MYCN Amplification Research Articles

SMARCE1 promotes neuroblastoma tumorigenesis through assisting MYCN-mediated transcriptional activation.

SMARCE1 gene, encoding a core subunit of SWI/SNF chromatin remodeling complex, is situated on chromosome 17q21-ter region that is frequently gained in neuroblastoma. However, its role in the tumorigenesis remains unknown. Here, we showed that high expression of SMARCE1 was associated with poor prognosis of patients with neuroblastoma, especially those with MYCN amplification. Knockdown of SMARCE1 reduced proliferation, colony formation, and tumorigenicity of neuroblastoma cells. Mechanistically, SMARCE1 directly interacted with MYCN, which was necessary for MYCN-mediated transcriptional activation of downstream target genes including PLK1, ODC1, and E2F2. Overexpression of PLK1, ODC1 or E2F2 significantly reversed the inhibiting effect of SMARCE1 knockdown on the proliferation, colony formation, and tumorigenicity of MYCN-amplified neuroblastoma cells. Moreover, we revealed that MYCN directly regulated SMARCE1 transcription through binding to a non-canonical E-box of SMARCE1 promoter, thus enhancing SMARCE1-MYCN cooperativity. These findings establish SMARCE1 is a critical oncogenic factor in neuroblastoma and provide a new potential target for treatment of neuroblastoma with 17q21-ter gain and MYCN amplification.

PRECISION MEDICINE IN NEUROBLASTOMA: EXPERIENCE WITH NEXT GENERATION SEQUENCING

Objectives: The objective of this study is to examine neuroblastoma patients that were examined by next generation sequencing (NGS) for targeted therapy related specific gene mutations including Anaplastic Lymphoma Kinase (ALK) Materials and Methods: Molecular examinations of neuroblastoma according to Turkish Pediatric Oncology Group protocols (Nmyc amplification, 11q23 deletion, 1p36 deletion, DNA ploidy by Cell cycle kit in flow cytometer) is studied in our department in all cases collected from Turkey. Among these cases, the patients that recurred even after multi model therapies, are requested NGS to evaluate for targeted therapy decision. We study single nucleated variations after DNA isolation using Pillar Onco/Reveal Multicancer v4 with CNV Panel with 60 genes (ALK, BRAF, ERB2, PIK3CA, EGFR, KRAS, MET, ….) on İllumina Miniseq platform. Results: We especially reported ALK mutations which has indication for crizotinib or alectinib. Out of our investigated patients only 22% (9/41) had ALK mutations. Two patients (22%) had R1275Q mutation, six patients (66,7%) had F1174L mutation and one patient (11%) had L1226F, S1189F, V1135A and G1225S mutations. Conclusion: Our patient cohort seems to be showing ALK mutations and F1174L (sensitive to alectinib) mutation more often than R1275Q mutation (sensitive to crizotinib) in neuroblastoma cases. The reason that ALK mutation ratio is higher than literature. The reason of this might be because of selection of patients by the clinicians. These patients received targeted therapies and had longer survival.

Interferon signalling contributes to therapy‐resistant prostate cancer progression

Interferon signalling contributes to therapy‐resistant prostate cancer progression

Amplification of CDK4 and MDM2: a detailed study of a high-risk neuroblastoma subgroup

In neuroblastoma, MYCN amplification and 11q-deletion are important, although incomplete, markers of high-risk disease. It is therefore relevant to characterize additional alterations that can function as prognostic and/or predictive markers. Using SNP-microarrays, a group of neuroblastoma patients showing amplification of one or multiple 12q loci was identified. Two loci containing CDK4 and MDM2 were commonly co-amplified, although amplification of either locus in the absence of the other was observed. Pharmacological inhibition of CDK4/6 with ribociclib or abemaciclib decreased proliferation in a broad set of neuroblastoma cell lines, including CDK4/MDM2-amplified, whereas MDM2 inhibition by Nutlin-3a was only effective in p53wild-type cells. Combined CDK4/MDM2 targeting had an additive effect in p53wild-type cell lines, while no or negative additive effect was observed in p53mutated cells. Most 12q-amplified primary tumors were of abdominal origin, including those of intrarenal origin initially suspected of being Wilms’ tumor. An atypical metastatic pattern was also observed with low degree of bone marrow involvement, favoring other sites such as the lungs. Here we present detailed biological data of an aggressive neuroblastoma subgroup hallmarked by 12q amplification and atypical clinical presentation for which our in vitro studies indicate that CDK4 and/or MDM2 inhibition also could be beneficial.

