FLT3-ITD Activates Cytoplasmic Drosha-Dependent Non-Canonical Mechanisms of Mir-155 Biogenesis in Acute Myeloid Leukemia

MiRNAs are small noncoding RNAs that control gene expression by binding to cognate sites in the 3′ untranslated region (3′ UTR) of target messenger RNAs (mRNAs). MiRNAs are transcribed as primary (pri) mRNAs and processed into precursor (pre) miRNAs by nuclear DROSHA before being exported into the cytoplasm via the Exportin 5 (XPO5)/RAN-GTP complex. Once in the cytoplasm, pre-miRNAs are further processed by Dicer into mature miRNAs. The FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is one of the most common mutations in acute myeloid leukemia (AML) and confers growth and survival advantages to leukemia blasts. Among miRNAs, miR-155 has emerged as one of the most significantly upregulated in the FLT3-ITD+ AML and has been shown to play a pivotal role in uncontrolled blast hyperproliferation and survival. Herein, we report a previously unrecognized activity of FLT3-ITD that leads to deregulation of miR-155 expression by providing the evidence for the existence of FLT3-dependent non-canonical mechanisms of miRNA biogenesis. We initially observed that FLT3-ITD blocked the biogenesis of intronic miRNAs via inhibition of the "gatekeeper" XPO5/RAN-GTP complex that allow nucleus-to-cytoplasm pre-miRNA transport. By using in vitro phosphorylation assay with [32P]-ATP labeling, we demonstrated that FLT3-ITD phosphorylates a Sprouty related EVH1 domain-containing protein 1 (SPRED1), and that phospho-SPRED1 in turn inhibited the XPO5/RAN-GTP complex thereby halting transportation of pre-miRNAs from the nucleus to cytoplasm. This resulted in a decrease of several intronic miRNAs involved in normal hematopoiesis (i.e., miR-29b, miR-181a, miR-146b, miR-126). Accordingly, knocking down of SPRED1 expression in FLT3-ITD+ AML cells resulted in increased production of mature intronic miRNAs. Since the XPO5/RAN-GTP complex is the main gatekeeper for miRNA biogenesis, these results appeared in contradiction with the miR-155 upregulation which has been invariably observed in FLT3-ITD+ AML blasts. Differently from intronic miRNAs, miR-155 is hosted at the genomic site of long non-coding RNA (lnc-RNA) and we therefore hypothesized that it follows a different path of biogenesis. In fact forced expression of FLT3-ITD in lin-Sca1+kit+ (LSK) cells decreased intronic miRNA biogenesis (i.e., miR-126) and increase production of lnc-RNA hosted miRNAs (i.e., miR-155). Accordingly, we demonstrated that upon FLT3-ITD activation, AKT phosphorylated DDX3X, a DEAD-box RNA helicase protein thereby reducing its ability to bind heterogeneous nuclear Ribonucleoprotein U (hnRNP U), a component of the hnRNP complex associated with pre-mRNA splicing. Consequently, in FLT3-ITD cells, BIC-155 splicing is decreased. In fact, we measured significant increased levels of phospho-DDX3X and unspliced BIC-155 in primary FLT3-ITD+ blasts compared with FLT3-ITD- AML blasts. In consistent, DDX3X knock-down (KO) in FLT3-ITD- cells decreased BIC-155 splicing whereas DDX3X re-expression reversed these effects. The excess of unspliced BIC-155 RNA then bound to nuclear RNA export factor 1 (NXF1), a shuttle protein that transports poly A+ RNAs from the nucleus to the cytoplasm. In consistent, NXF1 KO blocked unspliced BIC-155 nucleus-to-cytoplasm transportation. In FLT3-ITD+ blasts, once in the cytoplasm, the unspliced BIC-155 RNA was then processed by cytoplasmic isoforms of DROSHA, as demonstrated using immunostaining, RNA Immunoprecipitation (RIP), and other gain- and loss-of-function experiments. Furthermore, overexpression of cytoplasmic DROSHA but not nuclear DROSHA isoform increased the processing of unspliced BIC-155 to mature miR-155. None of the intronic miRNAs studies interacted with cytoplasmic DROSHA. The bound of BIC-155 RNA with cytoplasmic DROSHA was unique of FLT3-ITD+ blasts, as it was not observed in FLT3-ITD- blasts. Thus, our results indicate a two-fold activity of FLT3-ITD that leads to decreased levels of intronic miRNAs via XPO5/RAN-GTP blockage and upregulation of other lnc-RNA hosted miRNAs via cytoplasmic DROSHA activation. The net result is suppression of intronic miRNAs that participates to the regulation of normal hematopoiesis and upregulation of lnc-RNA-hosted miRNA, especially miR-155 that contribute to aberrant blast hyperproliferation in the FLT3-ITD AML phenotype (Figure 1). Disclosures No relevant conflicts of interest to declare.

