A distinct core regulatory module enforces oncogene expression in KMT2A-rearranged leukemia.

Acute myeloid leukemia with KMT2A (MLL) rearrangements is characterized by specific patterns of gene expression and enhancer architecture, implying unique core transcriptional regulatory circuitry. Here, we identified the transcription factors MEF2D and IRF8 as selective transcriptional dependencies of KMT2A-rearranged AML, where MEF2D displays partially redundant functions with its paralog, MEF2C. Rapid transcription factor degradation followed by measurements of genome-wide transcription rates and superresolution microscopy revealed that MEF2D and IRF8 form a distinct core regulatory module with a narrow direct transcriptional program that includes activation of the key oncogenes MYC, HOXA9, and BCL2. Our study illustrates a mechanism of context-specific transcriptional addiction whereby a specific AML subclass depends on a highly specialized core regulatory module to directly enforce expression of common leukemia oncogenes.

Discovery of potent BET bromodomain 1 stereoselective inhibitors using DNA-encoded chemical library selections.

BRDT, BRD2, BRD3, and BRD4 comprise the bromodomain and extraterminal (BET) subfamily which contain two similar tandem bromodomains (BD1 and BD2). Selective BD1 inhibition phenocopies effects of tandem BET BD inhibition both in cancer models and, as we and others have reported of BRDT, in the testes. To find novel BET BD1 binders, we screened >4.5 billion molecules from our DNA-encoded chemical libraries with BRDT-BD1 or BRDT-BD2 proteins in parallel. A compound series enriched only by BRDT-BD1 was resynthesized off-DNA, uncovering a potent chiral compound, CDD-724, with >2,000-fold selectivity for inhibiting BRDT-BD1 over BRDT-BD2. CDD-724 stereoisomers exhibited remarkable differences in inhibiting BRDT-BD1, with the R-enantiomer (CDD-787) being 50-fold more potent than the S-enantiomer (CDD-786). From structure­activity relationship studies, we produced CDD-956, which maintained picomolar BET BD1 binding potency and high selectivity over BET BD2 proteins and had improved stability in human liver microsomes over CDD-787. BROMOscan profiling confirmed the excellent pan-BET BD1 affinity and selectivity of CDD-787 and CDD-956 on BD1 versus BD2 and all other BD-containing proteins. A cocrystal structure of BRDT-BD1 bound with CDD-956 was determined at 1.82 Å and revealed BRDT-BD1­specific contacts with the αZ and αC helices that explain the high affinity and selectivity for BET BD1 versus BD2. CDD-787 and CDD-956 maintain cellular BD1-selectivity in NanoBRET assays and show potent antileukemic activity in acute myeloid leukemia cell lines. These BET BD1-specific and highly potent compounds are structurally unique and provide insight into the importance of chirality to achieve BET specificity.

Novel CIC variants identified in individuals with neurodevelopmental phenotypes.

Heterozygous pathogenic variants in CIC, which encodes a transcriptional repressor, have been identified in individuals with neurodevelopmental phenotypes. To date, 11 CIC variants have been associated with the CIC-related neurodevelopmental syndrome. Here, we describe three novel and one previously reported CIC variants in four individuals with neurodevelopmental delay. Notably, we report for the first time a de novo frameshift variant specific to the long isoform of CIC (CIC-L, NM_001304815.1:c.1100dup, p.Pro368AlafsTer16) in an individual with speech delay, intellectual disability, and autism spectrum disorder. Our investigation into the function of CIC-L reveals that partial loss of CIC-L leads to transcriptional derepression of CIC target genes. We also describe a missense variant (NM_015125.3:c.683G>A, p.Arg228Gln) in an individual with a history of speech delay and relapsed pre-B acute lymphoblastic leukemia. Functional studies of this variant suggest a partial loss of CIC transcriptional repressor activity. Our study expands the list of CIC pathogenic variants and contributes to the accumulating evidence that CIC haploinsufficiency or partial loss of function is a pathogenic mechanism causing neurodevelopmental phenotypes.

Synchronous occurrence of acute lymphoblastic leukemia and wilms tumor in two patients: underlying etiology and combined treatment plan.

