic tumor cells following treatment, we examined the genetic landscape in multiple clinical PRE and POST tumor samples from nine neuroblastoma patients revealing a significant correlation between an increased amount of copy number aberrations (CNA) and macrophage infiltration in POST MNA+ tumor samples. Using an in vivo neuroblastoma patient-derived xenograft (PDX) chemotherapy model, we further show that inhibition of macrophage recruitment with anti-CSF1R treatment prevents the regrowth of MNA+ tumors following chemotherapy. Taken together, our work supports a therapeutic strategy for fighting the relapse of MNA+ neuroblastoma by targeting the immune microenvironment.

Inhibition of PIM Kinases Promotes Neuroblastoma Cell Differentiation to a Neuronal Phenotype

BackgroundNeuroblastoma arises from aberrancies in neural stem cell differentiation. PIM kinases contribute to cancer formation, but their precise role in neuroblastoma tumorigenesis is poorly understood. In the current study, we evaluated the effects of PIM kinase inhibition on neuroblastoma differentiation. MethodsVersteeg database query assessed the correlation between PIM gene expression and the expression of neuronal stemness markers and relapse free survival. PIM kinases were inhibited with AZD1208. Viability, proliferation, motility were measured in established neuroblastoma cells lines and high-risk neuroblastoma patient-derived xenografts (PDXs). qPCR and flow cytometry detected changes in neuronal stemness marker expression after AZD1208 treatment. ResultsDatabase query showed increased levels of PIM1, PIM2, or PIM3 gene expression were associated with higher risk of recurrent or progressive neuroblastoma. Increased levels of PIM1 were associated with lower relapse free survival rates. Higher levels of PIM1 correlated with lower levels of neuronal stemness markers OCT4, NANOG, and SOX2. Treatment with AZD1208 resulted in increased expression of neuronal stemness markers. ConclusionsInhibition of PIM kinases differentiated neuroblastoma cancer cells toward a neuronal phenotype. Differentiation is a key component of preventing neuroblastoma relapse or recurrence and PIM kinase inhibition provides a potential new therapeutic strategy for this disease.

Analysis of Serial Neuroblastoma PDX Passages in Mice Allows the Identification of New Mediators of Neuroblastoma Aggressiveness.

Neuroblastoma is a neural crest cell-derived pediatric tumor characterized by high inter- and intra-tumor heterogeneity, and by a poor outcome in advanced stages. Patient-derived xenografts (PDXs) have been shown to be useful models for preserving and expanding original patient biopsies in vivo, and for studying neuroblastoma biology in a more physiological setting. The maintenance of genetic, histologic, and phenotypic characteristics of the original biopsy along serial PDX passages in mice is a major concern regarding this model. Here we analyze consecutive PDX passages in mice, at both transcriptomic and histological levels, in order to identify potential changes or highlight similarities to the primary sample. We studied temporal changes using mRNA and miRNA expression and correlate those with neuroblastoma aggressiveness using patient-derived databases. We observed a shortening of tumor onset and an increase in proliferative potential in the PDXs along serial passages. This behavior correlates with changes in the expression of genes related to cell proliferation and neuronal differentiation, including signaling pathways described as relevant for neuroblastoma malignancy. We also identified new genes and miRNAs that can be used to stratify patients according to survival, and which could be potential new players in neuroblastoma aggressiveness. Our results highlight the usefulness of the PDX neuroblastoma model and reflect phenotypic changes that might be occurring in the mouse environment. These findings could be useful for understanding the progression of tumor aggressiveness in this pathology.

Engineering human mini-bones for the standardized modeling of healthy hematopoiesis, leukemia, and solid tumor metastasis.

