Loss of FBXO11 establishes a stem cell program in acute myeloid leukemia by dysregulating LONP1
Hayle Kincross, Ya-Chi Angela Mo, Xuan Wang, Linda Chang, Gerben Duns, Franziska Mey, Jihong Jiang, Zurui Zhu, Naomi Isak, Harwood Kwan, Tammy T.Y. Lau, T. Roderick Docking, Pranav Garg, Jessica Tran, Shane Colborne, Se-Wing Grace Cheng, Shujun Huang, Nadia Gharaee, Elijah Willie, Jeremy D.K. Parker, Joshua Bridgers, Davis Wood, Ramon I. Klein Geltink, Gregg B. Morin, Aly Karsan
Hayle Kincross, Ya-Chi Angela Mo, Xuan Wang, Linda Chang, Gerben Duns, Franziska Mey, Jihong Jiang, Zurui Zhu, Naomi Isak, Harwood Kwan, Tammy T.Y. Lau, T. Roderick Docking, Pranav Garg, Jessica Tran, Shane Colborne, Se-Wing Grace Cheng, Shujun Huang, Nadia Gharaee, Elijah Willie, Jeremy D.K. Parker, Joshua Bridgers, Davis Wood, Ramon I. Klein Geltink, Gregg B. Morin, Aly Karsan
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Research Article Hematology

Loss of FBXO11 establishes a stem cell program in acute myeloid leukemia by dysregulating LONP1

Abstract

Acute myeloid leukemia (AML) is an aggressive cancer with very poor outcomes. To identify additional drivers of leukemogenesis, we analyzed sequencing data from 1,727 unique individual patients with AML, which revealed mutations in ubiquitin ligase family genes in 11.2% of samples from adult patients with AML with mutual exclusivity. The SKP1/CUL1/F-box (SCF) E3 ubiquitin ligase complex gene, FBXO11, was the most significantly downregulated gene of the SCF complex in AML. We found that FBXO11 interacts with and catalyzes K63-linked ubiquitination of LONP1 in the cytosol, to promote LONP1 entry into mitochondria. We show that depletion of FBXO11 or LONP1 reduced mitochondrial respiration through impaired LONP1 chaperone activity to assemble electron transport chain Complex IV. Reduced mitochondrial respiration secondary to FBXO11 or LONP1 depletion imparted myeloid-biased stem cell properties in primary CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro. In a human xenograft model, depletion of FBXO11 cooperated with AML1-ETO and mutant KRASG12D to generate serially transplantable AML. Our findings suggest that reduced FBXO11 cooperates to initiate AML by priming HSPC for myeloid-biased self renewal through attenuation of LONP1-mediated regulation of mitochondrial respiration.

Authors

Hayle Kincross, Ya-Chi Angela Mo, Xuan Wang, Linda Chang, Gerben Duns, Franziska Mey, Jihong Jiang, Zurui Zhu, Naomi Isak, Harwood Kwan, Tammy T.Y. Lau, T. Roderick Docking, Pranav Garg, Jessica Tran, Shane Colborne, Se-Wing Grace Cheng, Shujun Huang, Nadia Gharaee, Elijah Willie, Jeremy D.K. Parker, Joshua Bridgers, Davis Wood, Ramon I. Klein Geltink, Gregg B. Morin, Aly Karsan

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Figure 6

FBXO11 deficiency impairs LONP1 maintenance of mitochondrial respiration in AML.

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FBXO11 deficiency impairs LONP1 maintenance of mitochondrial respiration...
(A) Oxygen consumption rate (OCR) measured by the Agilent Seahorse XF Cell Mito Stress Test in K562 FBXO11-KO cells expressing empty vector (EV) or FLAG-FBXO11 with quantification of (B) basal and (C) maximal respiration (N = 6). (D) Mitochondrial membrane potential (MMP) of K562 FBXO11-KO cells measured by flow cytometry (N = 3). P values represent 2-tailed t tests. MMP of (E) K562 cells and (F) CD34+ HSPCs expressing shRNA targeting FBXO11 or LONP1 or a nontargeting control (shCTR) (N = 4–5). (G) MMP measured in K562 cells expressing WT FBXO11 or FBXO11 lacking the F-box domain (FBXO11-ΔFbox) (N = 3). (H) MMP measured in K562 cells expressing EV with shCTR, or FLAG-FBXO11 coexpressed with shCTR or shLONP1 (N = 3). (I) MMP measured in K562 cells expressing LONP1-FLAG or LONP1del22–66-FLAG and LONP1del551–602-FLAG (N = 3). (J) Immunoblots of whole cell FBXO11 and LONP1 levels in OCI-AML3 cells expressing shFBXO11, shLONP1 or shCTR, and assembled ETC complexes (II–V) assayed by blue native page electrophoresis followed by immunoblotting of OCI-AML3 mitochondrial lysates. (K) Immunoblots of K63-linked polyubiquitination (K63-Ub) of LONP1 immunoprecipitated from K562 FBXO11-KO lines expressing empty vector or FLAG-FBXO11 (N = 3). (L) MMP measured in K562 cells expressing FLAG-FBXO11 treated with vehicle (DMSO), enzyme inhibitors of neddylation (MLN4924 or TAS4464), or ubiquitination (PYR-41 or TAK-243) (N = 3). (M) MMP of CD34+ HSPC from a 25-participant cord blood pool and 9 bone marrow samples from patients with AML presented in Figure 2C (N = 3-6). P value represents 2-tailed t test. (N) Correlation of AML sample MMP versus relative FBXO11 protein abundance of the sample or (O) reported bone marrow blast percentage at the time of clinical sampling. P and R values represent simple linear regression. All other P values represent 1-way ANOVA with Dunnett’s test, error bars represent SD.