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1.
Res Sq ; 2024 Jun 04.
Article in English | MEDLINE | ID: mdl-38883758

ABSTRACT

A deep learning model using attention-based multiple instance learning (aMIL) and self-supervised learning (SSL) was developed to perform pathologic classification of neuroblastic tumors and assess MYCN-amplification status using H&E-stained whole slide digital images. The model demonstrated strong performance in identifying diagnostic category, grade, mitosis-karyorrhexis index (MKI), and MYCN-amplification on an external test dataset. This AI-based approach establishes a valuable tool for automating diagnosis and precise classification of neuroblastoma tumors.

2.
Cell Rep ; 43(5): 114165, 2024 May 28.
Article in English | MEDLINE | ID: mdl-38691450

ABSTRACT

The N6-methyladenosine (m6A) RNA modification is an important regulator of gene expression. m6A is deposited by a methyltransferase complex that includes methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14). High levels of METTL3/METTL14 drive the growth of many types of adult cancer, and METTL3/METTL14 inhibitors are emerging as new anticancer agents. However, little is known about the m6A epitranscriptome or the role of the METTL3/METTL14 complex in neuroblastoma, a common pediatric cancer. Here, we show that METTL3 knockdown or pharmacologic inhibition with the small molecule STM2457 leads to reduced neuroblastoma cell proliferation and increased differentiation. These changes in neuroblastoma phenotype are associated with decreased m6A deposition on transcripts involved in nervous system development and neuronal differentiation, with increased stability of target mRNAs. In preclinical studies, STM2457 treatment suppresses the growth of neuroblastoma tumors in vivo. Together, these results support the potential of METTL3/METTL14 complex inhibition as a therapeutic strategy against neuroblastoma.


Subject(s)
Cell Differentiation , Cell Proliferation , Methyltransferases , Neuroblastoma , Methyltransferases/metabolism , Methyltransferases/antagonists & inhibitors , Neuroblastoma/pathology , Neuroblastoma/metabolism , Neuroblastoma/drug therapy , Neuroblastoma/genetics , Humans , Cell Differentiation/drug effects , Cell Proliferation/drug effects , Cell Line, Tumor , Animals , Mice , Gene Expression Regulation, Neoplastic/drug effects , Adenosine/analogs & derivatives , Adenosine/metabolism , Adenosine/pharmacology
3.
Clin Cancer Res ; 2024 Mar 20.
Article in English | MEDLINE | ID: mdl-38506712

ABSTRACT

PURPOSE: The importance of cellular context to the synergy of DNA Damage Response (DDR) targeted agents is important for tumors with mutations in DDR pathways, but less well-established for tumors driven by oncogenic transcription factors. In this study, we exploit the widespread transcriptional dysregulation of the EWS-FLI1 transcription factor to identify an effective DDR targeted combination therapy for Ewing Sarcoma (ES). EXPERIMENTAL DESIGN: We used matrix drug screening to evaluate synergy between a DNA-PK inhibitor (M9831) or an ATR inhibitor (berzosertib) and chemotherapy. The combination of berzosertib and cisplatin was selected for broad synergy, mechanistically evaluated for ES selectivity, and optimized for in vivo schedule. RESULTS: Berzosertib combined with cisplatin demonstrates profound synergy in multiple ES cell lines at clinically achievable concentrations. The synergy is due to loss of expression of the ATR downstream target CHEK1, loss of cell cycle checkpoints, and mitotic catastrophe. Consistent with the goals of the project, EWS-FLI1 drives the expression of CHEK1 and five other ATR pathway members. The loss of CHEK1 expression is not due to transcriptional repression and instead caused by degradation coupled with suppression of protein translation. The profound synergy is realized in vivo with a novel optimized schedule of this combination in subsets of ES models leading to durable complete responses in 50% of animals bearing two different ES xenografts. CONCLUSION: These data exploit EWS-FLI1 driven alterations in cell context to broaden the therapeutic window of berzosertib and cisplatin to establish a promising combination therapy and a novel in vivo schedule.