The bone marrow microenvironment provides indispensable factors to sustain blood production throughout life. It is also a hotspot for the progression of hematologic disorders and the most frequent site of solid tumor metastasis. Preclinical research relies on xenograft mouse models, but these models preclude the human-specific functional interactions of stem cells with their bone marrow microenvironment. Instead, human mesenchymal cells can be exploited for the in vivo engineering of humanized niches, which confer robust engraftment of human healthy and malignant blood samples. However, mesenchymal cells are associated with major reproducibility issues in tissue formation. Here, we report the fast and standardized generation of human mini-bones by a custom-designed human mesenchymal cell line. These resulting humanized ossicles (hOss) consist of fully mature bone and bone marrow structures hosting a human mesenchymal niche with retained stem cell properties. As compared to mouse bones, we demonstrate superior engraftment of human cord blood hematopoietic cells and primary acute myeloid leukemia samples and also validate hOss as a metastatic site for breast cancer cells. We further report the engraftment of neuroblastoma patient-derived xenograft cells in a humanized model, recapitulating clinically described osteolytic lesions. Collectively, our human mini-bones constitute a powerful preclinical platform to model bone-developing tumors using patient-derived materials.

Evaluation of the DLL3-targeting antibody-drug conjugate rovalpituzumab tesirine in preclinical models of neuroblastoma.

Neuroblastomas have neuroendocrine features and often show similar gene expression patterns to small cell lung cancer including high expression of delta-like ligand 3 (DLL3). Here we determine the efficacy of rovalpituzumab tesirine (Rova-T), an antibody drug conjugated (ADC) with a pyrrolobenzodiazepine (PBD) dimer toxin targeting DLL3, in preclinical models of human neuroblastoma. We evaluated DLL3 expression in RNA sequencing data sets and performed immunohistochemistry (IHC) on neuroblastoma patient derived xenograft (PDX), human neuroblastoma primary tumor and normal childhood tissue microarrays (TMAs). We then evaluated the activity of Rova-T against 11 neuroblastoma PDX models using varying doses and schedules and compared anti-tumor activity to expression levels. DLL3 mRNA was differentially overexpressed in neuroblastoma at comparable levels to small cell lung cancer, as well as Wilms and rhabdoid tumors. DLL3 protein was robustly expressed across the neuroblastoma PDX array, but membranous staining was variable. The human neuroblastoma array, however, showed staining in only 44% of cases, whereas no significant staining was observed in the normal childhood tissue array. Rova-T showed a clear dose response effect across the 11 models tested, with a single dose inducing a complete or partial response in 3/11 and stable disease in another 3/11 models. No overt signs of toxicity were observed, and there was no treatment-related mortality. Strong membranous staining was necessary, but not sufficient, for anti-tumor activity. Rova-T has activity in a subset of neuroblastoma preclinical models, but heterogeneous expression in these models and the near absence of expression seen in human tumors suggests that any DLL3-targeting clinical trial should be only performed with a robust companion diagnostic to evaluate DLL3 expression for patient selection.

Abstract 2006: Preclinical evaluation of dual AURKA (LY3295668) and BCL2 (venetoclax) inhibition in neuroblastoma (NB)

Abstract Introduction: Deregulated expression of the MYCN oncogene is associated with the development of many neural-derived pediatric cancers including NB, medulloblastoma and atypical teratoid rhabdoid tumors. Amplification of the MYCN oncogene is present in over 40% of high-risk NBs that remain lethal in most patients, thus developing better therapies for these patients is essential. While direct therapeutic targeting of MYCN protein remains challenging, we previously demonstrated synergistic activity of dual targeting of BCL-2 (venetoclax) and AURKA (MLN8237) in MYCN-amplified NB (Ham Cancer Cell, 2016). With MLN8237 no longer being developed due to toxicities related to AURKB inhibition, testing of a highly selective AURKA inhibitor, LY3295668, is warranted. Methods: LY3295668 (Chemie Tek) and venetoclax (Selleck Chem) were tested alone and in combination in six NB PDX models using 2-3 mice/arm. Five Patient Derived Xenograft (PDX) models tested (COG-N-424x, COG-N-453x, NB-1643, Obelix-Rx and Obelix-2x) were MYCN-amplified and one xenograft model (SK-N-AS) was MYCN-non-amplified. First, a comprehensive toxicity study, testing LY3295668 at 30mg/kg with venetoclax at 100mg/kg, 75mg/kg, 50mg/kg, and 25mkg/kg, was performed. Based on tolerability of combination, an optimal dose of 30mg/kg for LY3295668 and 50mg/kg for venetoclax was selected and administered once daily, seven days a week, via oral gavage for 60 days. These were the maximum tolerated doses when tested in combination, that did not result in weight loss greater than 15% or other overt signs of toxicity. Results: LY3295668 (30mg/kg) and venetoclax (50mg/kg) were well tolerated as monotherapy, and when administered in combination showed a maximum weight loss of 13%. Single agent therapy resulted in minimal if any growth delay. Four of five MYCN-amplified PDX models demonstrated maintained complete responses and one showed significant tumor growth delay. No effect was observed in the MYCN-non-amplified model with single agent or combination treatment compared to vehicle. Conclusions: Dual targeting of AURKA and BCL2 with LY3295668 and venetoclax is tolerable and demonstrates compelling anti-tumor activity in MYCN-amplified NB PDX models, validating our earlier findings (Ham et al). These preclinical results provisionally support clinical development of this combination for NB patients with MYCN amplification. Assessment of additional PDX models is currently ongoing and will be reported. Citation Format: David Groff, Colleen Casey, Kateryna krytska, Matthew Tsang, Christina Acholla, John M. Maris, Yael P. Mosse. Preclinical evaluation of dual AURKA (LY3295668) and BCL2 (venetoclax) inhibition in neuroblastoma (NB) [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 2006.

