DDRE-24. ARF4-MEDIATED RETROGRADE TRAFFICKING PROMOTES CHEMORESISTANCE IN GBM

Glioblastoma (GBM) is the most common type of adult malignant brain tumor, with a median survival of only 21 months. This is partly due to the high rate of resistance to conventional therapy, including temozolomide (TMZ), leading to recurrence rates close to 100%. To identify the unknown genes driving the development of this resistance, we performed a genome-wide CRISPR knockout screen comparing a DMSO-treated population with a TMZ-treated population over 14 days. Results showed significant enrichment of ~200 novel genes and pathways. From this list, we identified 4 previously unstudied genes showing significant elevations in RNA expression (p< 0.05) when comparing recurrent and primary tumors in patient datasets, along with significant survival benefits corresponding to low gene expression (p< 0.05). Validation experiments in vitro showed significant elevations in RNA and protein expression in multiple patient-derived xenografts (PDX) lines during TMZ-treated conditions, while knocking out these genes also resulted in significantly heightened sensitivity to TMZ (p< 0.01). We investigated one particularly enriched gene, ARF4, known to be involved in retrograde trafficking. With previous studies showing that ARF4 is upregulated under ER stress, we first confirmed the increased expression of ER stress markers during TMZ treatment to explain the increased expression of ARF4 during treatment. Further investigation via live-cell imaging also showed a consequent increase in retrograde trafficking in TMZ-treated cells, as evidenced by significantly increased trafficking of transferrin receptors, a retrograde transport marker, as well as EGFR, known to play a role in promoting chemoresistance through strengthened DNA repair response. ARF4-overexpressed GBM cells similarly showed increased trafficking of transferrin receptors and EGFR to the nucleus, while ARF4-knockdowns showed decreased trafficking and nuclear EGFR expression. Ultimately, our CRISPR-Cas9 screen has identified a promising therapeutic target, ARF4, which may drive GBM’s robust resistance to chemotherapy through increased retrograde trafficking of chemoresistance-promoting nuclear EGFR.

α-Synuclein inhibits Snx3-retromer retrograde trafficking of the conserved membrane-bound proprotein convertase Kex2 in the secretory pathway of Saccharomyces cerevisiae

We tested the ability of alpha-synuclein (α-syn) to inhibit Snx3-retromer mediated retrograde trafficking of Kex2 and Ste13 between late endosomes and the trans-Golgi (TGN) using a Saccharomyces cerevisiae model of Parkinson’s disease (PD). Kex2 and Ste13 are a conserved, membrane-bound proprotein convertase and dipeptidyl aminopeptidase, respectively, that process pro-α-factor and pro-killer toxin. Each of these proteins contains a cytosolic tail that binds to sorting nexin Snx3. Using a combination of techniques, including fluorescence microscopy, western blotting and a yeast mating assay, we found that α-syn disrupts Snx3-retromer trafficking of Kex2-GFP and GFP-Ste13 from the late endosome to the TGN, resulting in these two proteins transiting to the vacuole by default. Using three α-syn variants (A53T, A30P, and α-synΔC, which lacks residues 101–140), we further found that A53T and α-synΔC, but not A30P, reduce Snx3-retromer trafficking of Kex2-GFP, which is likely to be due to weaker binding of A30P to membranes. Degradation of Kex2 and Ste13 in the vacuole should result in the secretion of unprocessed, inactive forms of α-factor, which will reduce mating efficiency between MATa and MATα cells. We found that wild-type α-syn but not A30P significantly inhibited the secretion of α-factor. Collectively, our results support a model in which the membrane-binding ability of α-syn is necessary to disrupt Snx3-retromer retrograde recycling of these two conserved endopeptidases.