An Unfavorable Metastatic Site at Initial Diagnosis in a 2-year-old Boy with Neuroblastoma

Neuroblastoma (NB) is considered one of the malignant tumors of the nervous system which originates from primordial neural crest cell and is known as the most common extra-cranial solid tumor in childhood. This tumor which is often intra-abdominal, metastases in various sites including the skull, long bones, liver, bone marrow and lymph nodes, but lung metastasis at initial diagnosis is rare (0.2-3.7%). Here, the authors reported a two-year-old boy who referred to Imam Hussein Children's Hospital affiliated to Isfahan University of Medical Sciences, Iran in April 2017 with complaint of abdominal pain and distension along with diarrhea, vomiting and fever. In physical examination, a palpable mass was observed at the right side of the abdomen, that in further studies, NB with lung metastasis along with poorly differentiated, low MKI, positive MYCN amplification was reported. This is the first documented case report of NB with lung metastasis from Iran. In conclusion, although lung is considered as an unfavorable metastatic site for NB, in high grade and poor prognosis cases, this organ should also be examined in metastatic site examinations. Therefore, lung High-resolution computed tomography (HRCT) is introduced as the gold standard modality for this work.

A Random Forest-based Classifier for MYCN Status Prediction in Neuroblastoma using CT Images.

Neuroblastoma (NB) is the most common extracranial solid tumor in childhood. Genomic amplification of MYCN is associated with poor outcomes and is detected in 16% of all NB cases. CT scans and MRI are the imaging techniques recommended for diagnosis and disease staging. The assessment of imaging features such as tumor volume, shape, and local extension represent relevant prognostic information. Radiogenomics have shown powerful results in the assessment of the genotype based on imaging findings automatically extracted from medical images. In this work, random forest was used to classify the MYCN amplification using radiomic features extracted from CT slices in a population of 46 NB patients. The learning model showed an area under the curve (AUC) of 0.85 ± 0.13, suggesting that radiomic-based methodologies might be helpful in the extraction of information that is not accessible by human naked eyes but could aid the clinicians on the diagnosis and treatment plan definition. Clinical relevance - This approach represents a random forest-based model to predict the MYCN amplification in NB patients that could give a faster, earlier, and repeatable analysis of the tumor along the time.

MYCN protein stability is a better prognostic indicator in neuroblastoma

ObjectiveMYCN oncogene amplification is associated with treatment failure and poor prognosis in neuroblastoma. To date, most detection methods of MYCN focus on DNA copy numbers instead of protein expression, which is the real one performing biological function, for poor antibodies. The current investigation was to explore a fast and reliable way to detect MYCN protein expression and evaluate its performance in predicting prognosis.MethodsSeveral MYCN antibodies were used to detect MYCN protein expression by immunohistochemistry (IHC), and one was chosen for further study. We correlated the IHC results of MYCN from 53 patients with MYCN fluorescence in situ hybridization (FISH) and identified the sensitivity and specificity of IHC. The relationship between patient prognosis and MYCN protein expression was detected from this foundation.ResultsMYCN amplification status detected by FISH was most valuable for INSS stage 3 patients. In the cohort of 53 samples, IHC test demonstrated 80.0–85.7% concordance with FISH results. Further analyzing those cases with inconsistent results, we found that patients with MYCN amplification but low protein expression tumors always had a favorable prognosis. In contrast, if patients with MYCN non-amplified tumors were positive for MYCN protein, they had a poor prognosis.ConclusionMYCN protein level is better than MYCN amplification status in predicting the prognosis of neuroblastoma patients. Joint of FISH and IHC could confirm MYCN protein stability and achieve better prediction effect than the singular method.

An immature, dedifferentiated, and lineage-deconstrained cone precursor origin of N-Myc–initiated retinoblastoma

Most retinoblastomas develop from maturing cone precursors in response to biallelic RB1 loss and are dependent on cone maturation-related signaling. Additionally, ∼2% lack RB1 mutations but have MYCN amplification (MYCNA), N-Myc protein overexpression, and more rapid and invasive growth, yet the MYCNA retinoblastoma cell of origin and basis for its responses to deregulated N-Myc are unknown. Here, using explanted cultured retinae, we show that ectopic N-Myc induces cell cycle entry in cells expressing markers of several retinal types yet induces continuous proliferation and tumorigenesis only in cone precursors. Unlike the response to RB1 loss, both immature cone arrestin-negative (ARR3-) and maturing ARR3+ cone precursors proliferate, and maturing cone precursors rapidly dedifferentiate, losing ARR3 as well as L/M-opsin expression. N-Myc-overexpressing retinal cells also lose cell lineage constraints, occasionally coexpressing the cone-specific RXRγ with the rod-specific NRL or amacrine-specific AP2α and widely coexpressing RXRγ with the progenitor and Müller cell-specific SOX9 and retinal ganglion cell-specific BRN3 and GAP43. Mechanistically, N-Myc induced Cyclin D2 and CDK4 overexpression, pRB phosphorylation, and SOX9-dependent proliferation without a retinoma-like stage that characterizes pRB-deficient retinoblastoma, despite continuous p16INK4A expression. Orthotopic xenografts of N-Myc-overexpressing retinal cells formed tumors with retinal cell marker expression similar to those in MYCN-transduced retinae and MYCNA retinoblastomas in patients. These findings demonstrate the MYCNA retinoblastoma origin from immature and lineage-deconstrained cone precursors, reveal their opportunistic use of an undifferentiated retinal progenitor cell feature, and illustrate that different cancer-initiating mutations cooperate with distinct developmental stage-specific cell signaling circuitries to drive retinoblastoma tumorigenesis.

