Chronic myeloid leukemia in chronic phase (CML-CP) is induced by BCR-ABL1 oncogenic tyrosine kinase. Tyrosine kinase inhibitors eliminate the bulk of CML-CP cells, but fail to eradicate leukemia stem cells (LSCs) and leukemia progenitor cells (LPCs) displaying innate and acquired resistance, respectively. These cells may accumulate genomic instability, leading to disease relapse and/or malignant progression to a fatal blast phase. In the present study, we show that Rac2 GTPase alters mitochondrial membrane potential and electron flow through the mitochondrial respiratory chain complex III (MRC-cIII), thereby generating high levels of reactive oxygen species (ROS) in CML-CP LSCs and primitive LPCs. MRC-cIII–generated ROS promote oxidative DNA damage to trigger genomic instability, resulting in an accumulation of chromosomal aberrations and tyrosine kinase inhibitor–resistant BCR-ABL1 mutants. JAK2(V617F) and FLT3(ITD)–positive polycythemia vera cells and acute myeloid leukemia cells also produce ROS via MRC-cIII. In the present study, inhibition of Rac2 by genetic deletion or a small-molecule inhibitor and down-regulation of mitochondrial ROS by disruption of MRC-cIII, expression of mitochondria-targeted catalase, or addition of ROS-scavenging mitochondria-targeted peptide aptamer reduced genomic instability. We postulate that the Rac2-MRC-cIII pathway triggers ROS-mediated genomic instability in LSCs and primitive LPCs, which could be targeted to prevent the relapse and malignant progression of CML.

Charcot-Marie-Tooth disease (CMT) is a length-dependent peripheral neuropathy. The aminoacyl-tRNA synthetases constitute the largest protein family implicated in CMT. Aminoacyl-tRNA synthetases are predominantly cytoplasmic, but are also present in the nucleus. Here we show that a nuclear function of tyrosyl-tRNA synthetase (TyrRS) is implicated in a Drosophila model of CMT. CMT-causing mutations in TyrRS induce unique conformational changes, which confer capacity for aberrant interactions with transcriptional regulators in the nucleus, leading to transcription factor E2F1 hyperactivation. Using neuronal tissues, we reveal a broad transcriptional regulation network associated with wild-type TyrRS expression, which is disturbed when a CMT-mutant is expressed. Pharmacological inhibition of TyrRS nuclear entry with embelin reduces, whereas genetic nuclear exclusion of mutant TyrRS prevents hallmark phenotypes of CMT in the Drosophila model. These data highlight that this translation factor may contribute to transcriptional regulation in neurons, and suggest a therapeutic strategy for CMT.

Here, we established the Drosophila Giant Fiber neurons (GF) as a novel model to study axonal trafficking of L1-type Cell Adhesion Molecules (CAM) Neuroglian (Nrg) in the adult CNS using live imaging. L1-type CAMs are well known for their importance in nervous system development and we previously demonstrated a role for Nrg in GF synapse formation. However, in the adult they have also been implicated in synaptic plasticity and regeneration. In addition, to its canonical role in organizing cytoskeletal elements at the plasma membrane, vertebrate L1CAM has also been shown to regulate transcription indirectly as well as directly via its import to the nucleus. Here, we intend to determine if the sole L1CAM homolog Nrg is retrogradley transported and thus has the potential to relay signals from the synapse to the soma. Live imaging of c-terminally tagged Nrg in the GF revealed that there are at least two populations of retrograde vesicles that differ in speed, and either move with consistent or varying velocity. To determine if endogenous Nrg is retrogradely transported, we inhibited two key regulators, Lissencephaly-1 (Lis1) and Dynactin, of the retrograde motor protein Dynein. Similar to previously described phenotypes for expression of poisonous subunits of Dynactin, we found that developmental knock down of Lis1 disrupted GF synaptic terminal growth and that Nrg vesicles accumulated inside the stunted terminals in both mutant backgrounds. Moreover, post mitotic Lis1 knock down in mature GFs by either RNAi or Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) induced mutations, resulted in normal length terminals with fully functional GF synapses which also exhibited severe accumulation of endogenous Nrg vesicles. Thus, our data suggests that accumulation of Nrg vesicles is due to failure of retrograde transport rather than a failure of terminal development. Together with the finding that post mitotic knock down of Lis1 also disrupted retrograde transport of tagged Nrg vesicles in GF axons, it demonstrates that endogenous Nrg protein is transported from the synapse to the soma in the adult central nervous system in a Lis1-dependent manner.

