Abstract c-MYC is an important driver of high-risk neuroblastoma. A lack of c-MYC–driven genetically engineered mouse models (GEMM) has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and to develop effective therapies. In this study, we showed that conditional c-MYC induction via Cre recombinase driven by a tyrosine hydroxylase promoter led to a preponderance of PDX1+ somatostatinoma, a type of pancreatic neuroendocrine tumor. However, c-MYC activation via an improved Cre recombinase driven by a dopamine β-hydroxylase promoter resulted in neuroblastoma development. The c-MYC murine neuroblastoma tumors recapitulated the pathologic and genetic features of human neuroblastoma and responded to anti-GD2 immunotherapy and difluoromethylornithine, an FDA-approved inhibitor targeting the MYC transcriptional target ODC1. Thus, c-MYC overexpression results in different but related tumor types depending on the targeted cell. The GEMMs represent valuable tools for testing immunotherapies and targeted therapies for these diseases. Significance: The development of c-MYC–driven genetically engineered neuroblastoma and somatostatinoma mouse models provides useful tools for understanding the tumor cell origin and investigating treatment strategies.

The MYC proto-oncogenes (c-MYC, MYCN , MYCL ) are among the most deregulated oncogenic drivers in human malignancies including high-risk neuroblastoma, 50% of which are MYCN -amplified. Genetically engineered mouse models (GEMMs) based on the MYCN transgene have greatly expanded the understanding of neuroblastoma biology and are powerful tools for testing new therapies. However, a lack of c-MYC-driven GEMMs has hampered the ability to better understand mechanisms of neuroblastoma oncogenesis and therapy development given that c-MYC is also an important driver of many high-risk neuroblastomas. In this study, we report two transgenic murine neuroendocrine models driven by conditional c-MYC induction in tyrosine hydroxylase (Th) and dopamine β-hydroxylase (Dbh)-expressing cells. c-MYC induction in Th-expressing cells leads to a preponderance of Pdx1 + somatostatinomas, a type of pancreatic neuroendocrine tumor (PNET), resembling human somatostatinoma with highly expressed gene signatures of δ cells and potassium channels. In contrast, c-MYC induction in Dbh-expressing cells leads to onset of neuroblastomas, showing a better transforming capacity than MYCN in a comparable C57BL/6 genetic background. The c-MYC murine neuroblastoma tumors recapitulate the pathologic and genetic features of human neuroblastoma, express GD2, and respond to anti-GD2 immunotherapy. This model also responds to DFMO, an FDA-approved inhibitor targeting ODC1, which is a known MYC transcriptional target. Thus, establishing c-MYC-overexpressing GEMMs resulted in different but related tumor types depending on the targeted cell and provide useful tools for testing immunotherapies and targeted therapies for these diseases.

The cellular plasticity of neuroblastoma is defined by a mixture of two major cell states, adrenergic (ADRN) and mesenchymal (MES), which may contribute to therapy resistance. However, how neuroblastoma cells switch cellular states during therapy remains largely unknown and how to eradicate neuroblastoma regardless of their cell states is a clinical challenge. To better understand the lineage switch of neuroblastoma in chemoresistance, we comprehensively defined the transcriptomic and epigenetic map of ADRN and MES types of neuroblastomas using human and murine models treated with indisulam, a selective RBM39 degrader. We showed that cancer cells not only undergo a bidirectional switch between ADRN and MES states, but also acquire additional cellular states, reminiscent of the developmental pliancy of neural crest cells. The lineage alterations are coupled with epigenetic reprogramming and dependency switch of lineage-specific transcription factors, epigenetic modifiers and targetable kinases. Through targeting RNA splicing, indisulam induces an inflammatory tumor microenvironment and enhances anticancer activity of natural killer cells. The combination of indisulam with anti-GD2 immunotherapy results in a durable, complete response in high-risk transgenic neuroblastoma models, providing an innovative, rational therapeutic approach to eradicate tumor cells regardless of their potential to switch cell states.