ACBD3 modulates KDEL receptor interaction with PKA for its trafficking via tubulovesicular carrier

Background KDEL receptor helps establish cellular equilibrium in the early secretory pathway by recycling leaked ER-chaperones to the ER during secretion of newly synthesized proteins. Studies have also shown that KDEL receptor may function as a signaling protein that orchestrates membrane flux through the secretory pathway. We have recently shown that KDEL receptor is also a cell surface receptor, which undergoes highly complex itinerary between trans-Golgi network and the plasma membranes via clathrin-mediated transport carriers. Ironically, however, it is still largely unknown how KDEL receptor is distributed to the Golgi at steady state, since its initial discovery in late 1980s. Results We used a proximity-based in vivo tagging strategy to further dissect mechanisms of KDEL receptor trafficking. Our new results reveal that ACBD3 may be a key protein that regulates KDEL receptor trafficking via modulation of Arf1-dependent tubule formation. We demonstrate that ACBD3 directly interact with KDEL receptor and form a functionally distinct protein complex in ArfGAPs-independent manner. Depletion of ACBD3 results in re-localization of KDEL receptor to the ER by inducing accelerated retrograde trafficking of KDEL receptor. Importantly, this is caused by specifically altering KDEL receptor interaction with Protein Kinase A and Arf1/ArfGAP1, eventually leading to increased Arf1-GTP-dependent tubular carrier formation at the Golgi. Conclusions These results suggest that ACBD3 may function as a negative regulator of PKA activity on KDEL receptor, thereby restricting its retrograde trafficking in the absence of KDEL ligand binding. Since ACBD3 was originally identified as PAP7, a PBR/PKA-interacting protein at the Golgi/mitochondria, we propose that Golgi-localization of KDEL receptor is likely to be controlled by its interaction with ACBD3/PKA complex at steady state, providing a novel insight for establishment of cellular homeostasis in the early secretory pathway.

An Extended Coatomer Binding Motif in the SARS-CoV-2 Spike Protein

β-Coronaviruses such as SARS-CoV-2 hijack coatomer protein-I (COPI) for spike protein retrograde trafficking to the progeny assembly site in endoplasmic reticulum-Golgi intermediate compartment (ERGIC). However, limited residue-level details are available into how the spike interacts with COPI. Here we identify a novel extended COPI binding motif in the spike that encompasses the canonical K-x-H dibasic sequence. This motif demonstrates selectivity for αCOPI subunit. Guided by an in silico analysis of dibasic motifs in the human proteome, we employ mutagenesis and binding assays to show that the spike motif terminal residues are critical modulators of complex dissociation, which is essential for spike release in ERGIC. αCOPI residues critical for spike motif binding are elucidated by mutagenesis and crystallography and found to be conserved in the zoonotic reservoirs, bats, pangolins, camels, and in humans. Collectively, our investigation on the spike motif identifies key COPI binding determinants with implications for retrograde trafficking.

ARF4-mediated Retrograde Trafficking Drives Chemoresistance in Glioblastoma

Glioblastoma (GBM) is the most common type of adult malignant brain tumor, with a median survival of only 21 months. This is partly due to the high rate of resistance to conventional therapy, including temozolomide (TMZ), leading to recurrence rates close to 100%. It still remains unknown what drives the development of this resistance. To identify the unknown genes driving the development of this resistance, we performed a genome-wide CRISPR knockout screen comparing a DMSO-treated population with a TMZ-treated population over 14 days. We identified 4 previously unstudied genes – ARF4, PLAA, SPTLC1, and PIGK – that showed significant elevations in expression in recurrent tumors in patient datasets, along with significant survival benefits corresponding to low gene expression. Further investigation of ARF4, known to be involved in retrograde trafficking, allowed us to identify a mechanism of resistance that is mediated by increased retrograde transport of EGFR into the nucleus. Ultimately, our CRISPR-Cas9 screen has identified a promising therapeutic target, ARF4, which may drive GBM's high resistance to chemotherapy.

Tamoxifen Derivatives Alter Retromer-Dependent Endosomal Tubulation and Sorting to Block Retrograde Trafficking of Shiga Toxins