Molecular landscape of pediatric type IDH wildtype, H3 wildtype hemispheric glioblastomas

The WHO (2021) Classification classified a group of pediatric-type high-grade gliomas as IDH wildtype, H3 wildtype but as of currently, they are characterized only by negative molecular features of IDH and H3. We recruited 35 cases of pediatric IDH wildtype and H3 wildtype hemispheric glioblastomas. We evaluated them with genome-wide methylation profiling, targeted sequencing, RNAseq, TERT promoter sequencing, and FISH. The median survival of the cohort was 27.6 months. With Capper et al.'s36 methylation groups as a map, the cases were found to be epigenetically heterogeneous and were clustered in proximity or overlay of methylation groups PXA-like (n = 8), LGG-like (n = 10), GBM_MYCN (n = 9), GBM_midline (n = 5), and GBM_RTKIII (n = 3). Histology of the tumors in these groups was not different from regular glioblastomas. Methylation groups were not associated with OS. We were unable to identify groups specifically characterized by EGFR or PDGFRA amplification as proposed by other authors. EGFR, PDGFRA, and MYCN amplifications were not correlated with OS. 4/9 cases of the GBM_MYCN cluster did not show MYCN amplification; the group was also enriched for EGFR amplification (4/9 cases) and the two biomarkers overlapped in two cases. Overall, PDGFRA amplification was found in only four cases and they were not restricted to any groups. Cases in proximity to GBM_midline were all hemispheric and showed loss of H3K27me3 staining. Fusion genes ALK/NTRK/ROS1/MET characteristic of infantile glioblastomas were not identified in 17 cases successfully sequenced. BRAF V600E was only found in the PXA group but CDKN2A deletion could be found in other methylation groups. PXA-like cases did not show PXA histological features similar to findings by other authors. No case showed TERT promoter mutation. Mutations of mismatch repair (MMR) genes were poor prognosticators in single (p ≤ 0.001) but not in multivariate analyses (p = 0.229). MGMT had no survival significance in this cohort. Of the other common biomarkers, only TP53 and ATRX mutations were significant poor prognosticators and only TP53 mutation was significant after multivariate analyses (p = 0.024). We conclude that IDH wildtype, H3 wildtype pediatric hemispheric glioblastomas are molecularly heterogeneous and in routine practice, TP53, ATRX, and MMR status could profitably be screened for risk stratification in laboratories without ready access to methylation profiling.

Prognostic analysis of E2F transcription factors E2F1 and E2F3 in four independent pediatric neuroblastoma cohorts

BackgroundPreviously, we had analyzed the prognosis of E2F transcription factors across adult tumor types. However, the expressions and prognosis of E2F transcription factors in pediatric neuroblastoma have not yet been fully studied.MethodsThe prognosis of E2F transcription factors was determined in four independent pediatric neuroblastoma cohorts from Therapeutically Applicable Research to Generate Effective Treatments (TARGET), Gene Expression Omnibus (GEO) and European ArrayExpres datasets using Kaplan–Meier and cox regression analysis.ResultsE2F regulated gene set was associated with the event free survival and the overall survival of neuroblastoma. E2F1 and E2F3 were prognostic factors in all four independent pediatric neuroblastoma cohorts. Over-expressions of E2F1 or E2F3 were correlated with the shorted event free survival and overall survival of neuroblastoma. Expression levels of E2F1 and E2F3 were higher in neuroblastoma patients with MYCN amplification or age at diagnosis ≥ 18 months. Moreover, the prognostic significance of E2F1 or E2F3 in neuroblastoma was independent of MYCN amplification and age of diagnosis. Combinations of E2F1, E2F3 with MYCN amplification or age of diagnosis achieved better prognosis of neuroblastoma. Identification of 234 genes were associated with E2F1 and E2F3 expressions in neuroblastoma and those genes were significantly enriched in cell cycle signaling pathway. Also, higher scores of cell cycle signaling pathway were correlated with the adverse prognosis of neuroblastoma.ConclusionsE2F transcription factors E2F1 and E2F3 were prognostic makers of neuroblastoma.