Abstract Disclosure: E.M. Pinto: None. R. Epperly: None. T. Santiago: None. H. Sheppard: None. M. Clay: None. C. Morton: None. P. Nguyen: None. Y. Zhou: None. M.A. Woolard: None. C. O'Reilly: None. J. Justice: None. W. Akers: None. M. Johnson: None. C. Cheng: None. G. Zambetti: None. A.M. Davidoff: None. R. Ribeiro: None. C. DeRenzo: None. Over the past decade, significant progress has been achieved in understanding the underlying molecular mechanisms driving adrenocortical tumorigenesis, with the identification of several genetic and molecular drivers of this disease. However, a lack of preclinical models has hindered the discovery of new therapeutic treatment modalities, particularly in the pediatric population. In this study we describe the development of three patient-derived xenograft models for pediatric adrenocortical tumors (SJ-ACC3, SJ-ACC4, and SJACT030812_X1), which recapitulate the molecular features and heterogeneity of these rare tumors. All three models were derived from aggressive tumors, harboring TP53 and ATRX variants. We evaluated the efficacy of various chemotherapeutic agents in two models. SJ-ACC3 tumors exhibited sensitivity to cisplatin and topotecan, while SJ-ACC4 demonstrated limited response to all evaluated drugs including cisplatin, doxorubicin, and etoposide. Consistent with adult adrenocortical tumors, we found that most primary pediatric adrenocortical tumors express the tumor associated antigen B7-H3 (CD276), regardless of their molecular subtype. Notably, B7-H3 was highly expressed in two of our models, SJ-ACC4 and SJACT030812_X1, with limited expression in SJ-ACC3. Critically, we demonstrated that B7-H3 CAR T cells exhibited significant antitumor activity in both B7-H3-positive models, clearing tumors in most mice. These findings suggested that CAR T cells therapy targeting B7-H3 might be a promising therapeutic strategy for pediatric patients with high-risk adrenocortical tumors. Presentation: 6/3/2024

Background: Acute lymphoblastic leukemia (ALL) is characterized by the involvement of B-cell or T-cell lineages and is classified into subtypes based on the immunophenotype of the cells, which is determined by the presence of specific antigens on the cells.The presence of the Philadelphia (Ph) chromosome is significant for the choice of treatment strategy and in the prognosis of the disease.Objective: To determine the frequency of Ph-negative ALL among adolescents and young adults (AYAs) aged 15-39 years.Setting: Hematology center after prof.R. Yeolyan, Yerevan, Armenia.Materials and Methods: Retrospective ALL cases were observed and diagnosed within 2017-2022 years.Within this time period, 15 cases were associated with AYAs and were identified as primary Ph-negative ALL.The average age of the patients was 24 years.The sex ratio was 9 males to 6 females.Results: Among them, 10 primary cases were associated with B-cell lineage and 5 primary cases with T-cell lineage ALL.Based on flow cytometry, the 15 cases were classified immunophenotypically: 3 cases of early precursor B-cell (early pre-B-cell) (20%), 7 cases of pre-B-cell (46.6%), 2 cases of early T-cell precursor (ETP) (13.3%), 1 case of early immature T-cell (6.6%) and 2 cases of cortical/thymic T-cell (13.3%)ALL.Conclusion: Among youth, as the age increases, the incidence of the disease decreases.B-cell lineage ALL is more common and is often represented by the pre-B-cell subtype.