Shiga toxin 1 and 2 (STx1 and STx2) undergo retrograde trafficking to reach the cytosol of cells where they target ribosomes. As retrograde trafficking is essential for disease, inhibiting STx1/STx2 trafficking is therapeutically promising. Recently, we discovered that the chemotherapeutic drug tamoxifen potently inhibits the trafficking of STx1/STx2 at the critical early endosome-to-Golgi step. We further reported that the activity of tamoxifen against STx1/STx2 is independent of its selective estrogen receptor modulator (SERM) property and instead depends on its weakly basic chemical nature, which allows tamoxifen to increase endolysosomal pH and alter the recruitment of retromer to endosomes. The goal of the current work was to obtain a better understanding of the mechanism of action of tamoxifen against the more disease-relevant toxin STx2, and to differentiate between the roles of changes in endolysosomal pH and retromer function. Structure activity relationship (SAR) analyses revealed that a weakly basic amine group was essential for anti-STx2 activity. However, ability to deacidify endolysosomes was not obligatorily necessary because a tamoxifen derivative that did not increase endolysosomal pH exerted reduced, but measurable, activity. Additional assays demonstrated that protective derivatives inhibited the formation of retromer-dependent, Golgi-directed, endosomal tubules, which mediate endosome-to-Golgi transport, and the sorting of STx2 into these tubules. These results identify retromer-mediated endosomal tubulation and sorting to be fundamental processes impacted by tamoxifen; provide an explanation for the inhibitory effect of tamoxifen on STx2; and have important implications for the therapeutic use of tamoxifen, including its development for treating Shiga toxicosis.

Halting Retrograde Transport Excludes ErbB-2 From the Nucleus Abrogating Tumor Growth in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) refers to a subtype of tumors with poor prognosis, devoid of hormone receptors and of membrane overexpression or gene amplification of ErbB-2. Due to its molecular heterogeneity, TNBC represents a major clinical challenge. In this regard, clinical biomarkers and targeted therapies remain elusive, and chemotherapy has been the standard of care for early and metastatic TNBC. ErbB-2, a member of the ErbB family of tyrosine kinase receptors, is a major player in the BC scenario. While it is a cell membrane-bound receptor, it migrates to the nucleus (NErbB-2) where it acts as a transcription factor or coactivator. We recently found that both the canonical (wild-type, WT) ErbB-2 and the alternative isoform c are located in the nucleus of TNBC, a scenario with an aggressive oncogenic potential. The route of intracellular transport from the plasma membrane to the trans Golgi network (TGN) and the endoplasmic reticulum (ER) is termed retrograde trafficking, and constitutes the pathway by which ErbB-2 migrates to the nucleus. The retrograde transport route is also hijacked by toxins and viruses to access the ER and exert their deleterious effects. Retro-2, a small molecule inhibitor, was shown to protect cells from toxin and virus effects by blocking their retrograde trafficking. Given the high levels of NErbB-2 in TNBC cells, we explored whether treatment with Retro-2 modulates localization of ErbB-2 and proliferation in TNBC. We found that Retro-2 treatment decreased the levels of both WT ErbB-2 and isoform c in the nucleus of TNBC cells demonstrating that Retro-2 effects are not limited to a particular ErbB-2 isoform. Indeed, immunofluorescence assays revealed accumulation of ErbB-2 in the Golgi after Retro-2 treatment further preventing its sorting to the ER. We previously demonstrated that growth factors induce ErbB-2 migration into the nucleus in ErbB-2-positive BC cells. Consistently, we observed that Retro-2 prevents growth factor-induced NErbB-2 in ErbB-2-positive BC cells. Retro-2 treatment resulted in a dose-dependent decrease in cell proliferation in a panel of TNBC cells, whilst did not inhibit cell proliferation in the ErbB-2-negative MCF10A normal breast cell line. Moreover, disruption of retrograde transport by Retro-2 decreased the expression of cell cycle related NErbB-2 target genes (i.e. Erk5 and cyclin D1) therefore inducing cell cycle arrest at the G0/G1 phase. Most importantly, Retro-2 excluded ErbB-2 from the nucleus and abrogated tumor growth in preclinical models of TNBC. Collectively, our findings reveal Retro-2, a non-toxic inhibitor of the retrograde transport route, as a candidate novel therapeutic agent for TNBC based on its ability to evict ErbB-2 from the nucleus and to abrogate TNBC growth.

Cooperation of the IFT-A complex with the IFT-B complex is required for ciliary retrograde protein trafficking and GPCR import

Little is known about how the IFT-A and IFT-B complexes of the intraflagellar transport machinery cooperate to mediate ciliary protein trafficking. We show that IFT144-IFT122 and IFT88-IFT52-IFT46 contribute to the IFT-A-IFT-B interface and that the interaction of IFT-A with IFT-B is required for retrograde trafficking of the IFT machinery and ciliary entry of G protein-coupled receptors.