Pre-Clinical Study Evaluating Novel Protein Phosphatase 2A Activators as Therapeutics for Neuroblastoma.

Simple SummaryDespite significant advancements in neuroblastoma therapy, over half of the children with high-risk neuroblastoma will suffer disease relapse. Protein phosphatase 2A (PP2A) is a tumor suppressor that is decreased in neuroblastoma. Previous studies with FTY720, an immunomodulatory agent that activates PP2A, led to a decreased malignant phenotype in neuroblastoma. To activate PP2A without issues associated with the use of an immunomodulator, we investigated two novel PP2A-activating compounds, ATUX-792 and DBK-1154, designed for improved efficacy and fewer side effects. We aimed to demonstrate that ATUX-792 and DBK-1154 would decrease neuroblastoma cell survival and tumor growth with the goal of providing pre-clinical data to support advancing these compounds into the clinical arena.Background: Protein phosphatase 2A (PP2A) functions as an inhibitor of cancer cell proliferation, and its tumor suppressor function is attenuated in many cancers. Previous studies utilized FTY720, an immunomodulating compound known to activate PP2A, and demonstrated a decrease in the malignant phenotype in neuroblastoma. We wished to investigate the effects of two novel PP2A activators, ATUX-792 (792) and DBK-1154 (1154). Methods: Long-term passage neuroblastoma cell lines and human neuroblastoma patient-derived xenograft (PDX) cells were used. Cells were treated with 792 or 1154, and viability, proliferation, and motility were examined. The effect on tumor growth was investigated using a murine flank tumor model. Results: Treatment with 792 or 1154 resulted in PP2A activation, decreased cell survival, proliferation, and motility in neuroblastoma cells. Immunoblotting revealed a decrease in MYCN protein expression with increasing concentrations of 792 and 1154. Treatment with 792 led to tumor necrosis and decreased tumor growth in vivo. Conclusions: PP2A activation with 792 or 1154 decreased survival, proliferation, and motility of neuroblastoma in vitro and tumor growth in vivo. Both compounds resulted in decreased expression of the oncogenic protein MYCN. These findings indicate a potential therapeutic role for these novel PP2A activators in neuroblastoma.

Targeting High-Risk Neuroblastoma Patient-Derived Xenografts with Oncolytic Virotherapy.