MYCN-driven fatty acid uptake is a metabolic vulnerability in neuroblastoma

Neuroblastoma (NB) is a childhood cancer arising from sympatho-adrenal neural crest cells. MYCN amplification is found in half of high-risk NB patients; however, no available therapies directly target MYCN. Using multi-dimensional metabolic profiling in MYCN expression systems and primary patient tumors, we comprehensively characterized the metabolic landscape driven by MYCN in NB. MYCN amplification leads to glycerolipid accumulation by promoting fatty acid (FA) uptake and biosynthesis. We found that cells expressing amplified MYCN depend highly on FA uptake for survival. Mechanistically, MYCN directly upregulates FA transport protein 2 (FATP2), encoded by SLC27A2. Genetic depletion of SLC27A2 impairs NB survival, and pharmacological SLC27A2 inhibition selectively suppresses tumor growth, prolongs animal survival, and exerts synergistic anti-tumor effects when combined with conventional chemotherapies in multiple preclinical NB models. This study identifies FA uptake as a critical metabolic dependency for MYCN-amplified tumors. Inhibiting FA uptake is an effective approach for improving current treatment regimens.

Asphericity of tumor [123 I]mIBG uptake as a prognostic factor in high-risk neuroblastoma.

In recent years, many research groups have attempted to identify a subgroup of "ultra-high risk" patients within the high-risk neuroblastoma (NB) category. The aim of our study was to evaluate the prognostic significance of parameters derived from pretherapeutic 123 I-meta-iodobenzylguanidine ([123 I]mIBG) integrated single photon emission computed tomography and computed tomography in high-risk patients with NB. The established parameters metabolic tumor volume (MTV), maximal standardized uptake value (SUVmax ) and the novel parameter tumor asphericity as well as clinical (age, stage) and genetic factors (1p/11q deletions and MYCN amplification) were analyzed in this single-center retrospective study of high-risk patients with newly diagnosed NB. Univariate/multivariable Cox regression and propensity score matching were performed for clinical and radiological parameters. Twenty-eight high-risk patients with NB were included (14 males, median age 28.8 (11.3-41.0), range 3-74months). Multivariable analysis of "full" cohort identified high asphericity (≥65%, adjusted hazard ratio [HR] 5.32, 95% confidence interval [CI]: 1.18-24.07, p=.03) and MTV (≥50ml, adjusted HR 4.31, 95% CI: 1.18-15.80, p=.027) as the only factors associated with worse event-free survival. In matched cohort, tumor asphericity was a significant predictor of relapse/progression (HR 3.83, 95% CI: 1.03-14.26, p=.046). In this exploratory study, imaging parameters related to tumor metabolic activity, tumor asphericity and MTV, provided prognostic value for event-free survival in high-risk NB patients. Asphericity ≥65% and MTV ≥50ml may serve as additional prognostic factors to those already used.

High-Level MYCN-Amplified RB1-Proficient Retinoblastoma Tumors Retain Distinct Molecular Signatures.

PurposeRetinoblastomas are malignant eye tumors diagnosed in young children. Most retinoblastomas are genetically characterized by biallelic inactivation of the RB1 gene. However, 1.5% of tumors demonstrate high-level amplification of the proto-oncogene MYCN. Patients with MYCN-amplified RB1-proficient retinoblastoma receive a diagnosis at an earlier age and show a clinically and histologically more malignant phenotype. This study aimed to identify genome-wide molecular features that distinguish this subtype from other retinoblastomas.DesignCohort study.ParticipantsForty-seven retinoblastoma tumors, comprising 36 RB1–/–, 4 RB1+/–, and 7 RB1+/+ tumors. In total, 5 retinoblastomas displayed high-level MYCN amplification, with 3 being RB1+/+, 1 being RB1+/–, and 1 being RB1–/–.MethodsIntegrated analysis, based on gene expression, methylation, and methylation-expression correlations, was performed to identify distinct molecular components of MYCN-amplified RB1-proficient retinoblastomas compared with other retinoblastoma subtypes. The methylation and methylation-expression correlation analysis was initially conducted within a subset of samples (n = 15) for which methylation profiles were available. The significant findings were cross-validated in the entire cohort (n = 47) and in publicly available data.Main Outcome MeasuresDifferentially expressed genes/pathways, differentially methylated genes, and methylation-driven differential gene expression.ResultsA large number of genes (n = 3155) were identified with distinct expression patterns in MYCN-amplified RB1-proficient retinoblastomas. The upregulated and downregulated genes were associated with translation and cell-cycle processes, respectively. Methylation analysis revealed distinct methylated patterns in MYCN-amplified RB1-proficient tumors, many of which showing significant impact on gene expression. Data integration identified a 40-gene expression signature with hypermethylated state resulting in a significant downregulation in MYCN-amplified RB1-proficient retinoblastomas. Cross-validation using the entire cohort and the public domain expression data verified the overall lower expression of these genes not only in retinoblastomas with a MYCN-amplified RB1-proficient background, but also in MYCN-amplified neuroblastomas. These include the metabolism-associated TSTD1 gene and the cyclin-dependent kinase inhibitor gene CDKN2C.ConclusionsMYCN-amplified RB1-proficient retinoblastomas display significantly distinct molecular features compared with other retinoblastomas, including a set of 40 hypermethylation-driven downregulated genes. This gene set can give insight into the biology of MYCN-amplified retinoblastomas and may help us to understand the more aggressive clinical behavior.