Outcomes for pediatric patients with unresectable, metastatic, or relapsed adrenocortical carcinoma (ACC) are dismal despite aggressive management with multimodality therapies. CAR T cell therapy (CAR T) is a promising approach to improve outcomes for patients with this disease. However, progress is hindered by a lack of targetable surface antigens and limited availability of relevant preclinical models. B7-H3 is a promising immunotherapeutic target but little is known about its expression in pediatric ACC. Thus, we sought to i) evaluate B7-H3 expression in primary pediatric ACC samples and ii) assess B7-H3-CAR T activity in two novel pediatric patient-derived xenograft (PDX) models. First, we assessed B7-H3 expression in 44 pediatric adrenocortical tumors (ACTs) using a CLIA-approved immunohistochemistry (IHC) assay. ACTs had a range of B7-H3 positivity, including 24 (55%) with expression above the threshold for eligibility on our active B7-H3-CAR T clinical trial (H-score ≥ 100; NCT04897321). Univariate analyses demonstrated B7-H3 expression was correlated with the presence of germline TP53 mutations and poor prognostic factors including the inability to achieve complete resection, Cushing phenotype, and the absence of TP53 by IHC. Given a dearth of in vivo models, we next established two clinically relevant pediatric ACC PDX models. SJ-ACC4 was derived from a metastatic tumor and had somatic mutations in TP53 (p.C135Lfs*14) and CTNNB1 (p.S45F). SJACT030812_X1 (www.stjude.org/cstn) was derived from a primary tumor and had a germline TP53 mutation (p.R110Pfs*39). Both had partial ATRX deletions (exons 11-30; 3-28), uniparental disomy at chromosome 11p15, and were B7-H3-positive. Notably, SJ-ACC4 was resistant to a battery of chemotherapeutic agents. To evaluate B7-H3-CAR T anti-ACC activity in vivo, SJ-ACC4 tumor fragments were implanted subcutaneously in NOD SCID gamma (NSG) mice and treated with B7-H3-CAR or control T cells. Strikingly, B7-H3-CAR T eradicated 100% of SJ-ACC4 tumors and mice survived long term compared to controls that met endpoints within 35 days (n=8 per group, p< 0.0001). Finally, we asked if B7-H3-CAR T had antitumor activity in an orthotopic pediatric ACC PDX model. SJACT030812_X1 cells were injected into the para-adrenal region of NSG mice under ultrasound guidance and monitored by serial volumetric ultrasonography. B7-H3-CAR T again had significant antitumor activity, and mice survived long-term compared to controls that met endpoints within 42 days (n=5 per group, p< 0.01). In conclusion, B7-H3 is expressed in a majority of pediatric ACTs and B7-H3-CAR T have potent anti-ACC activity. These findings provide a strong rationale for clinically evaluating B7-H3-CAR T for pediatric patients with chemotherapy-resistant ACC. Additionally, the newly established pediatric ACC PDX models provide a valuable resource for investigating different aspects of this rare and aggressive disease. Citation Format: Rebecca Epperly, Emilia M. Pinto, Teresa Santiago, Heather Sheppard, Michael R. Clay, Christopher Morton, Phuong Nguyen, Yinmei Zhou, Mary Woolard, Carla O'Reilly, Julie Justice, Walter Akers, Melissa Johnson, Asa Karlstrom, Cheng Cheng, Gerard Zambetti, Andrew Davidoff, Raul Ribeiro, Christopher DeRenzo. B7-H3-CAR T cells for the treatment of pediatric adrenocortical carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1197.

PDF file - 96K, ELISA data for individual markers in newly diagnosed samples separated based on stage at the time of diagnosis compared to matched controls. Only significant differences are demarcated. * indicates p<0.05, ** indicates p<0.01.

PDF file - 112K, Workflow of experiments and sample cohorts described in the manuscript.

PDF file - 70K, Correlations between different markers based on ELISA data of newly diagnosed samples.

PDF file - 225K, Histograms for case-to-control ratios for each of the 9 MS experiments used for normalization.

