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MOLECULAR TARGETS FOR THERAPY

Inhibition of NOTCH4 sensitizes FLT3/ITD acute myeloid leukemia cells to FLT3 tyrosine kinase inhibition

Leukemia volume 38, pages 1581–1591 (2024)Cite this article

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Abstract

Internal tandem duplication mutations of FLT3 (FLT3/ITD) confer poor prognosis in AML. FLT3 tyrosine kinase inhibitors (TKIs) alone have limited and transient clinical efficacy thus calling for new targets for more effective combination therapy. In a loss-of-function RNAi screen, we identified NOTCH4 as one such potential target whose inhibition proved cytotoxic to AML cells, and also sensitized them to FLT3 inhibition. Further investigation found increased NOTCH4 expression in FLT3/ITD AML cell lines and primary patient samples. Inhibition of NOTCH4 by shRNA knockdown, CRISPR-Cas9-based knockout or γ-secretase inhibitors synergized with FLT3 TKIs to kill FLT3/ITD AML cells in vitro. NOTCH4 inhibition sensitized TKI-resistant FLT3/ITD cells to FLT3 TKI inhibition. The combination reduced phospho-ERK and phospho-AKT, indicating inhibition of MAPK and PI3K/AKT signaling pathways. It also led to changes in expression of genes involved in regulating cell cycling, DNA repair and transcription. A patient-derived xenograft model showed that the combination reduced both the level of leukemic involvement of primary human FLT3/ITD AML cells and their ability to engraft secondary recipients. In summary, these results demonstrate that NOTCH4 inhibition synergizes with FLT3 TKIs to eliminate FLT3/ITD AML cells, providing a new therapeutic target for AML with FLT3/ITD mutations.

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Fig. 1: NOTCH4 is essential for the survival of FLT3/ITD cells and its inhibition sensitizes FLT3/ITD AML cells to FLT3 TKI.
Fig. 2: NOTCH4 knockdown sensitizes FLT3/ITD AML cells to TKI treatment.
Fig. 3: Gilteritinib and γ-secretase inhibitors have a synergistic effect on FLT3/ITD AML cells.
Fig. 4: Gilteritinib and γ-secretase inhibitors have a synergistic effect on primary AML sample cells with FLT3/ITD mutations.
Fig. 5: γ-secretase inhibitors reduce MAPK/ERK activity of FLT3/ITD cells and impact expression of cancer-related genes.
Fig. 6: Combination of FLT3 TKI and/or NOTCH4 inhibition eliminates FLT3/ITD AML cells and reduces disease progression in vivo.

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All data generated and analyzed in this study are included in this published article and its supplementary materials and figures.

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Acknowledgements

This work was supported by grants from the National Cancer Institute (R01 CA090668 (DS) and P30 CA006973, T32 CA60441 (CMS), Commonwealth Fund and the Giant Food Pediatric Cancer Research Fund (DS). RZ is supported by the China Scholarship Council and National Natural Science Foundation of China (No. 82200187). DS is also supported by the Kyle Haydock Professorship. DS has also received research funding and has served as a consultant to Pharos I, B&T for an unrelated project and serves on the SAB of In Silico Medicine, Inc. and Mitomed.

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Author notes

  1. S. Haihua Chu

    Present address: Beam Therapeutics, Cambridge, MA, USA

  2. These authors contributed equally: Ruiqi Zhu, Courtney M. Shirley.

Authors and Affiliations

  1. Department of Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Ruiqi Zhu, Courtney M. Shirley, S. Haihua Chu, Li Li, Bao H. Nguyen, Jaesung Seo, Min Wu, Tessa Seale, Mark Levis & Donald Small

  2. Department of Hematology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China

    Ruiqi Zhu & Yu Hu

  3. Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Amy S. Duffield

  4. Lymphoid Malignancies Branch, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA

    Louis M. Staudt

  5. Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, USA

    Donald Small

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Contributions

RZ, CMS and SHC designed experiments, performed experiments and analyzed data; LL, BHN, JS, MW and AD performed data analysis and provided technical support; LMS and YH provided technical support; ML provided patient samples; DS designed experiments and analyzed data. RZ wrote the first version of the manuscript; RZ, CMS, LL and DS revised the manuscript.

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Correspondence to Donald Small.

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Competing interests

The authors declare no competing financial interest. DS does serve on the SAB of InSilico Medicine and Mitomed and receives research support and serves as a consultant for an unrelated project from Pharos, Ltd. ML has received honoraria from Daiichi Sankyo, Novartis, and Agios; has served in a consulting or advisory role for Daiichi Sankyo, Novartis, and Agios; and has received research funding from Astellas and Novartis.

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Zhu, R., Shirley, C.M., Chu, S.H. et al. Inhibition of NOTCH4 sensitizes FLT3/ITD acute myeloid leukemia cells to FLT3 tyrosine kinase inhibition. Leukemia 38, 1581–1591 (2024). https://doi.org/10.1038/s41375-024-02292-w

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