Abstract 1678: Single-cell landscape of the TH-MYCN neuroblastoma mouse model

Abstract Background and aims: Neuroblastoma (NB) is the most common and deadly malignancy of infancy. It is an embryonal neuroendocrine tumor arising from the neural crest lineage. Amplification of MYCN is a frequent genetic abnormality, found in approximately 20% of NB tumors, and represents a critical stratifying prognostic marker. Given that MYCN plays a key role in NB tumorigenesis and aggressiveness, the TH-MYCN transgenic mouse model is widely used in NB research. This model is characterized by the overexpression of MYCN in neuroectodermal cells, driven by tyrosine hydroxylase (TH) promoter. This overexpression gives rise to tumors exclusively in the sympathoadrenal system, reflecting human NB features. We aimed to explore the transcriptional landscape of TH-MYCN tumors by single-cell RNA sequencing (scRNA-seq). Methods: Tumors from three homozygous and two hemizygous TH-MYCN mice were dissociated into single cells, profiled by scRNA-seq (Chromium 3’ v2 kit, 10x Genomics), sequenced on the NextSeq platform (Illumina). A total of 41666 single cells were analyzed using pagoda2 and conos. TH-MYCN tumor single-cell transciptomes were further aligned with previously published transcriptomes from the adrenal gland of three mice at embryonic day (E)13.5. To evaluate malignant tumor cells, copy number variants (CNV) were analyzed using inferCNV. Immunohistochemistry was used to validate the bioinformatically predicted cell types. Results: scRNA-seq of tumors from TH-MYCN mice revealed 16 major cell populations, spanning stromal, immune and tumor compartments. MYCN+ tumor cells predominantly resemble sympathetic neuroblasts (Isl1, Stmn1, Nefl, Nefm), while MYCN+ chromaffin-like tumor cells (Pnmt, Cdkn1c, Th, Dbh) were rare. Immunofluorescence stainings confirmed that tumors predominantly consist of proliferating sympathetic neuroblasts. Tumor cell nests were surrounded by supportive stromal and immune cells. The adrenergic tumor cell composition observed in TH-MYCN tumors is highly reminiscent of human NB. Interestingly, we discovered a cluster of immature adrenergic tumor cells aligning with normal fetal cells transitioning from bridge to chromaffin state (characterized by Sox11, Atrx, Xist, Ptprs, Tcf4). Distinct genomic aberrations, particularly unique chromosomal deletions, were found in the immature adrenergic tumor cells relative to their sympathoblast and chromaffin tumor cell counterparts. Conclusions: Examining the cellular composition of TH-MYCN tumors at a transcriptional level gives a detailed view of tumor cell heterogeneity at an unprecedented resolution. This comprehensive atlas of TH-MYCN tumors provides a resource for unmasking novel therapeutic targets. Citation Format: Bethel Tesfai Embaie, Adele Alchahin, Thale Kristin Olsen, Hirak Sarkar, Shenglin Mei, Ninib Baryawno. Single-cell landscape of the TH-MYCN neuroblastoma mouse model [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 1678.