Simple SummaryMore than half of children with high-risk neuroblastoma will not survive despite aggressive treatment regimens. The discovery of suitable novel therapies that target high-risk disease remain limited. Oncolytic virotherapy employs common viruses, such as herpes simplex virus. These viruses are modified to solely target cancer cells and avoid damage to healthy cells. The previous work of others and ourselves has shown oncolytic herpes simplex viruses (oHSVs) are effective in targeting cultured neuroblastoma cancer cell lines, but to our knowledge, no one has demonstrated the effects of oHSV in neuroblastoma patient-derived xenograft cells. Herein, we show that oHSV works well in high-risk neuroblastoma patient-derived xenografts and provide critical preclinical evidence to translate this therapy to a clinical setting.Cancer is the leading cause of death by disease in children, and over 15% of pediatric cancer-related mortalities are due to neuroblastoma. Current treatment options for neuroblastoma remain suboptimal as they often have significant toxicities, are associated with long-term side effects, and result in disease relapse in over half of children with high-risk disease. There is a dire need for new therapies, and oncolytic viruses may represent an effective solution. Oncolytic viruses attack tumor cells in two ways: direct infection of tumor cells leading to cytolysis, and production of a debris field that stimulates an anti-tumor immune response. Our group has previously shown that M002, an oncolytic herpes simplex virus (oHSV), genetically engineered to express murine interleukin-12 (mIL-12), was effective at targeting and killing long term passage tumor cell lines. In the current study, we investigated M002 in three neuroblastoma patient-derived xenografts (PDXs). PDXs better recapitulate the human condition, and these studies were designed to gather robust data for translation to a clinical trial. We found that all three PDXs expressed viral entry receptors, and that the virus actively replicated in the cells. M002 caused significant tumor cell death in 2D culture and 3D bioprinted tumor models. Finally, the PDXs displayed variable susceptibility to M002, with a more profound effect on high-risk neuroblastoma PDXs compared to low-risk PDX. These findings validate the importance of incorporating PDXs for preclinical testing of oncolytic viral therapeutics and showcase a novel technique, 3D bioprinting, to test therapies in PDXs. Collectively, our data indicate that oHSVs effectively target high-risk neuroblastoma, and support the advancement of this therapy to the clinical setting.

Label-free separation of neuroblastoma patient-derived xenograft (PDX) cells from hematopoietic progenitor cell products by acoustophoresis

BackgroundGraft-contaminating tumor cells correlate with inferior outcome in high-risk neuroblastoma patients undergoing hematopoietic stem cell transplantation and can contribute to relapse. Motivated by the potential therapeutic benefit of tumor cell removal as well as the high prognostic and diagnostic value of isolated circulating tumor cells from stem cell grafts, we established a label-free acoustophoresis-based microfluidic technology for neuroblastoma enrichment and removal from peripheral blood progenitor cell (PBPC) products.MethodsNeuroblastoma patient-derived xenograft (PDX) cells were spiked into PBPC apheresis samples as a clinically relevant model system. Cells were separated by ultrasound in an acoustophoresis microchip and analyzed for recovery, purity and function using flow cytometry, quantitative real-time PCR and cell culture.ResultsPDX cells and PBPCs showed distinct size distributions, which is an important parameter for efficient acoustic separation. Acoustic cell separation did not affect neuroblastoma cell growth. Acoustophoresis allowed to effectively separate PDX cells from spiked PBPC products. When PBPCs were spiked with 10% neuroblastoma cells, recoveries of up to 98% were achieved for PDX cells while more than 90% of CD34+ stem and progenitor cells were retained in the graft. At clinically relevant tumor cell contamination rates (0.1 and 0.01% PDX cells in PBPCs), neuroblastoma cells were depleted by more than 2-log as indicated by RT-PCR analysis of PHOX2B, TH and DDC genes, while > 85% of CD34+ cells could be retained in the graft.ConclusionThese results demonstrate the potential use of label-free acoustophoresis for PBPC processing and its potential to develop label-free, non-contact tumor cell enrichment and purging procedures for future clinical use.

Abstract 3034: Proteomic profiling identifies PTK7 as a novel immunotherapeutic candidate for neuroblastoma