Pregnant women and infants are considered high-risk groups for increased influenza disease severity. While influenza virus vaccines are recommended during pregnancy, infants cannot be vaccinated until at least six months of age. Passive transfer of maternal antibodies (matAbs) becomes vital for the infant’s protection. Here, we employed an ultrasound-based timed-pregnancy murine model and examined matAb responses to distinct influenza vaccine platforms and influenza A virus (IAV) infection in dams and their offspring. We demonstrate vaccinating dams with a live-attenuated influenza virus (LAIV) vaccine or recombinant hemagglutinin (rHA) proteins administered with adjuvant resulted in enhanced and long-lasting immunity and protection from influenza in offspring. In contrast, a trivalent split-inactivated vaccine (TIV) afforded limited protection in our model. By cross-fostering pups, we show the timing of antibody transfer from vaccinated dams to their offspring (prenatal versus postnatal) can shape the antibody profile depending on the vaccine platform. Our studies provide information on how distinct influenza vaccines lead to immunogenicity and efficacy during pregnancy, impact the protection of their offspring, and detail roles for IgG1 and IgG2c in the development of vaccine administration during pregnancy that stimulate and measure expression of both antibody subclasses.

PDF fiel - 144K, Peptide coverage for each of the 6 individual proteins in two colorectal cancer cell lines with different driver mutations.

PDF file - 48K, Peptide sequences from proteins identified in immune complexes.

PDF file - 54K, Coefficients from linear regression models used for ROC analysis.

PDF file - 117K, ELISA data for individual markers in newly diagnosed samples separated by gender. Only significant differences are demarcated. * indicates p<0.05, ** indicates p<0.01, *** indicates p<0.001.

Neuroblastoma is a common pediatric cancer, where preclinical studies suggest that a mesenchymal-like gene expression program contributes to chemotherapy resistance. However, clinical outcomes remain poor, implying we need a better understanding of the relationship between patient tumor heterogeneity and preclinical models. Here, we generated single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We developed an unsupervised machine learning approach ('automatic consensus nonnegative matrix factorization' (acNMF)) to compare the gene expression programs found in preclinical models to a large cohort of patient tumors. We confirmed a weakly expressed, mesenchymal-like program in otherwise adrenergic cancer cells in some pre-treated high-risk patient tumors, but this appears distinct from the presumptive drug-resistance mesenchymal programs evident in cell lines. Surprisingly however, this weak-mesenchymal-like program was maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 hours, suggesting an uncharacterized therapy-escape mechanism. Collectively, our findings improve the understanding of how neuroblastoma patient tumor heterogeneity is reflected in preclinical models, provides a comprehensive integrated resource, and a generalizable set of computational methodologies for the joint analysis of clinical and pre-clinical single-cell RNA-seq datasets.

Here, we showed that the combination of FLT3 (gilteritinib) and BCL2 (venetoclax) inhibitors is highly effective at reducing leukemia burden in a preclinical model of BCL11B-a lineage ambiguous leukemia. This combination blocked leukemic growth in vivo and was effective in highly engrafted animals. Ongoing studies in additional PDXs and pharmacodynamic work will identify mechanisms of synergy and characterize the residual combination-treated population to better understand putative drug resistance mechanisms.

Aberrant alternative pre-mRNA splicing plays a critical role in MYC-driven cancers and therefore may represent a therapeutic vulnerability. Here, we show that neuroblastoma, a MYC-driven cancer characterized by splicing dysregulation and spliceosomal dependency, requires the splicing factor RBM39 for survival. Indisulam, a "molecular glue"that selectively recruits RBM39 to the CRL4-DCAF15 E3 ubiquitin ligase for proteasomal degradation, is highly efficacious against neuroblastoma, leading to significant responses in multiple high-risk disease models, without overt toxicity. Genetic depletion or indisulam-mediated degradation of RBM39 induces significant genome-wide splicing anomalies and cell death. Mechanistically, the dependency on RBM39 and high-level expression of DCAF15 determine the exquisite sensitivity of neuroblastoma to indisulam. Our data indicate that targeting the dysregulated spliceosome by precisely inhibiting RBM39, a vulnerability in neuroblastoma, is a valid therapeutic strategy.