Abstract 2013: Integrated proteomic analysis identifies four distinct subtypes of high-risk neuroblastoma

Abstract Neuroblastoma is the most common solid extracranial tumor in children. Despite intensive multi-modal therapy, 5-year survival rates for patients with high-risk disease remain at approximately 50%. Neuroblastomas typically have low mutational burden and harbor few recurrent mutations making the identification of new therapeutic targets challenging. To characterize the proteomic landscape of high-risk neuroblastoma, we performed liquid chromatography-mass spectrometry-based deep expression proteomics and phosphoproteomics on 30 neuroblastoma tumors from 26 patients with high-risk disease. Our integrated analysis identified four distinct proteomic subgroups of high-risk neuroblastoma that were not otherwise apparent based on clinical or genomic features. The four subgroups were named based on their defining proteomic signature: C1-Mixed, C2-Neuronal, C3-Functional MYCN and C4-Stromal. Only one third of C3-Functional MYCN tumors had amplification of MYCN. Segmental chromosomal losses and gains were enriched in, but not exclusive to, C1-Mixed and C3-Functional MYCN tumors. The activities of multiple kinases including CDK2, CDK7 and MEK2 differed significantly between subgroups. C3-Functional MYCN and C4-Stromal tumors were enriched for immune and stromal cells, respectively. Focal adhesion signaling was specifically upregulated in C4-Stromal tumors suggesting increased extracellular matrix interactions in this tumor subgroup. C2-Neuronal tumors were enriched for axon guidance and neurotrophin signaling pathways. Rho family GTPase signaling was also evident in multiple tumor subgroups. C1-Mixed and C3-Functional MYCN tumors had elevated expression of RNA processing proteins which was associated with increased alternative splicing. Splicing analysis also identified multiple novel protein coding splice events that were shared amongst multiple neuroblastoma tumors and outliers compared to both the Genotype-Tissue Expression (GTEx) dataset and a panel of commercial reference tissues. Protein domain analysis of these novel splice variants suggested that these novel protein isoforms may have aberrant functions that contribute to tumorigenesis. In conclusion, phosphoproteomic analysis can identify candidate pathways for the development of new therapies for patients with high-risk neuroblastoma. Citation Format: Kristin L. Leskoske, Sara A. Byron, Seema Plaisier, Apurva M. Hegde, Krystine Garcia-Mansfield, Ritin Sharma, Genevieve Bergendahl, Abhinav Nagulapally, William Ferguson, Jaqueline Kraveka, Javier Oesterheld, William P. Hendricks, Giselle L. Saulnier Sholler, Jeffrey M. Trent, Patrick Pirrotte. Integrated proteomic analysis identifies four distinct subtypes of high-risk neuroblastoma [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 2013.

Abstract LB137: Orally available, CDK9 selective, small molecule inhibitors shows promise in n-Myc-driven neuroendocrine prostate cancers (NEPC)

Abstract Introduction/Purpose: Amplification of N-MYC causes its overexpression in Neuroendocrine Prostate Cancer (NEPC) patients is prevalent, and it is a rare lethal subtype of cancer detected in 2% of all Prostate Cancers (PC)and over 10-17% of mCRPC (Metastatic Castration-Resistant PC) patients. N-MYC and activated CDK9 act as oncogenic drivers sufficient to transform human-derived prostate cancer cells to take on NEPC phenotypic changes resulting in a more aggressive disease identified in late-stage human PC patients. Additionally, N-MYC is required for tumor resistance, and its downregulation through CDK9 inhibitors lessens the tumor burden. Our efforts in this study have proven that the targeting of N-NYC as a driver of NEPC with CDK9 inhibitors such as BLX-3030 and its series could be a viable approach for therapeutic intervention of mCRPC and NEPCs. Experimental Procedures: Utilizing our FIELDS fragment-based design strategies, we have developed and synthesized a series of novel small molecule CDK9 agents that reversibly bind to CDK9, competitively block the ATP site, and elicit pharmacological responses in N-MYC and other prostate cancer cells in vitro. CDK9 cell-free kinase binding, CDKs selectivity, pCDK9 inhibition assays were performed and confirmed its on-target efficacy and potency of lead CDK9 inhibitor BLX-3030. Results and Summary: In these studies, our lead CDK9 kinase inhibitor BLX-3030 from over 60 plus analogs demonstrated potent activity with an IC50 of <5 nM in inhibiting CDK9. CDK9 and N-MYC expression has been detected in PC-3, DU-145, LNCaP, and in LASCPC-01, NCI-H660 and 22RV1 cell lines. HCI-3030 exhibited potent cellular efficacy with an IC50 of 22 to 590 nM in PC cell lines and in N-MYC expressed cells had an activity of 76, 470, and 132 nM respectively and reduced pCDK9, C-MYC, Mcl-1, N-MYC and its downstream RNA Pol II (Ser-2) and pAR (Ser-81) dose-dependently. Our preliminary results support the hypothesis that inhibition of CDK9 reinstates anti-tumor and blocks tumor progression in both mCRPC and NEPCs cellular models. BLX-3030 and a few of its analogs had excellent PK properties in mouse species in the range of 28-94% F, low clearance, and >3 hours half-life. BLX-3030 is nominated as a pre-clinical agent for additional 3D, FACS/apoptosis, cell migration, live-cell imaging including immunofluorescent studies along with in vivo prostate cancer mouse model efficacy studies as well and these results will be presented. Conclusions: In summary, a series of BLX-3030, its analogs as CDK9 inhibitors, display a range of potency in prostate cancer and NEPC cells with ideal PK parameters, were identified. Within compounds of this profile, BLX-3030 was selected as a candidate CDK9 inhibitor upon in-depth evaluation of the in vitro and in Vivo PK properties further supporting our conclusion that BLX-3030 had the appropriate qualities for development as a pre-clinical candidate. Citation Format: Kyle Medley, Zhaoliang Li, Saisha Vankayalapati, Sowmya Paritala, Dongqing Yan, Kimberly Coffman, Neeraj Agarwal, David J. Bearss. Orally available, CDK9 selective, small molecule inhibitors shows promise in n-Myc-driven neuroendocrine prostate cancers (NEPC) [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 LB137.