Edited by Eric R. Fearon L1 cell adhesion molecule (L1CAM) is well-known for its importance in nervous system development and cancer progression. In addition to its role as a plasma membrane protein in cytoskeletal organization, recent in vitro studies have revealed that both transmembrane and cytosolic fragments of proteolytically cleaved vertebrate L1CAM translocate to the nucleus. In vitro studies indicate that nuclear L1CAM affects genes with functions in DNA post-replication repair, cell cycle control, and cell migration and differentiation, but its in vivo role and how its nuclear levels are regulated is less well-understood. Here, we report that mutations in the conserved ankyrin-binding domain affect nuclear levels of the sole Drosophila homolog neuroglian (Nrg) and that it also has a noncanonical role in regulating transcript levels of the oncogene Myc in the adult nervous system. We further show that altered nuclear levels of Nrg correlate with altered transcript levels of Myc in neurons, similar to what has been reported for human glioblastoma stem cells. However, whereas previous in vitro studies suggest that increased nuclear levels of L1CAM promote tumor cell survival, we found here that elevated levels of nuclear Nrg in neurons are associated with increased sensitivity to oxidative stress and reduced life span of adult animals. We therefore conclude that these findings are of potential relevance to the management of neurodegenerative diseases associated with oxidative stress and cancer.L1-type cell adhesion molecules (CAMs) 4 are single-pass transmembrane glycoproteins belonging to the immunoglobu-lin family of receptors that are highly conserved from invertebrates to vertebrates (1). The structure of L1CAM consists of extracellular immunoglobulin and fibronectin type III domains, a transmembrane domain, and a cytoplasmic tail harboring an ezrin-binding FERM domain as well as an ankyrinbinding FIGQY domain (Fig. 1A). In its unphosphorylated state, the highly conserved FIGQY domain reversibly binds to ankyrin (Fig. 1A), which couples L1-type CAMs to actin. This interaction is known to mediate neuritogenesis, synapse growth, and stability (2-4). In contrast, phosphorylation of the tyrosine in the FIGQY domain inhibits ankyrin binding (5,6).In addition to its function as a cytoskeleton-organizing protein at the plasma membrane, vertebrate L1CAM can be proteolytically cleaved with fragments translocating to the nucleus (7-11). The 200-kDa full-length L1CAM is cleaved proximal to the plasma membrane by metalloproteases to a 32-kDa fragment (12,13). The 32-kDa fragment is further cleaved by ␥-secretase/presenilin, releasing a 28-kDa cytosolic fragment containing the intracellular domain (ICD), which translocates to the nucleus (10, 11). Similar to full-length L1CAM, recombinant expression of L1-ICD in nonneuronal cell lines affects gene expression of CRABPII and ␤3-integrin (11). In addition, it was shown that nuclear L1-ICD also led to up-regulation of NBS1 (Nijmegen breakage syndrome gene) via c-Myc, w...

The role of the Amyloid Precursor Protein (APP) in the pathology of Alzheimer’s disease (AD) has been well studied. However, the normal function of APP in the nervous system is poorly understood. Here, we characterized the role of the Drosophila homolog (APPL) in the adult giant fiber (GF) neurons. We find that endogenous APPL is transported from the synapse to the soma in the adult. Live-imaging revealed that retrograde moving APPL vesicles co-traffic with L1-type cell adhesion molecule Neuroglian (Nrg). In APPL null mutants, stationary Nrg vesicles were increased along the axon, and the number of Nrg vesicles moving in retrograde but not anterograde direction was reduced. In contrast, trafficking of endo-lysosomal vesicles, which did not co-localize with APPL in GF axons, was not affected. This suggests that APPL loss of function does not generally disrupt axonal transport but that APPL has a selective role in the effectiveness of retrograde transport of proteins it co-traffics with. While the GF terminals of APPL loss of function animals exhibited pruning defects, APPL gain of function had no disruptive effect on GF morphology and function, or on retrograde axonal transport of Nrg. However, cell-autonomous developmental expression of a secretion-deficient form of APPL (APPL-SD), lacking the α-, β-, and, γ-secretase cleavage sites, resulted in progressive retraction of the GF terminals. Conditional expression of APPL-SD in mature GFs caused accumulation of Nrg in normal sized synaptic terminals, which was associated with severely reduced retrograde flux of Nrg labeled vesicles in the axons. Albeit β-secretase null mutants developed GF terminals they also exhibited Nrg accumulations. This suggests that cleavage defective APPL has a toxic effect on retrograde trafficking and that β-secretase cleavage has a function in Nrg sorting in endosomal compartments at the synapse. In summary, our results suggest a role for APPL and its proteolytic cleavage sites in retrograde trafficking, thus our findings are of relevance to the understanding of the endogenous role of APP as well as to the development of therapeutic treatments of Alzheimer’s disease.

