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Acute myeloid leukemia

Notch blockade overcomes endothelial cell-mediated resistance of FLT3/ITD-positive AML progenitors to AC220 treatment

Leukemia volume 35, pages 601–605 (2021)Cite this article

Subjects

AML patients harboring FLT3/ITD are at risk for relapse with conventional therapies, especially those with a high ratio of the FLT3/ITD vs. wild-type allele (FLT3-AR), and even the addition of FLT3 inhibitors provides only modest improvements [1]. As the tumor microenvironment (TME) has been implicated in drug resistance [2], we reasoned that AML-TME interactions might be critical for leukemic progenitor survival and drug resistance, and that targeting AML-TME interactions might be crucial to improving survival in FLT3/ITD-positive AML patients. Endothelial cells (ECs), a component of the TME, provide the critical microenvironmental support for leukemic progression and resistance to chemotherapy [3] and are implicated in survival, dormancy, and pro-metastatic properties in other cancers [4]. We previously showed that ECs derived from human umbilical cord vein transduced with E4ORF1 virus (E4 ECs) establish Notch-dependent growth and expansion of hematopoietic stem cells (HSCs, ref. [5]) and provide efficient conditions for culturing pre-leukemic and leukemic precursors long term [6]. In this study, we inquire whether E4 ECs confer drug resistance to FLT3/ITD-positive AML in the presence of AC220, a clinically relevant inhibitor of FLT3. We show that E4 ECs promoted the survival of FLT3/ITD-positive progenitors and implicate the Notch pathway in this protection for AML with high FLT3-AR. We further provide evidence that Notch inhibition sensitizes the FLT3/ITD-positive CD34+CD33− fraction to AC220 treatment for a patient sample exhibiting high FLT3-AR, demonstrating the therapeutic potential of inhibiting Notch signaling.

In this study, we used frozen aliquots of mononuclear CD45+ cells isolated from FLT3/ITD-positive AML diagnostic bone marrow samples obtained from the Children’s Oncology Group. Cells were placed in liquid culture or EC coculture containing SFEM II (+50 ng/mL huSCF) and incubated at 37 °C in 3% O2 and 5% CO2. Treatment with AC220 and inhibitory antibodies specific to the negative regulatory region (NRR) of Notch1 and Notch2 receptors (anti-NRR1 and anti-NRR2 were kindly provided by Chris Siebel, Genentech, ref. [7]), colony-forming cell (CFC) assay and RNA-seq analysis are detailed in Supplementary Materials and Methods. The research was approved by the FHCRC Institutional Review Board (IRB9950).

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Fig. 1: EC coculture improved survival of FLT3/ITD-positive progenitors treated with AC220.
Fig. 2: Inhibiting Notch signaling reduced EC-mediated protection of FLT3/ITD-positive CFCs in the presence of AC220 for patient samples with high FLT3-AR.

Data availability

RNA-seq data are available at GEO, accession GSE138340.

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Acknowledgements

This study was supported by The Hartwell Fellowship Foundation to QL. We thank Chris Siebel (Genentech) for the anti-NRR1 and anti-NRR2 antibodies and Shahin Rafii (Weill Cornell Medical College) for the E4ORF1 lentivirus construct.

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Authors and Affiliations

  1. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Quy Le, Brandon Hadland, Soheil Meshinchi & Irwin Bernstein

  2. Department of Pediatrics, University of Washington, Seattle, WA, USA

    Soheil Meshinchi & Irwin Bernstein

  3. Children’s Oncology Group, Monrovia, CA, USA

    Soheil Meshinchi & Irwin Bernstein

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  1. Quy Le
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  2. Brandon Hadland
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  3. Soheil Meshinchi
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  4. Irwin Bernstein
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Contributions

QL and IB designed the experiments; QL performed the experiments and analyzed the data; QL, IB, BH, and SM wrote the paper.

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Correspondence to Irwin Bernstein.

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Le, Q., Hadland, B., Meshinchi, S. et al. Notch blockade overcomes endothelial cell-mediated resistance of FLT3/ITD-positive AML progenitors to AC220 treatment. Leukemia 35, 601–605 (2021). https://doi.org/10.1038/s41375-020-0893-y

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