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Epigenetically dysregulated NOTCH-Delta-HES signaling cascade can serve as a subtype classifier for acute lymphoblastic leukemia

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Abstract

The NOTCH-Delta-HES signaling cascade is regarded as a double-edged sword owing to its dual tumor-suppressor and oncogenic roles, in different cellular environments. In the T-cells, it supports leukemogenesis by promoting differentiation while in B-cells, it controls leukemogenesis by inhibiting early differentiation/inducing growth arrest in the lead to apoptosis. The present study was undertaken to assess if this bi-faceted behavior of NOTCH family can be exploited as a diagnostic biomarker or subtype classifier of acute lymphoblastic leukemia (ALL). In this pursuit, expression of seven NOTCH cascade genes was analyzed in bone marrow (BM) biopsy and blood plasma (BP) of pediatric ALL patients using quantitative PCR (qPCR). Further, promoter DNA methylation status of the differentially expressed genes (DEGs) was assessed by methylation-specific qMSP and validated through bisulphite amplicon sequencing. Whereas hypermethylation of JAG1, DLL1, and HES-2, HES-4, and HES-5 was observed in all patients, NOTCH3 was found hypermethylated specifically in Pre-B ALL cases while DLL4 in Pre-T ALL cases. Aberrant DNA methylation strongly correlated with downregulated gene expression, which restored at complete remission stage as observed in “follow-up/post-treatment” subjects. The subtype-specific ROC curve analysis and Kaplan–Meier survival analysis predicted a clinically applicable diagnostic and prognostic potential of the panel. Moreover, the logistic regression model (Pre-B vs Pre-T ALL) was found to be the best-fitted model (McFadden’s R2 = 0.28, F1 measure = 0.99). Whether analyzed in BM-aspirates or blood plasma, the NOTCH epigenetic signatures displayed comparable results (p < 0.001), advocating the potential of NOTCH-Delta-HES cascade, as a subtype classifier, in minimally invasive diagnosis of ALL.

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Data Availability

The data that support the findings of this study are available on appropriate request from the corresponding author, [SS].

Abbreviations

ALL :

Acute lymphoblastic leukemia

3′ UTR :

3′ Untranslated region

BCL2 :

B-cell leukemia/lymphoma 2

BM :

Bone marrow

BMA :

Bone marrow aspirate

BP :

Blood plasma

cDNA :

Complementary DNA

Cq :

Quantitative cycle

DAPK :

Death-associated protein kinase

DEGs :

Differentially expressed genes

DLL1 :

Delta-like ligand 1

DLL4 :

Delta-like ligand 4

DNA :

Deoxyribonucleic acid

FC :

Fold change (normalized)

GAPDH :

Glyceraldehyde-3-phosphate dehydrogenase

HC :

Healthy control

HES2 :

Hairy and enhancer of split 2

HES4 :

Hairy and enhancer of split 4

HES5 :

Hairy and enhancer of split 5

JAG1 :

Jagged 1

MI :

Methylation index

NICD :

NOTCH intracellular domain

NOTCH3 :

Neurogenic locus NOTCH homolog protein 3

NRT :

No reverse transcriptase

NTC :

No template control

qMSP :

Quantitative methylation specific PCR

qPCR :

Quantitative real-time PCR

RNA :

Ribonucleic acid

ROC :

Receiver operator characteristic

SMRT :

Silencing mediator of retinoic acid and thyroid hormone receptor

SNVs :

Single-nucleotide variants

TSGs :

Tumor suppressor genes

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Acknowledgements

The authors are thankful to all study subjects and guardians who participated in this study and voluntarily gave their BM and/or BP samples. Thanks are due to the paramedical staff of the Leukemia Center, Children’s Hospital Lahore, for facilitation in sample collections. We are also thankful to Dr. Tahir Shamsi (Late) for providing the bone marrow samples of healthy donors.

Funding

The work was supported by a grant from PakPat World Intellectual Property Protection Services (Grant No. 23974/2023) and partially by the University of the Punjab, Lahore, Pakistan.

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Authors and Affiliations

  1. Biopharmaceuticals and Biomarkers Discovery Lab., School of Biochemistry and Biotechnology, University of the Punjab, Lahore, 54590, Pakistan

    Syeda Saliah Hussan, Muhammad Shrafat Ali, Mishal Fatima, Memoona Altaf & Saima Sadaf

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  1. Syeda Saliah Hussan
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Contributions

SSH performed experiments and acquired, analyzed, and interpreted all the datasets in the present study. MSA, MA, and MF facilitated sample collection, cataloging, and setting-up the runs. SSH and MSA confirmed authenticity of all the raw data and wrote the draft manuscript. Project conception, study design, research supervision, and critical revision of manuscript for important intellectual contents were done by SS. All authors have read and approved the final manuscript.

Corresponding author

Correspondence to Saima Sadaf.

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Ethics approval and consent to participate

All experiments were performed according to the Declaration of Helsinki. Study design was approved by the University Advanced Studies and Research Board besides approval Ethics Committee (Ref. 268/FIMS-22–06-2022).

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The authors declare no competing interests.

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Highlights

• NOTCH-Delta-HES signaling cascade, a double edge sword, plays both tumor suppressor and oncogenic roles.

• Distinct expression patterns of NOTCH pathway members in Pre-B ALL and Pre-T-ALL subjects are derived by differential promoter DNA methylation.

• Dysregulated expression of NOTCH3 and DLL4 can serve as an ALL subtype classifier in bone marrow and liquid-biopsy samples.

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Hussan, S.S., Ali, M.S., Fatima, M. et al. Epigenetically dysregulated NOTCH-Delta-HES signaling cascade can serve as a subtype classifier for acute lymphoblastic leukemia. Ann Hematol 103, 511–523 (2024). https://doi.org/10.1007/s00277-023-05515-9

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