Synchronous cancers are extraordinarily rare in pediatric patients and present a therapeutic challenge. Patient A presented with synchronous unilateral Wilms tumor (WT) and standard-risk (SR) B-precursor acute lymphoblastic leukemia (ALL). Genetic testing revealed bialleleic BRCA2/FANCD1 mutations. Patient B, after SR B-precursor ALL induction therapy, was noted on fever workup to have a renal mass; pathology demonstrated lesion indeterminate between WT and nephrogenic rest. Therapy was customized for each patient to treat both cancers. Both patients have ongoing remission from their cancers, without excessive toxicity. We report two regimens for treating synchronous WT and ALL and recommend screening such patients for cancer predisposition.

A Prospective Cohort Quality Improvement Study to Reduce the Time to Antibiotics for New Fever in Neutropenic Pediatric Oncology Inpatients.

BACKGROUND: Fever and neutropenia (F&N) is a pediatric oncology emergency due to the risk of disseminated infection. Quality improvement (QI) efforts to improve time to antibiotics for F&N in the emergency department have been documented, but the issue has not been studied in the established inpatient setting. PROCEDURE: We undertook a prospective cohort QI study to decrease time to antibiotics for neutropenic pediatric oncology inpatients with new fever to <60 min. Our key intervention was discussion of a plan in case of new fever, including antibiotic(s) to be started, for each patient on rounds. Timing for each step in the process, from fever identification to antibiotic administration, was measured through the electronic medical record for each fever event. RESULTS: The median time to antibiotics during the 3-three month intervention study period was 76.0 min, although the distribution was skewed due to several long outliers (mean 142.5, interquartile range 51-206, range 47-593 min). Time to antibiotics was significantly shorter when a fever contingency plan was documented in the most recent note than not (mean 102 vs. 254 min, P = 0.039). Over the total 2.75 year data-collection period, the quarterly percentage of patients receiving antibiotics within 60 min has improved from 35 to 65, whereas quarterly mean time to antibiotics has improved from 99 to 50 min. CONCLUSIONS: Daily discussion of a fever contingency plan appears effective in decreasing the time to antibiotics for neutropenic pediatric oncology inpatients with new fever, likely by circumventing the need for multi-level discussion of the antibiotic plan when fever is identified.

A novel APC gene mutation associated with a severe phenotype in a patient with Turcot syndrome.

Turcot syndrome is a rare inherited condition of colonic polyposis associated with central nervous system tumors. We report a patient with a novel adenomatous polyposis coli gene mutation leading to a severe phenotype including medulloblastoma, low-grade fibromyxoid sarcoma following cranial radiation, pilomatrixomas, colonic adenomas, and abdominal desmoid tumor following colectomy, all of which were successfully treated. Multiple tumors may be seen in patients with Turcot syndrome but the occurrence of sarcomas is rare. This case highlights the importance of close follow-up for patients with Turcot syndrome and the importance of a broad differential diagnosis in evaluating a condition in which multiple tumors are frequently seen.

EZH2-driven immune evasion defines high-risk pediatric AML with t(16;21) FUS::ERG gene fusion.

Despite decades of research, acute myeloid leukemia (AML) remains a remarkably lethal malignancy. While pediatric AML (pAML) carries a more favorable prognosis than adult AML, the past 25 years of large clinical trials have produced few improvements in pAML survival. Nowhere is this more evident than in patients carrying a t(16;21)(p11;q22) translocation, which yields the FUS::ERG fusion transcript. Patients with FUS::ERG-positive AML are often primary refractory, and most responders quickly relapse. In COG clinical trials, allogeneic stem cell transplantation was of no benefit to FUS::ERG pAML patients; 100% of transplanted patients succumbed to their disease. Expression of major histocompatibility complex (MHC) class I & II and costimulatory molecules is absent at diagnosis in FUS::ERG AML, mirroring the epigenetic mechanism of post-transplant relapse seen in adult AML and its associated dismal outcomes. Here we show that this class-defining immune-repressive phenotype is driven by overexpression of the EZH2 histone lysine methyltransferase in vitro and in multiple clinical cohorts. We show that treatment with the FDA-approved EZH2 inhibitor tazemetostat along with IFN-γ reverses this phenotype, re-establishes MHC presentation, and severely impairs the viability of FUS::ERG AML cells. EZH2 inhibitors may thus provide the first targeted therapeutic option for patients with this high-risk subtype of pAML, with particular benefit as a bridge to successful allogeneic stem cell transplantation.

KMT2A oncoproteins induce epigenetic resistance to targeted therapies.