Abstract Anti-GD2 antibody therapy significantly improved high-risk neuroblastoma (NB) patient survival yet is associated with toxicity due to GD2 expression on pain fibers. Also, GD2 may be downregulated in NB following chemotherapy and relapse. It is paramount to identify novel tumor-associated antigens that are upregulated on the cell surface throughout therapy with minimal normal tissue expression. We utilized a novel cell surface capturing technology to define the NB cell surfaceome before and after chemotherapy and nominate potential targets for cell-mediated immunotherapy. Two NB patient derived xenografts (PDXs) and one cell line xenograft (IMR5) were treated with topotecan/cyclophosphamide in vivo. Treated and untreated tumors were disaggregated and N-glycoproteins on intact cells were oxidized, selectively labeled with biocytin-hydrazide, lysed, proteins digested, and cell surface peptides enriched using NeutrAvidin beads, followed by LC-MS analysis. We prioritized cell surface proteins that were abundant on all NB models and did not change under the selective pressure of chemotherapy. Our top hit was confirmed for cell surface expression by flow, evaluated for primary tumor and normal cell expression using gene expression datasets, and evaluated for functional relevance using CRISPR/CAS9 and immunotherapy targeting via chimeric antigen receptor (CAR). In two replicates, 292 cell surface proteins were identified as abundant and unchanged following chemotherapy. 34/292 proteins had high abundance of ≥70 a.u. across all xenografts, including NB surface proteins ALK and NCAM1. One protein identified was PTK7, a member of the WNT signaling pathway that is overexpressed in adult tumors. PTK7 is highly expressed in primary NB tumors with very low normal tissue expression. PTK7 protein is present on the cell surface in 17/17 NB cell lines and PDXs. While high PTK7 expression in primary NB is associated with poor patient prognosis, CRISPR/CAS9 knockout of PTK7 shows no functional effect on cell survival, proliferation or chemotherapy response. We developed a codon-optimized, second-generation CAR construct specific to PTK7. Our CAR-engineered T cells specifically bound to recombinant human-PTK7 Fc chimera. PTK7 CAR Jurkat T cells became activated in the presence of PTK7-expressing KELLY, IMR-5, and NB-1643 NB cells, and activation was not seen in the presence of PTK7 negative CMK leukemia cells or NB-1643 PTK7 knockout cells, confirming activation is specific to the CAR recognizing PTK7. Primary T cell PTK7 CAR studies are ongoing to assess cytotoxicity against NB. Multiple surface proteins remain stable on NB tumors after chemotherapy treatment. PTK7 is a potential cell surface antigen that could be utilized for immunotherapy approaches in NB while minimizing damage to normal tissues. PTK7 is robustly expressed in other pediatric and adult cancers, suggesting broader clinical relevance. Citation Format: Arhanti Sadanand, Victor Maximov, Jasmine Lee, Suttipong Suttapitugsakul, Jenny Shim, Chris Doering, Ronghu Wu, Robert Schnepp, Trent Spencer, Kelly Goldsmith. Proteomic profiling identifies PTK7 as a novel immunotherapeutic candidate for neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3034.

Probenecid increases renal retention and antitumor activity of DFMO in neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in children. Interference with the polyamine biosynthesis pathway by inhibition of MYCN-activated ornithine decarboxylase (ODC) is a validated approach. The ODC inhibitor α-difluoromethylornithine (DFMO, or Eflornithine) has been FDA-approved for the treatment of trypanosomiasis and hirsutism and has advanced to clinical cancer trials including NB as well as cancer-unrelated human diseases. One key challenge of DFMO is its rapid renal clearance and the need for high and frequent drug dosing during treatment. We performed in vivo pharmacokinetic (PK), antitumorigenic, and molecular studies with DFMO/probenecid using NB patient-derived xenografts (PDX) in mice. We used LC-MS/MS, HPLC, and immunoblotting to analyze blood, brain tissue, and PDX tumor tissue samples collected from mice. The organic anion transport 1/3 (OAT 1/3) inhibitor probenecid reduces the renal clearance of DFMO and significantly increases the antitumor activity of DFMO in PDX of NB (P < 0.02). Excised tumors revealed that DFMO/probenecid treatment decreases polyamines putrescine and spermidine, reduces MYCN protein levels and dephosphorylates retinoblastoma (Rb) protein (p-RbSer795), suggesting DFMO/probenecid-induced cell cycle arrest. Addition of probenecid as an adjuvant to DFMO therapy may be suitable to decrease overall dose and improve drug efficacy in vivo.

