Conditional c-MYC activation in catecholaminergic cells drives distinct neuroendocrine tumors: neuroblastoma vs somatostatinoma.

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 ß-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.

An integrated single-cell RNA-seq map of human neuroblastoma tumors and preclinical models uncovers divergent mesenchymal-like gene expression programs.

BACKGROUND: 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. RESULTS: Here, we generate single-cell RNA-seq maps of neuroblastoma cell lines, patient-derived xenograft models (PDX), and a genetically engineered mouse model (GEMM). We develop 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 confirm 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 is maintained in PDX and could be chemotherapy-induced in our GEMM after only 24 h, suggesting an uncharacterized therapy-escape mechanism. CONCLUSIONS: 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.

An integrated single-cell RNA-seq map of human neuroblastoma tumors and preclinical models uncovers divergent mesenchymal-like gene expression programs.

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.

RBM39 degrader invigorates natural killer cells to eradicate neuroblastoma despite cancer cell plasticity.

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.

Maternal immunization with distinct influenza vaccine platforms elicits unique antibody profiles that impact the protection of offspring.

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.

Targeting the spliceosome through RBM39 degradation results in exceptional responses in high-risk neuroblastoma models.

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.