Elucidating the Mechanisms of Leukemogenesis Driven By FBXO11 Depletion
Authors: Mo Angela , Chang Linda Ya-Ting , Duns Gerben , Wang Xuan , Morin Gregg , Lau Tammy , Docking Rod , Colborne Shane , Cheng Se-Wing Grace , Huang Shujun , Kincross Hayle , Gharaee Nadia , Willie Elijah , Jiang Jihong , Parker Jeremy , Karsan Aly 
Published: November 2021

Mutations in SKP1 and CUL1 (Zhang et. al. Oncol Lett 2018), which encode components of the SKP1-CUL1-F-BOX (SCF) ubiquitin E3-ligase complex, have previously been reported or characterized in AML. FBXO11, which encodes the substrate recognizing component, however, has not been studied in AML. We performed whole exome sequencing and RNA-seq on140 clinical AML samples and identified recurrent inactivating mutations in FBXO11. Of the components of the SCF FBXO11 complex, FBXO11 transcript expression is most significantly reduced in AML samples compared to normal. We show that loss of FBXO11 drives leukemogenesis through dysregulation of the novel target, LONP1, by reducing mitochondrial potential and promoting self-renewal.

We found that UPS mutations co-occur with AML1-ETO (RUNX1-RUNX1T1) fusions and RAS mutations. Fbxo11 knockdown in mouse hematopoietic stem/progenitor cells (HSPC) cooperated with AML1-ETO to generate serially transplantable AML in mice. FBXO11 depletion in human cord-blood derived CD34+ cells (CD34+ CB), combined with AML1-ETO and a KRAS mutant, promoted stem cell maintenance and myeloid malignancy in a human xenotransplant model.

Mass spectrometry analysis of FLAG-FBXO11 co-immunoprecipitating proteins in K562 cells identified mitochondrial protease, LONP1, as a top target. LONP1 protein expression did not vary with FBXO11 loss or overexpression, suggesting that LONP1 is not a degradation target of the SCF FBXO11complex.

Knockdown of either FBXO11 or LONP1 resulted in myeloid bias in CD34+ CB in vitro, pointing to an activating role of FBXO11 on LONP1. Both FBXO11 and LONP1 depletion reduced mitochondrial membrane potential (MMP) in CD34+ CB and myeloid cell lines, aligning with the stemness phenotypes observed with FBXO11 depletion, as long-term hematopoietic stem cells (LT-HSCs) are characterized by low MMP (Mansell et. al. Cell Stem Cell 2021), and disruption of MMP promotes self-renewal in HSCs (Vannini et. al. Nat Commun 2016).

As FBXO11 neddylates p53 to regulate transcription (Abida et. al. J. Biol. Chem 2007), we examined protein neddylation, and detected increased neddylation in immunoprecipitated LONP1 from FLAG-FBXO11-expressing K562 cells. As, neddylation regulates protein activation (Wu et. al. Nature 2005), our findings suggest that FBXO11 neddylation of LONP1 activates LONP1 to maintain mitochondrial function. Consequently, loss of FBXO11 function primes HSPC for self-renewal by reduction of MMP.

To clarify the regulatory relationship between FBXO11 and LONP1, we performed RNA-seq on CD34+ CB cells expressing combinations of shRNAs targeting FBXO11 or LONP1, with overexpression of FLAG -FBXO11 or LONP1. Unsupervised clustering revealed that LONP1-overexpressing samples clustered with controls, suggesting that LONP1 requires modification by FBXO11 for functional effects. Using gene set enrichment analysis, we found that both FBXO11 and LONP1 depletion enriched for HSC and LSC (leukemic stem cell) gene sets. Knockdown of LONP1 reversed the effect of FLAG-FBXO11 overexpression, supporting a model of LONP1 being a downstream mediator of FBXO11 function. Both FBXO11 and LONP1 depletion enriched for a gene set composed of mitochondrial electron transport chain complex (ETC) genes, potentially reflecting a transcriptional response to loss of functional ETC activity, as suggested by accumulation of misfolded ETC proteins with knockdown of LONP1 (Ghosh et. al. Oncogene 2019).

In this work, we demonstrate the leukemogenic effects of FBXO11 loss. We draw a novel connection between the UPS and the mitochondrial protease system with the identification of LONP1 as an FBXO11 target that regulates hematopoiesis.

Disclosures

No relevant conflicts of interest to declare.