Novel Second Generation Rexinoid Induces Growth Arrest and Reduces Cancer Cell Stemness in Human Neuroblastoma Patient-Derived Xenografts

Novel Second Generation Rexinoid Induces Growth Arrest and Reduces Cancer Cell Stemness in Human Neuroblastoma Patient-Derived Xenografts

Novel second-generation rexinoid induces growth arrest and reduces cancer cell stemness in human neuroblastoma patient-derived xenografts

IntroductionThe poor therapeutic efficacy seen with current treatments for neuroblastoma may be attributed to stem cell-like cancer cells (SCLCCs), a subpopulation of cancer cells associated with poor prognosis and disease recurrence. Retinoic acid (RA) is a differentiating agent used as maintenance therapy for high-risk neuroblastoma but nearly half of children treated with RA relapse. We hypothesized that 6-Methyl-UAB30 (6-Me), a second-generation rexinoid recently developed with a favorable toxicity profile compared to RA, would reduce cancer cell stemness in human neuroblastoma patient-derived xenografts (PDXs). MethodsCells from three neuroblastoma PDXs were treated with 6-Me and proliferation, viability, motility, and cell-cycle progression were assessed. CD133 expression, sphere formation, and mRNA abundance of stemness and differentiation markers were evaluated using flow cytometry, in vitro extreme limiting dilution analysis, and real-time PCR, respectively. ResultsTreatment with 6-Me decreased proliferation, viability, and motility, and induced cell-cycle arrest and differentiation in all three neuroblastoma PDXs. In addition, 6-Me treatment led to decreased CD133 expression, decreased sphere-forming ability, and decreased mRNA abundance of Oct4, Nanog, and Sox2, indicating decreased cancer cell stemness. Conclusions6-Me decreased oncogenicity and reduced cancer cell stemness of neuroblastoma PDXs, warranting further exploration of 6-Me as potential novel therapy for neuroblastoma.

Rewiring Tumor Cell State in Cancer Therapy: Comment on ‘9-cis-UAB30, a Novel Rexinoid Agonist, Decreases Tumorigenicity and Cancer Cell Stemness of Human Neuroblastoma Patient-Derived Xenografts’ by ‘Elizabeth Beierle et al.’

Rewiring Tumor Cell State in Cancer Therapy: Comment on ‘9-cis-UAB30, a Novel Rexinoid Agonist, Decreases Tumorigenicity and Cancer Cell Stemness of Human Neuroblastoma Patient-Derived Xenografts’ by ‘Elizabeth Beierle et al.’

Novel Second-Generation Rexinoid Reduces Cancer Cell Stemness in Human Neuroblastoma Patient-Derived Xenografts

Marayati, Raoud MD; Williams, Adele P. MD; Bownes, Laura V. MD; Quinn, Colin H. BS; Atigadda, Venkatram R. PhD; Stewart, Jerry E. BS; Aye, Jamie M. MD; Yoon, Karina J. PhD; Beierle, Elizabeth A. MD Author Information

9-cis-UAB30, a novel rexinoid agonist, decreases tumorigenicity and cancer cell stemness of human neuroblastoma patient-derived xenografts

Retinoic acid (RA) therapy has been utilized as maintenance therapy for high-risk neuroblastoma, but over half of patients treated with RA relapse. Neuroblastoma stem cell-like cancer cells (SCLCCs) are a subpopulation of cells characterized by the expression of the cell surface marker CD133 and are hypothesized to contribute to drug resistance and disease relapse. A novel rexinoid compound, 9-cis-UAB30 (UAB30), was developed having the same anti-tumor effects as RA but a more favorable toxicity profile. In the current study, we investigated the efficacy of UAB30 in neuroblastoma patient-derived xenografts (PDX). Two PDXs, COA3 and COA6, were utilized and alterations in the malignant phenotype were assessed following treatment with RA or UAB30. UAB30 significantly decreased proliferation, viability, and motility of both PDXs. UAB30 induced cell-cycle arrest as demonstrated by the significant increase in percentage of cells in G1 (COA6: 33.7 ± 0.7 vs. 43.3 ± 0.7%, control vs. UAB30) and decrease in percentage of cells in S phase (COA6: 44.7 ± 1.2 vs. 38.6 ± 1%, control vs. UAB30). UAB30 led to differentiation of PDX cells, as evidenced by the increase in neurite outgrowth and mRNA abundance of differentiation markers. CD133 expression was decreased by 40% in COA6 cells after UAB30. The ability to form tumorspheres and mRNA abundance of known stemness markers were also significantly decreased following treatment with UAB30, further indicating decreased cancer cell stemness. These results provide evidence that UAB30 decreased tumorigenicity and cancer cell stemness in neuroblastoma PDXs, warranting further exploration as therapy for high-risk neuroblastoma.