Ran-GTP is non-essential to activate NuMA for spindle pole focusing, but dynamically polarizes HURP to control mitotic spindle length

During mitosis, a bipolar spindle is assembled around chromosomes to efficiently capture chromosomes. Previous work proposed that a chromosome-derived Ran-GTP gradient promotes spindle assembly around chromosomes by liberating spindle assembly factors (SAFs) from inhibitory importins. However, Ran’s dual functions in interphase nucleocytoplasmic transport and mitotic spindle assembly have made it difficult to assess its mitotic roles in somatic cells. Here, using auxin-inducible degron technology in human cells, we developed acute mitotic degradation assays to dissect Ran’s mitotic roles systematically and separately from its interphase function. In contrast to the prevailing model, we found that the Ran pathway is not essential for spindle assembly activities that occur at sites spatially separated from chromosomes, including activating NuMA for spindle pole focusing or for targeting TPX2. In contrast, Ran-GTP is required to localize HURP and HSET specifically at chromosome-proximal regions. We demonstrated that Ran-GTP and importin-β coordinately promote HURP’s dynamic microtubule binding-dissociation cycle near chromosomes, which results in stable kinetochore-fiber formation. Intriguingly, this pathway acts to establish proper spindle length preferentially during prometaphase, rather than metaphase. Together, we propose that the Ran pathway is required to activate SAFs specifically near chromosomes, but not generally during human mitotic spindle assembly. Ran-dependent spindle assembly is likely coupled with parallel pathways to activate SAFs, including NuMA, for spindle pole focusing away from chromosomes.HighlightsUsing auxin-inducible degron technology, we developed mitotic degradation assays for the Ran pathway in human cells.The Ran pathway is non-essential to activate NuMA for spindle pole focusing.The Ran pathway dynamically polarizes HURP and defines mitotic spindle length preferentially during prometaphase.Ran-GTP is required to activate SAFs specifically near chromosomes, but not generally, in human mitotic cells.

F-Actin nucleated on chromosomes coordinates their capture by microtubules in oocyte meiosis

Capture of each and every chromosome by spindle microtubules is essential to prevent chromosome loss and aneuploidy. In somatic cells, astral microtubules search and capture chromosomes forming lateral attachments to kinetochores. However, this mechanism alone is insufficient in large oocytes. We have previously shown that a contractile F-actin network is additionally required to collect chromosomes scattered in the 70-µm starfish oocyte nucleus. How this F-actin–driven mechanism is coordinated with microtubule capture remained unknown. Here, we show that after nuclear envelope breakdown Arp2/3-nucleated F-actin “patches” form around chromosomes in a Ran-GTP–dependent manner, and we propose that these structures sterically block kinetochore–microtubule attachments. Once F-actin–driven chromosome transport is complete, coordinated disassembly of F-actin patches allows synchronous capture by microtubules. Our observations indicate that this coordination is necessary because early capture of chromosomes by microtubules would interfere with F-actin–driven transport leading to chromosome loss and formation of aneuploid eggs.

F-actin patches nucleated on chromosomes coordinate capture by microtubules in oocyte meiosis

Capture of each and every chromosome by spindle microtubules is essential to prevent chromosome loss and aneuploidy. In somatic cells, astral microtubules search and capture chromosomes forming lateral attachments to kinetochores. However, this mechanism alone is insufficient in large oocytes. We have previously shown that a contractile F-actin network is additionally required to collect chromosomes scattered in the 70-μm starfish oocyte nucleus. How this F-actin-driven mechanism is coordinated with microtubule capture remained unknown. Here, we show that after nuclear envelope breakdown Arp2/3-nucleated F-actin patches form around chromosomes in a Ran-GTP-dependent manner, and we propose that these structures sterically block kinetochore-microtubule attachments. Once F-actin-driven chromosome transport is complete, coordinated disassembly of these F-actin patches allows synchronous capture by microtubules. Our observations indicate that this coordination is necessary, as early capture of chromosomes by microtubules would interfere with F-actin-driven transport leading to chromosome loss and formation of aneuploid eggs.

Structural insight into TPX2-stimulated microtubule assembly

During mitosis and meiosis, microtubule (MT) assembly is locally upregulated by the chromatin-dependent Ran-GTP pathway. One of its key targets is the MT-associated spindle assembly factor TPX2. The molecular mechanism of how TPX2 stimulates MT assembly remains unknown because structural information about the interaction of TPX2 with MTs is lacking. Here, we determine the cryo-electron microscopy structure of a central region of TPX2 bound to the MT surface. TPX2 uses two flexibly linked elements (’ridge’ and ‘wedge’) in a novel interaction mode to simultaneously bind across longitudinal and lateral tubulin interfaces. These MT-interacting elements overlap with the binding site of importins on TPX2. Fluorescence microscopy-based in vitro reconstitution assays reveal that this interaction mode is critical for MT binding and facilitates MT nucleation. Together, our results suggest a molecular mechanism of how the Ran-GTP gradient can regulate TPX2-dependent MT formation.