Chromosomal translocations involving the Lysine-Methyl-Tansferase-2A ( KMT2A ) locus generate potent oncogenes that cause highly aggressive acute leukemias 1 . KMT2A and the most frequent translocation partners encode proteins that interact with DNA to regulate developmental gene expression 2 . KMT2A-oncogenic fusion proteins (oncoproteins) contribute to the epigenetic mechanisms that allow KMT2A -rearranged leukemias to evade targeted therapies. By profiling the oncoprotein-target sites of 34 KMT2A -rearranged leukemia samples, we find that the genomic enrichment of oncoprotein binding is highly variable between samples. At high levels of expression, the oncoproteins preferentially activate either the lymphoid or myeloid lineage program depending on the fusion partner. These fusion-partner-dependent binding sites correspond to the frequencies of each mutation in acute lymphoid leukemia versus acute myeloid leukemia. By profiling a sample that underwent a lymphoid-to-myeloid lineage switching event in response to lymphoid-directed treatment, we find the global oncoprotein levels are reduced and the oncoprotein-target gene network changes. At lower levels of expression, the oncoprotein shifts to a non-canonical regulatory program that favors the myeloid lineage, and in a subset of resistant patients, the Menin inhibitor Revumenib induces a similar response. The dynamic shifts in KMT2A oncoproteins we describe likely contribute to epigenetic resistance of KMT2A -rearranged leukemias to targeted therapies.

Tazemetostat for tumors harboring SMARCB1/SMARCA4 or EZH2 alterations: results from NCI-COG pediatric MATCH APEC1621C.

BACKGROUND: National Cancer Institute-Children's Oncology Group Pediatric Molecular Analysis for Therapy Choice assigns patients aged 1-21 years with refractory solid tumors, brain tumors, lymphomas, and histiocytic disorders to phase II trials of molecularly targeted therapies based on detection of predefined genetic alterations. Patients whose tumors harbored EZH2 mutations or loss of SMARCB1 or SMARCA4 by immunohistochemistry were treated with EZH2 inhibitor tazemetostat. METHODS: Patients received tazemetostat for 28-day cycles until disease progression or intolerable toxicity (max 26 cycles). The primary endpoint was objective response rate; secondary endpoints included progression-free survival and tolerability of tazemetostat. RESULTS: Twenty patients (median age = 5 years) enrolled, all evaluable for response and toxicities. The most frequent diagnoses were atypical teratoid rhabdoid tumor (n = 8) and malignant rhabdoid tumor (n = 4). Actionable alterations consisted of SMARCB1 loss (n = 16), EZH2 mutation (n = 3), and SMARCA4 loss (n = 1). One objective response was observed in a patient with non-Langerhans cell histiocytosis with SMARCA4 loss (26 cycles, 1200 mg/m2/dose twice daily). Four patients with SMARCB1 loss had a best response of stable disease: epithelioid sarcoma (n = 2), atypical teratoid rhabdoid tumor (n = 1), and renal medullary carcinoma (n = 1). Six-month progression-free survival was 35% (95% confidence interval [CI] = 15.7% to 55.2%) and 6-month overall survival was 45% (95% CI = 23.1% to 64.7%). Treatment-related adverse events were consistent with prior tazemetostat reports. CONCLUSIONS: Although tazemetostat did not meet its primary efficacy endpoint in this population of refractory pediatric tumors (objective response rate = 5%, 90% CI = 1% to 20%), 25% of patients with multiple histologic diagnoses experienced prolonged stable disease of 6 months and over (range = 9-26 cycles), suggesting a potential effect of tazemetostat on disease stabilization.

Venetoclax for Acute Myeloid Leukemia in Pediatric Patients: A Texas Medical Center Experience.