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.

Abstract 699: NF-kB is a master regulator of resistance to therapy in high-risk neuroblastoma

Abstract BACKGROUND High-risk neuroblastoma is a pediatric cancer arising from the developing sympathetic nervous system with a 50% relapse rate that is typically fatal. At least two subpopulations of neuroblastoma cells exist that can transdifferentiate, adrenergic and mesenchymal, the latter being more resistant to chemotherapy. Mechanisms of therapy resistance are largely unknown and the cells responsible for relapse have not been identified. METHODS We used single nucleus RNA and ATAC sequencing to identify and characterize the cells that survive chemotherapy, termed here “persister cells”, from a cohort of 20 matched diagnostic and post induction chemotherapyhigh-risk neuroblastoma patients and two patient derived xenograft (PDX) models from diagnostic tumors. Eight representative cell lines derived from neuroblastomas at diagnosis were treated with standard-of-care chemotherapy, and flow cytometry was used to sort for live cells. ML120B and CRISPR-CAS9 were used to modulate NF-kB signaling. An RNA-seq dataset of 153 high-risk neuroblastoma patients was used to determine differentially activated pathways between adrenergic and mesenchymal tumors. RESULTS Residual malignant cells in the post-chemotherapy tumor samples clustered into three main groups separated by the response to therapy. The most prevalent group of persister cells in responders (N=16/20) displayed low MYC(N) activity even in the presence of MYCN amplification. This group also demonstrated decreased expression of the adrenergic core regulatory circuit genes including PHOX2B, ISL1, HAND2, along with marked activation of TNF-alpha via NF-kB signaling. High NF-kB activity was found in a subpopulation of diagnostic cells in two chemo-refractory patients. We validated decreased expression of MYCN (2-fold decrease, p<0.0001) and PHOX2B (3.13-fold decrease, p<0.0001) in PDXs following chemotherapy. MYCN protein levels were decreased and nuclear p65 levels were increased in cell lines treated with chemotherapy. Pharmacologic inhibition of NF-kB signaling and genetic depletion of p65 resulted in increased killing (3.58-fold increase, p=0.0012) of neuroblastoma cell lines in response to chemotherapy. Finally, we classified 153 diagnostic high-risk neuroblastomas as predominantly adrenergic or mesenchymal using RNA-seq, showing that mesenchymal tumors were enriched with NF-kB pathway activation signatures. We then validated high nuclear p65 levels in 3 mesenchymal cell lines. We tested 6 adrenergic lines, 4 of which had no detectable nuclear p65. Notably, the 2 cell lines with detectable nuclear p65 were derived from diagnostic specimens that showed de novo chemotherapy resistance. CONCLUSIONS NF-kB activation is a major mediator of de novo and acquired chemotherapy resistance in high-risk neuroblastoma. We postulate that concomitant silencing of this pathway could eliminate persister cells and prevent disease relapse. Citation Format: Liron D. Grossmann, Yasin Uzun, Jarrett Lindsay, Chia-Hui Chen, Catherine Wingrove, Peng Gao, Anusha Thadi, Quinlen Marshall, Nathan M. Kendsersky, Lea Surrey, Daniel Martinez, Emily Mycek, Colleen Casey, Kateryna Krytska, Matthew Tsang, Adam Wolpaw, David N. Groff, Erin Runbeck, Jayne McDevitt, Dinh Diep, Tasleema Patel, Kathrin M. Bernt, Chi Dang, Kun Zhang, Yael P. Mosse, Kai Tan, John M. Maris. NF-kB is a master regulator of resistance to therapy in high-risk neuroblastoma [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 699.