4.
Trends Cancer ; 9(7): 528-542, 2023 07.
Article in English | MEDLINE | ID: mdl-37147166

ABSTRACT

Numerous strategies are employed by cancer cells to control gene expression and facilitate tumorigenesis. In the study of epitranscriptomics, a diverse set of modifications to RNA represent a new player of gene regulation in disease and in development. N6-methyladenosine (m6A) is the most common modification on mammalian messenger RNA and tends to be aberrantly placed in cancer. Recognized by a series of reader proteins that dictate the fate of the RNA, m6A-modified RNA could promote tumorigenesis by driving protumor gene expression signatures and altering the immunologic response to tumors. Preclinical evidence suggests m6A writer, reader, and eraser proteins are attractive therapeutic targets. First-in-human studies are currently testing small molecule inhibition against the methyltransferase-like 3 (METTL3)/methyltransferase-like 14 (METTL14) methyltransferase complex. Additional modifications to RNA are adopted by cancers to drive tumor development and are under investigation.


Subject(s)
Methyltransferases , Neoplasms , Animals , Humans , Methyltransferases/genetics , Methyltransferases/metabolism , RNA/genetics , RNA/metabolism , Methylation , RNA, Messenger/genetics , Neoplasms/drug therapy , Neoplasms/genetics , Carcinogenesis , Mammals/genetics , Mammals/metabolism
5.
Nat Commun ; 10(1): 1757, 2019 04 15.
Article in English | MEDLINE | ID: mdl-30988284

ABSTRACT

Cyclin-dependent kinase 12 (CDK12) modulates transcription elongation by phosphorylating the carboxy-terminal domain of RNA polymerase II and selectively affects the expression of genes involved in the DNA damage response (DDR) and mRNA processing. Yet, the mechanisms underlying such selectivity remain unclear. Here we show that CDK12 inhibition in cancer cells lacking CDK12 mutations results in gene length-dependent elongation defects, inducing premature cleavage and polyadenylation (PCPA) and loss of expression of long (>45 kb) genes, a substantial proportion of which participate in the DDR. This early termination phenotype correlates with an increased number of intronic polyadenylation sites, a feature especially prominent among DDR genes. Phosphoproteomic analysis indicated that CDK12 directly phosphorylates pre-mRNA processing factors, including those regulating PCPA. These results support a model in which DDR genes are uniquely susceptible to CDK12 inhibition primarily due to their relatively longer lengths and lower ratios of U1 snRNP binding to intronic polyadenylation sites.


Subject(s)
Cyclin-Dependent Kinases/genetics , DNA Damage , DNA Repair/genetics , Cell Line, Tumor , Chromatography, High Pressure Liquid , Chromatography, Liquid , Humans , Models, Molecular , Phosphorylation , Polyadenylation , RNA Processing, Post-Transcriptional , Tandem Mass Spectrometry
6.
J Biol Chem ; 290(23): 14462-75, 2015 Jun 05.
Article in English | MEDLINE | ID: mdl-25903125

ABSTRACT

The retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin-proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study we explored the mechanisms connecting proteasome-mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130, were diminished during embryonic stem cell differentiation concomitant with their target gene acquisition. Proteasome-dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C-terminal regulatory domains in a process that is sensitive to cyclin-dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F-DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposition of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regulation of RB family repressor potency and the ubiquitin-proteasome system.


Subject(s)
Retinoblastoma Protein/analysis , Retinoblastoma Protein/metabolism , Amino Acid Sequence , Animals , Cell Line , Conserved Sequence , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , Evolution, Molecular , Humans , Mice , Models, Molecular , Molecular Sequence Data , Proteasome Endopeptidase Complex/metabolism , Protein Stability , Protein Structure, Tertiary , Sequence Alignment , Transcription, Genetic
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