Document Type

Article

Publication Date

6-13-2025

Publication Title

Journal of Biological Chemistry

Volume

301

Issue

5

Pages

1

Publisher Name

Elsevier Inc.

Abstract

BACKGROUND: Acute myeloid leukemia (AML) is an aggressive hematologic cancer with a notoriously bleak prognosis; for non-M3 AML, the overall 5-year survival rate is ∼30%. While 60-70% of newly diagnosed AML patients will achieve complete remission (CR), half of these patients will experience relapse (secondary resistance) by three years from their diagnosis. Moreover, 30-40% of AML patients present with refractory disease (primary resistance) and cannot respond to frontline treatments. Leukemia stem cells (LSCs) are implicated in both primary and secondary resistance, and their eradication is necessary to maintain CR. LSCs have unique transcriptomes and immunophenotypes, thus can be identified relatively accurately using RNA-Seq (e.g., HOXA9 and MEIS1) and flow cytometry (e.g., CD123, CD244, CLL1, c-KIT, and FLT3). However, despite our being able to identify LSCs, agents directed specifically against LSCs do not yet exist. Moreover, being non-selective in terms of the cells targeted, conventional chemotherapy comes with significant toxicity and offers long-term survival to fewer than 1 out of every 3 patients. Thus, there is a need for LSC-specific agents that are as effective as they are tolerable. OBJECTIVE: Considering previous data from our group implicating innate immunity-associated signaling (the TLR-TAK1/TICAM-1 axes) in the survival of LSCs, we sought to characterize TANK-binding kinase 1 (TBK1), a Ser/Thr kinase of the innate immune system, in normal hematopoiesis and MLL-AF9-driven (MLL-AF9+) murine leukemia, to determine if TBK1 may be a feasible drug target for the treatment of AML. METHODS: We generated a tamoxifen-inducible, global Tbk1-knockout (Tbk1NULL) C57BL/6 mouse model. Tbk1fx/fx mice were purchased from Lexicon Pharmaceuticals and crossed with Rosa26-CreERT2+ C57BL/6 mice purchased from The Jackson Laboratory. Isolated HSPCs were transduced with an MSCV retrovirus harboring the MLL-AF9 leukemic oncogene. All procedures were performed in accordance with the NIH's Guide for the Care and Use of Laboratory Animals and an Institutional Animal Care and Use Committee (IACUC)-approved protocol (#2020010) at Loyola University Chicago. RESULTS: In normal murine physiology, we found that Tbk1NULL C57BL/6 mice are viable and grossly normal; the genetic loss of Tbk1 does not significantly impede homeostatic hematopoiesis. However, we discovered that global deletion of Tbk1 leads to an increase in the size of the pool of MPP3-type HSPCs in the bone marrow and the number of neutrophils in peripheral blood. Moreover, we report that while TBK1 does not seem to regulate the engraftment nor reconstitution abilities of transplanted bone marrow cells, TBK1 does negatively regulate monocyte production in an HSPC-intrinsic fashion, as wild-type mice lethally conditioned with busulfan and given Tbk1NULL bone marrow displayed increased monocytes in peripheral blood at endpoint compared to mice transplanted with control marrow. In murine leukemia, we found that the genetic loss of Tbk1 strongly reduces the size of the c-Kit+Flt3+ compartment of MLL-AF9+ HSPCs in vitro and in vivo. We also learned that TBK1 negatively regulates the expression of Csf1r on MLL-AF9+ HSPCs and that some mice given Tbk1NULL MLL-AF9+ HSPCs develop a subcutaneous mass of AML cells (chloroma). DISCUSSION: Murine TBK1 appears to be required by specific LSCs yet dispensable in homeostatic hematopoiesis. While our study requires much further investigation and escalation to better-powered models, we are enthusiastic to present some preliminary data that implicate TBK1 as a drug target in the treatment of MLL-AF9+ AML. This work was supported by the NIH's National Heart, Lung, and Blood Institute (NHLBI; R01 HL133560-01 [Jiwang Zhang] & T35 HL120835 [Austin P. Runde, LUC SSOM's STAR/T35 Program]), National Cancer Institute (NCI; R01 CA223194-01 [Jiwang Zhang]), National.

Comments

Author Posting © The American Society for Biochemistry and Molecular Biology, Inc., 2025. This article is posted here by permission of Elsevier for personal use and redistribution. This article was published open access in Journal of Biological Chemistry, Vol. 301, Iss. 5, Supplement, (May 2025), 6, https://doi.org/10.1016/j.jbc.2025.108696.

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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