Abstract 2500: Metastatic retinoblastoma exhibit MYCN alterations, recurrent CNAs and hypomethylation as molecular characteristics of aggressive disease

Abstract Retinoblastoma is a rare pediatric malignancy but the most frequent tumor of the eye. Metastatic disease is the most common cause of disease-related death and is rare in high-income countries. Therefore, little is known about the genomics, transcriptomics and methylomics of extraocular retinoblastoma. Here we present a series of extraocular retinoblastoma cases analyzed at DNA, RNA and methylation level to elucidate the landscape of the metastatic disease. These samples were analyzed by OncoScan CNV, CytoScan HD or whole-exome sequencing, depending on sample availability, for CNA (clonal and subclonal) and mutation analysis; RNAseq for fusion and expression data, and Infinium MethylationEPIC assay for methylation examination. Hierarchical clustering and subtype classification was performed using previously published expression and methylation signatures. Cell cultures were successfully established for 4 tumor specimens. In vitro pharmacological sensitivity of those cell cultures was determined by MTT assay. In total, 11 patients and 14 samples were studied (55% male, 64% unilateral). One patient was RB+/+ and harbored a MYCN amplification. Dissemination sites included orbit (n=8), systemic (n=3), and central nervous system (n=5). Median age at diagnosis was 27 months (5-48); 36% were already metastatic. All samples were classified as subtype 2 retinoblastoma according to Liu et. al., 2021, both at the expression and methylation level. Among recurrent CNAs in all samples, common retinoblastoma alterations were observed: 1q (86%), 2p (71%, 29% MYCN amplified), 6p (93%) gains and 16q (64%) loss. Other less frequent alterations such as 17q gain (21%) and 11q (43%) and 19q (29%) loss were found. MYCN pathway alteration was the most outstanding feature, evidenced by frequent gains/amplification on that gene and in gene-expression clustering based on MYCN expression signatures. Global hypomethylation was also observed when comparing metastatic to intraocular samples and it was consistent with the general gene overexpression seen in metastatic samples. Metastasis-derived in vitro cultures treated either with melphalan, topotecan, idarubicin, doxorubicin or carboplatin showed various degrees of sensitivity (i.e. EC50 in the nM to mM range), with no clear association to MYCN overexpression. In conclusion, even considering the caveats regarding our current sample size, we could detect the alteration of the MYCN pathway as a distinct characteristic of extraocular retinoblastoma. Global hypomethylation has been related to the activation of oncogenic drivers and is consistent with metastatic behavior. These results suggest a pattern of molecular features that may have been selected during tumor evolution. Our results do not support a straightforward relationship between MYCN activation and resistance to therapy. Citation Format: Daiana Ganiewich, Santiago Zugbi, Rosario Aschero, Daniela Ottaviani, María B. Cancela, Fabiana Lubieniecki, Claudia Sampor, Cinzia Lavarino, François Radvanyi, Guillermo L. Chantada, Paula S. Schaiquevich, Andrea S. Llera. Metastatic retinoblastoma exhibit MYCN alterations, recurrent CNAs and hypomethylation as molecular characteristics of aggressive disease [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 2500.

The immune landscape of solid pediatric tumors

BackgroundLarge immunogenomic analyses have demonstrated the prognostic role of the functional orientation of the tumor microenvironment in adult solid tumors, this variable has been poorly explored in the pediatric counterpart.MethodsWe performed a systematic analysis of public RNAseq data (TARGET) for five pediatric tumor types (408 patients): Wilms tumor (WLM), neuroblastoma (NBL), osteosarcoma (OS), clear cell sarcoma of the kidney (CCSK) and rhabdoid tumor of the kidney (RT). We assessed the performance of the Immunologic Constant of Rejection (ICR), which captures an active Th1/cytotoxic response. We also performed gene set enrichment analysis (ssGSEA) and clustered more than 100 well characterized immune traits to define immune subtypes and compared their outcome.ResultsA higher ICR score was associated with better survival in OS and high risk NBL without MYCN amplification but with poorer survival in WLM. Clustering of immune traits revealed the same five principal modules previously described in adult tumors (TCGA). These modules divided pediatric patients into six immune subtypes (S1-S6) with distinct survival outcomes. The S2 cluster showed the best overall survival, characterized by low enrichment of the wound healing signature, high Th1, and low Th2 infiltration, while the reverse was observed in S4. Upregulation of the WNT/Beta-catenin pathway was associated with unfavorable outcomes and decreased T-cell infiltration in OS.ConclusionsWe demonstrated that extracranial pediatric tumors could be classified according to their immune disposition, unveiling similarities with adults’ tumors. Immunological parameters might be explored to refine diagnostic and prognostic biomarkers and to identify potential immune-responsive tumors.