The BCL-2 inhibitor venetoclax improves survival for adult patients with acute myeloid leukemia (AML) in combination with lower-intensity therapies, but its benefit in pediatric patients with AML remains unclear. We retrospectively reviewed two Texas Medical Center institutions' experience with venetoclax in 43 pediatric patients with AML; median age 17 years (range, 0.6-21). This population was highly refractory; 44% of patients (n = 19) had ≥3 prior lines of therapy, 37% (n = 16) had received a prior bone marrow transplant, and 81% (n = 35) had unfavorable genetics KMT2A (n = 17), WT1 (n = 13), FLT3-ITD (n = 10), monosomy 7 (n = 5), TP53 (n = 3), Inv(3) (n = 3), IDH1/2 (n = 2), monosomy 5 (n = 1), NUP98 (n = 1) and ASXL1 (n = 1). The majority (86%) received venetoclax with a hypomethylating agent. Grade 3 or 4 adverse events included febrile neutropenia in 37% (n = 16), non-febrile neutropenia in 12% (n = 5), anemia in 14% (n = 6), and thrombocytopenia in 14% (n = 6). Of 40 patients evaluable for response, 10 patients (25%) achieved complete response (CR), 6 patients (15%) achieved CR with incomplete blood count recovery (CRi), and 2 patients (5%) had a partial response, (CR/CRi composite = 40%; ORR = 45%). Eleven (25%) patients received a hematopoietic stem cell transplant following venetoclax combination therapy, and six remain alive (median follow-up time 33.6 months). Median event-free survival and overall survival duration was 3.7 months and 8.7 months, respectively. Our findings suggest that in pediatric patients with AML, venetoclax is well-tolerated, with a safety profile similar to that in adults. More studies are needed to establish an optimal venetoclax-based regimen for the pediatric population.

An oncogenic enhancer encodes selective selenium dependency in AML.

Deregulation of transcription is a hallmark of acute myeloid leukemia (AML) that drives oncogenic expression programs and presents opportunities for therapeutic targeting. By integrating comprehensive pan-cancer enhancer landscapes with genetic dependency mapping, we find that AML-enriched enhancers encode for more selective tumor dependencies. We hypothesized that this approach could identify actionable dependencies downstream of oncogenic driver events and discovered a MYB-regulated AML-enriched enhancer regulating SEPHS2, a key component of the selenoprotein production pathway. Using a combination of patient samples and mouse models, we show that this enhancer upregulates SEPHS2, promoting selenoprotein production and antioxidant function required for AML survival. SEPHS2 and other selenoprotein pathway genes are required for AML growth in vitro. SEPHS2 knockout and selenium dietary restriction significantly delay leukemogenesis in vivo with little effect on normal hematopoiesis. These data validate the utility of enhancer mapping in target identification and suggest that selenoprotein production is an actionable target in AML.

Phase II Trial of Inotuzumab Ozogamicin in Children and Adolescents With Relapsed or Refractory B-Cell Acute Lymphoblastic Leukemia: Children's Oncology Group Protocol AALL1621.

PURPOSE: Children's Oncology Group trial AALL1621 was conducted to prospectively determine the safety and efficacy of inotuzumab ozogamicin (InO) in pediatric and adolescent patients with relapsed or refractory (R/R) B-cell acute lymphoblastic leukemia (B-ALL). PATIENTS AND METHODS: This single-arm phase II trial enrolled patients age 1-21 years with R/R CD22-positive B-ALL. In cycle 1, InO dosing was 0.8 mg/m2 intravenously on day 1 and 0.5 mg/m2 on days 8 and 15 of a 28-day cycle with response evaluation at day 28. Using a two-stage design, the trial was continuously monitored for dose-limiting toxicities and sinusoidal obstruction syndrome (SOS). CD22 expression was retrospectively evaluated by central flow cytometry. RESULTS: Forty-eight patients were evaluable for response and toxicity; 19 had complete response (CR) and nine CR with incomplete count recovery (CRi) after cycle 1 (CR/CRi rate: 58.3%; two-sided 90% CI, 46.5 to 69.3). Twenty-seven of 28 patients with CR or CRi had minimal residual disease measured by flow cytometry; 18 (66.7%) had minimal residual disease < 0.01%. Seven of 28 patients (25%) with CR or CRi had delayed count recovery past day 42 in cycle 1. Three (6.3%) patients had grade 3 ALT elevation and one patient had grade 3 hyperbilirubinemia in cycle 1. Of 21 patients undergoing hematopoietic stem-cell transplantation after InO, 6 (28.6%) developed grade 3 SOS. Partial CD22 expression and lower CD22 site density were associated with lower likelihood of response to InO. CONCLUSION: InO is effective and well tolerated in heavily pretreated children and adolescents with R/R CD22-positive B-ALL. SOS after hematopoietic stem-cell transplantation and prolonged cytopenias were notable. CD22 modulation was identified as a mechanism of resistance. Expanded study of InO combined with chemotherapy is underway.

The synergy of BET inhibitors with aurora A kinase inhibitors in MYCN-amplified neuroblastoma is heightened with functional TP53.