2736 BCR-ABL1 –positive chronic myeloid leukemia in chronic phase (CML-CP) is a leukemia stem cell (LSC)-derived but leukemia progenitor cell (LPC)-driven disease, which may eventually develop resistance to the tyrosine kinase inhibitors (TKIs) and progress to fatal CML blast phase (CML-BP). In CML-CP, LSCs and LPCs reside in the CD34+CD38- and CD34+CD38+ populations, respectively. In addition, majority of LSCs and LPCs belong to quiescent (CFSEmax) and proliferative (CFSElow) populations, respectively. Quiescent LSCs are intrinsically insensitive to TKIs, and LPCs can acquire resistance to TKIs. In the TKI era, these cells may eventually initiate the disease relapse and progression to CML-BP, which is associated with genomic instability manifested by accumulation of a new or additional TKI-resistant BCR-ABL1 kinase mutations and chromosomal aberrations. We reported that BCR-ABL1 –positive leukemia cells contain high levels of the reactive oxygen species (ROS)-induced oxidative DNA damage resulting in genomic instability (Nowicki et al., Blood, 2005; Koptyra et al., Blood, 2006; Koptyra et al., Leukemia, 2008). These studies highlighted the importance of identification of the origin of leukemia cell lineage accumulating genomic instability and the mechanisms responsible for generation of ROS-mediated oxidative DNA damage. Here we show that LSC-enriched CD34+CD38- cells and quiescent LSCs, and also LPC-enriched CD34+CD38+ and proliferating CML-CP cells contain higher levels of ROS (superoxide anion, hydrogen peroxide, and hydroxyl radical) and oxidative DNA lesions (8-oxoG and DNA double-strand breaks) than corresponding cells from healthy donors. Surprisingly, the most primitive quiescent LSCs accumulated the highest levels of ROS and oxidative DNA damage. On the basis of these observations and the studies in murine hematopoietic 32Dcl3 cells expressing TKI-resistant BCR-ABL1 kinase variants (Y253F, T315I, H396P), we would predict that primitive CML-CP cells carrying these mutations would also contain high levels of ROS and oxidative DNA damage. Moreover, inhibition of BCR-ABL1 kinase with imatinib exerted only modest, if any, effect on ROS and oxidative DNA damage in LSCs/LPCs in the presence of growth factors (GFs). Among numerous signaling proteins activated in CML cells, Rac GTPases were potential candidates to regulate production of ROS. Importantly, Rac was stimulated in leukemia cells expressing non-mutated BCR-ABL1 and TKI-resistant kinase mutants and it remained active in CML-CP cells treated with imatinib in the presence of GFs. We used Rac dominant-negative mutant (RacT17N), Rac specific inhibitors (NSC23766 and EHT1864) and Rac1, Rac2 and Rac3 knockout cells to document that Rac2 GTPase is responsible for elevation of ROS and oxidative DNA damage in LSC-enriched CD34+CD38- cells, quiescent LSCs, and also in LPCs. Active Rac2 reduced mitochondrial membrane potential (ΔΨm) and slowed the electron flow between mitochondrial respiratory chain (MRC) complexes I-II and I-III leading to overproduction of ROS. Using cells depleted of functional mitochondria (Rho0 cells), applying specific probes to measure mitochondrial ROS (MitosoxRed and mitochondria matrix-targeted circularly permuted yellow fluorescence protein = mt-cpYFP) and employing a specific inhibitor of mitochondrial ROS (MitoQ) we determined that mitochondria are the main source of ROS causing oxidative DNA damage in CD34+CD38- and quiescent LSCs and in LPCs. Furthermore, using selective inhibitors of various MRC complexes we pinpointed complex III as major producer of ROS in LSCs and LPCs. This conclusion is supported by the observation that BCR-ABL1 –positive cells with genetically inactivated complex III, but not complex I, displayed diminished capability to generate ROS. Targeting Rac2 GTPase by RacT17N and reduction of mitochondrial ROS by mitochondrial-targeted catalase and by mitochondrial-targeted ROS-scavenging peptide aptamers prevented genomic instability. Altogether, Rac2 - MRC-cIII pathway is a major source of ROS-mediated oxidative DNA damage resulting in genomic instability in LSCs and LPCs, which could be targeted to prevent the relapse and malignant progression of CML. We also postulate that similar mechanisms cause genomic instability in FLT3(ITD)-positive acute myeloid leukemia cells and in JAK2(V617F)-positive polycythemia vera cells. Disclosures: Holyoake: Novartis: Consultancy, Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding. Hochhaus:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hughes:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.