Discovery of epi-Enprioline as a Novel Drug for the Treatment of Vincristine Resistant Neuroblastoma.

Neuroblastoma is a childhood solid tumour originating from undifferentiated neural progenitor cells of the sympathetic nervous system. Drug resistance of childhood cancer neuroblastoma is a serious clinical problem. In the present study, we aimed to identify novel drugs that can inhibit the growth and survival of chemoresistant neuroblastoma. High-throughput screening identified a small molecule, epi-enprioline that was able to induce apoptosis of vincristine-resistant neuroblastoma cells via the mitochondrial apoptotic pathway. Epi-enprioline reduced tumour growth in multiple preclinical models, including an orthotopic neuroblastoma patient-derived xenograft model in vivo. In summary, our data suggest that epi-enprioline can be considered as a lead compound for the treatment of vincristine-resistant neuroblastoma uncovering a novel strategy, which can be further explored as a treatment for drug-resistant neuroblastoma.

Abstract 1293: MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma

Abstract MYCN genomic amplification is one of the risk factors in neuroblastoma. 13-cis retinoic acid (13-cisRA), a differentiating agent, down-regulates MYCN protein and is part of neuroblastoma maintenance therapy. Despite the improvement in clinical outcome with 13-cisRA, anti-GD2 monoclonal antibody plus cytokine immunotherapy given in first response ~40% of high-risk neuroblastoma patients still die of recurrent disease. Although MYC genomic amplification is rare in neuroblastoma (~1%), 11% of neuroblastoma primary tumors collected at diagnosis (Dx) have high c-MYC protein suggesting that MYC transcriptional activation rather than its gene amplification drives such tumors. Here, we sought to investigate the role of MYC oncogene in progressive disease (PD) and to molecularly characterize mechanisms of MYC expression in neuroblastoma. We report transcriptional activation of MYC medicated by the OCT4 (encoded by POU5F1), functionally replacing MYCN in 13-cisRA-resistant progressive disease neuroblastoma. In large panels of neuroblastoma patient-derived cell lines (19 Dx and 16 PD) and patient-derived xenograft PDX models (8 Dx and 9 PD), we confirmed that c-MYC expression levels were higher in PD relative to Dx lines (P = 0.0005). We identified OCT4 and TCF3 as transcription factors highly expressed in neuroblastoma cells with high c-MYC. Subsequently, we confirmed two novel OCT4-binding sites (including OBS1 and OBS2) located in the MYC promoter/enhancer region: -1209 to -1140 and found that OCT4 NH2-terminal domain (NTD) and POU specific domain (POUs) are critical for MYC transcriptional activation. To identify kinases that is associated with OCT4-induced c-MYC activation, we used mass spectrometry and PhosphoMotif Finder® and identified MAPKAPK2 (MK2) as one of the upstream kinases that can bind to and directly regulate the OCT4 biological function by phosphorylation at its amino acid Ser111 residue to transcriptionally activate MYC expression. The data in 175 MYCN non-amplified high-risk primary tumors (TARGET database) showed that MAPKAPK2 positively correlated with MYC expression (P &amp;lt; 0.001) and overall survival was lower (P &amp;lt; 0.001) for patients with high MAPKAPK2. Also, OCT4, MK2, and c-MYC were higher in PD relative to Dx neuroblastomas models. Functional studies by gene knockdown of the POU5F1 or MAPKAPK2 using shRNAs showed decreased c-MYC expression, inhibition of cell proliferation, and restoring neurite outgrowth in response to 13-cisRA. In conclusion, high c-MYC independent of genomic amplification, not MYCN amplification, is associated with disease progression in neuroblastoma. The MK2-mediated OCT4 transcriptional activation is a novel mechanism for MYC activation in PD neuroblastoma and provides a potential novel therapeutic target. Citation Format: Sung Jen Wei, Thinh H. Nguyen, Dustin G. Mook, Monish R. Makena, Dattesh Verlekar, Ashly Hindle, Gloria Martinez, Shengping Yang, Hiroyuki Shimada, C. Patrick Reynolds, Min H. Kang. MYC transcription activation mediated by OCT4 as a mechanism of resistance to 13-cisRA-mediated differentiation in neuroblastoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1293.