Amplification of MYCN is a poor prognostic feature in neuroblastoma (NBL) indicating aggressive disease. We and others have shown BET bromodomain inhibitors (BETi) target MYCN indirectly by downregulating its transcription. Here we sought to identify agents that synergize with BETi and to identify biomarkers of resistance. We previously performed a viability screen of ∼1,900 oncology-focused compounds combined with BET bromodomain inhibitors against MYCN-amplified NBL cell lines. Reanalysis of our screening results prominently identified inhibitors of aurora kinase A (AURKAi) to be highly synergistic with BETi. We confirmed the anti-proliferative effects of several BETi+AURKAi combinations in MYCN-amplified NBL cell lines. Compared to single agents, these combinations cooperated to decrease levels of N-myc. We treated both TP53-wild type and mutant, MYCN-amplified cell lines with the BETi JQ1 and the AURKAi Alisertib. The combination had improved efficacy in the TP53-WT context, notably driving apoptosis in both genetic backgrounds. JQ1+Alisertib combination treatment of a MYCN-amplified, TP53-null or TP53-restored genetically engineered mouse model of NBL prolonged survival better than either single agent. This was most profound with TP53 restored, with marked tumor shrinkage and apoptosis induction in response to combination JQ1+Alisertib. BETi+AURKAi in MYCN-amplified NBL, particularly in the context of functional TP53, provided anti-tumor benefits in preclinical models. This combination should be studied more closely in a pediatric clinical trial.

Defining the transcriptional control of pediatric AML highlights RARA as a superenhancer-regulated druggable dependency.

Somatic mutations are rare in pediatric acute myeloid leukemia (pAML), indicating that alternate strategies are needed to identify targetable dependencies. We performed the first enhancer mapping of pAML in 22 patient samples. Generally, pAML samples were distinct from adult AML samples, and MLL (KMT2A)-rearranged samples were also distinct from non-KMT2A-rearranged samples. Focusing specifically on superenhancers (SEs), we identified SEs associated with many known leukemia regulators. The retinoic acid receptor alpha (RARA) gene was differentially regulated in our cohort, and a RARA-associated SE was detected in 64% of the study cohort across all cytogenetic and molecular subtypes tested. RARA SE+ pAML cell lines and samples exhibited high RARA messenger RNA levels. These samples were specifically sensitive to the synthetic RARA agonist tamibarotene in vitro, with slowed proliferation, apoptosis induction, differentiation, and upregulated retinoid target gene expression, compared with RARA SE- samples. Tamibarotene prolonged survival and suppressed the leukemia burden of an RARA SE+ pAML patient-derived xenograft mouse model compared with a RARA SE- patient-derived xenograft. Our work shows that examining chromatin regulation can identify new, druggable dependencies in pAML and provides a rationale for a pediatric tamibarotene trial in children with RARA-high AML.

Bromodomain and extra-terminal inhibitors-A consensus prioritisation after the Paediatric Strategy Forum for medicinal product development of epigenetic modifiers in children-ACCELERATE.

Based on biology and pre-clinical data, bromodomain and extra-terminal (BET) inhibitors have at least three potential roles in paediatric malignancies: NUT (nuclear protein in testis) carcinomas, MYC/MYCN-driven cancers and fusion-driven malignancies. However, there are now at least 10 BET inhibitors in development, with a limited relevant paediatric population in which to evaluate these medicinal products. Therefore, a meeting was convened with the specific aim to develop a consensus among relevant biopharmaceutical companies, academic researchers, as well as patient and family advocates, about the development of BET inhibitors, including prioritisation and their specific roles in children. Although BET inhibitors have been in clinical trials in adults since 2012, the first-in-child study (BMS-986158) only opened in 2019. In the future, when there is strong mechanistic rationale or pre-clinical activity of a class of medicinal product in paediatrics, early clinical evaluation with embedded correlative studies of a member of the class should be prioritised and rapidly executed in paediatric populations. There is a strong mechanistic and biological rationale to evaluate BET inhibitors in paediatrics, underpinned by substantial, but not universal, pre-clinical data. However, most pan-BET inhibitors have been challenging to administer in adults, since monotherapy results in only modest anti-tumour activity and provides a narrow therapeutic index due to thrombocytopenia. It was concluded that it is neither scientifically justified nor feasible to undertake simultaneously early clinical trials in paediatrics of all pan-BET inhibitors. However, there is a clinical need for global access to BET inhibitors for patients with NUT carcinoma, a very rare malignancy driven by bromodomain fusions, with proof of concept of clinical benefit in a subset of patients treated with BET inhibitors. Development and regulatory pathway in this indication should include children and adolescents as well as adults. Beyond NUT carcinoma, it was proposed that further clinical development of other pan-BET inhibitors in children should await the results of the first paediatric clinical trial of BMS-986158, unless there is compelling rationale based on the specific agent of interest. BDII-selective inhibitors, central nervous system-penetrant BET inhibitors (e.g. CC-90010), and those dual-targeting BET/p300 bromodomain are of particular interest and warrant further pre-clinical investigation. This meeting emphasised the value of a coordinated and integrated strategy to drug development in paediatric oncology. A multi-stakeholder approach with multiple companies developing a consensus with academic investigators early in the development of a class of compounds, and then engaging regulatory agencies would improve efficiency, productivity, conserve resources and maximise potential benefit for children with cancer.

ZFTA-RELA Dictates Oncogenic Transcriptional Programs to Drive Aggressive Supratentorial Ependymoma.

More than 60% of supratentorial ependymomas harbor a ZFTA-RELA (ZRfus) gene fusion (formerly C11orf95-RELA). To study the biology of ZRfus, we developed an autochthonous mouse tumor model using in utero electroporation (IUE) of the embryonic mouse brain. Integrative epigenomic and transcriptomic mapping was performed on IUE-driven ZRfus tumors by CUT&RUN, chromatin immunoprecipitation sequencing, assay for transposase-accessible chromatin sequencing, and RNA sequencing and compared with human ZRfus-driven ependymoma. In addition to direct canonical NFκB pathway activation, ZRfus dictates a neoplastic transcriptional program and binds to thousands of unique sites across the genome that are enriched with PLAGL family transcription factor (TF) motifs. ZRfus activates gene expression programs through recruitment of transcriptional coactivators (Brd4, Ep300, Cbp, Pol2) that are amenable to pharmacologic inhibition. Downstream ZRfus target genes converge on developmental programs marked by PLAGL TF proteins, and activate neoplastic programs enriched in Mapk, focal adhesion, and gene imprinting networks. SIGNIFICANCE: Ependymomas are aggressive brain tumors. Although drivers of supratentorial ependymoma (ZFTA- and YAP1-associated gene fusions) have been discovered, their functions remain unclear. Our study investigates the biology of ZFTA-RELA-driven ependymoma, specifically mechanisms of transcriptional deregulation and direct downstream gene networks that may be leveraged for potential therapeutic testing.This article is highlighted in the In This Issue feature, p. 2113.

Homozygous alpha-thalassemia treated with intrauterine transfusions and unrelated donor hematopoietic cell transplantation.

Recently, intrauterine transfusions and hematopoietic cell transplantation (HCT) have changed homozygous alpha-thalassemia from a frequently fatal disease to a potentially survivable condition. We present a patient with Hemoglobin Bart's disease who was cured after failing to engraft with 1 unrelated HCT, but engrafting after a second unrelated donor HCT.

Small molecule inhibitor regorafenib inhibits RET signaling in neuroblastoma cells and effectively suppresses tumor growth in vivo.

Neuroblastoma (NB), the most common extracranial pediatric solid tumor, continues to cause significant cancer-related morbidity and mortality in children. Dysregulation of oncogenic receptor tyrosine kinases (RTKs) has been shown to contribute to tumorigenesis in various human cancers and targeting these RTKs has had therapeutic benefit. RET is an RTK which is commonly expressed in NB, and high expression of RET correlates with poor outcomes in patients with NB. Herein we report that RET is required for NB cell proliferation and that the small molecule inhibitor regorafenib (BAY 73-4506) blocks glial cell derived neurotrophic factor (GDNF)-induced RET signaling in NB cells and inhibits NB growth both in vitro and in vivo. We found that regorafenib significantly inhibited cell proliferation and colony formation ability of NB cells. Moreover, regorafenib suppressed tumor growth in both an orthotopic xenograft NB mouse model and a TH-MYCN transgenic NB mouse model. Finally, regorafenib markedly improved the overall survival of TH-MYCN transgenic tumor-bearing mice. In summary, our study suggests that RET is a potential therapeutic target in NB, and that using a novel RET inhibitor, like regorafenib, should be investigated as a therapeutic treatment option for children with NB.