The CD33xCD123xCD70 Multispecific CD3-Engaging DARPin MP0533 Induces Selective T Cell-Mediated Killing of AML Leukemic Stem Cells.

The prognosis of patients with acute myeloid leukemia (AML) is limited, especially for elderly or unfit patients not eligible for hematopoietic stem cell (HSC) transplantation. The disease is driven by leukemic stem cells (LSCs), which are characterized by clonal heterogeneity and resistance to conventional therapy. These cells are therefore believed to be a major cause of progression and relapse. We designed MP0533, a multispecific CD3-engaging DARPin (designed ankyrin repeat protein) that can simultaneously bind to three antigens on AML cells (CD33, CD123, and CD70), aiming to enable avidity-driven T cell-mediated killing of AML cells co-expressing at least two of the antigens. In vitro, MP0533 induced selective T cell-mediated killing of AML cell lines, as well as patient-derived AML blasts and LSCs, expressing two or more target antigens, while sparing healthy HSCs, blood, and endothelial cells. The higher selectivity also resulted in markedly lower levels of cytokine release in normal human blood compared to single antigen-targeting T-cell engagers. In xenograft AML mouse models, MP0533 induced tumor-localized T-cell activation and cytokine release, leading to complete eradication of the tumors while having no systemic adverse effects. These studies show that the multispecific-targeting strategy used with MP0533 holds promise for improved selectivity towards LSCs and efficacy against clonal heterogeneity, potentially bringing a new therapeutic option to this group of patients with high unmet need. MP0533 is currently being evaluated in a dose-escalation phase 1 study in patients with relapsed or refractory AML (NCT05673057).

Circulating microRNA Analysis in a Prospective Co-clinical Trial Identifies MIR652-3p as a Response Biomarker and Driver of Regorafenib Resistance Mechanisms in Colorectal Cancer.

PURPOSE: The multi-kinase inhibitor (mKi) regorafenib has demonstrated efficacy in chemorefractory patients with metastatic colorectal cancer (mCRC). However, lack of predictive biomarkers and concerns over significant toxicities hamper the use of regorafenib in clinical practice. EXPERIMENTAL DESIGN: Serial liquid biopsies were obtained at baseline and monthly until disease progression in chemorefractory patients with mCRC treated with regorafenib in a phase II clinical trial (PROSPECT-R n = 40; NCT03010722) and in a multicentric validation cohort (n = 241). Tissue biopsies collected at baseline, after 2 months and at progression in the PROSPECT-R trial were used to establish patient-derived organoids (PDO) and for molecular analyses. MicroRNA profiling was performed on baseline bloods using the NanoString nCounter platform and results were validated by digital-droplet PCR and/or ISH in paired liquid and tissue biopsies. PDOs co-cultures and PDO-xenotransplants were generated for functional analyses. RESULTS: Large-scale microRNA expression analysis in longitudinal matched liquid and tissue biopsies from the PROSPECT-R trial identified MIR652-3p as a biomarker of clinical benefit to regorafenib. These findings were confirmed in an independent validation cohort and in a "control" group of 100 patients treated with lonsurf. Using ex vivo co-culture assays paired with single-cell RNA-sequencing of PDO established pre- and post-treatment, we modeled regorafenib response observed in vivo and in patients, and showed that MIR652-3p controls resistance to regorafenib by impairing regorafenib-induced lethal autophagy and by orchestrating the switch from neo-angiogenesis to vessel co-option. CONCLUSIONS: Our results identify MIR652-3p as a potential biomarker and as a driver of cell and non-cell-autonomous mechanisms of resistance to regorafenib.

Discovery of an In Vivo Chemical Probe for BCL6 Inhibition by Optimization of Tricyclic Quinolinones.

B-cell lymphoma 6 (BCL6) is a transcriptional repressor and oncogenic driver of diffuse large B-cell lymphoma (DLBCL). Here, we report the optimization of our previously reported tricyclic quinolinone series for the inhibition of BCL6. We sought to improve the cellular potency and in vivo exposure of the non-degrading isomer, CCT373567, of our recently published degrader, CCT373566. The major limitation of our inhibitors was their high topological polar surface areas (TPSA), leading to increased efflux ratios. Reducing the molecular weight allowed us to remove polarity and decrease TPSA without considerably reducing solubility. Careful optimization of these properties, as guided by pharmacokinetic studies, led to the discovery of CCT374705, a potent inhibitor of BCL6 with a good in vivo profile. Modest in vivo efficacy was achieved in a lymphoma xenograft mouse model after oral dosing.

Improved Binding Affinity and Pharmacokinetics Enable Sustained Degradation of BCL6 In Vivo.

The transcriptional repressor BCL6 is an oncogenic driver found to be deregulated in lymphoid malignancies. Herein, we report the optimization of our previously reported benzimidazolone molecular glue-type degrader CCT369260 to CCT373566, a highly potent probe suitable for sustained depletion of BCL6 in vivo. We observed a sharp degradation SAR, where subtle structural changes conveyed the ability to induce degradation of BCL6. CCT373566 showed modest in vivo efficacy in a lymphoma xenograft mouse model following oral dosing.

Targeting autophagy in disease: established and new strategies.

Macroautophagy/autophagy is an evolutionarily conserved pathway responsible for clearing cytosolic aggregated proteins, damaged organelles or invading microorganisms. Dysfunctional autophagy leads to pathological accumulation of the cargo, which has been linked to a range of human diseases, including neurodegenerative diseases, infectious and autoimmune diseases and various forms of cancer. Cumulative work in animal models, application of genetic tools and pharmacologically active compounds, has suggested the potential therapeutic value of autophagy modulation in disease, as diverse as Huntington, Salmonella infection, or pancreatic cancer. Autophagy activation versus inhibition strategies are being explored, while the role of autophagy in pathophysiology is being studied in parallel. However, the progress of preclinical and clinical development of autophagy modulators has been greatly hampered by the paucity of selective pharmacological agents and biomarkers to dissect their precise impact on various forms of autophagy and cellular responses. Here, we summarize established and new strategies in autophagy-related drug discovery and indicate a path toward establishing a more efficient discovery of autophagy-selective pharmacological agents. With this knowledge at hand, modern concepts for therapeutic exploitation of autophagy might become more plausible.Abbreviations: ALS: amyotrophic lateral sclerosis; AMPK: AMP-activated protein kinase; ATG: autophagy-related gene; AUTAC: autophagy-targeting chimera; CNS: central nervous system; CQ: chloroquine; GABARAP: gamma-aminobutyric acid type A receptor-associated protein; HCQ: hydroxychloroquine; LYTAC: lysosome targeting chimera; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NDD: neurodegenerative disease; PDAC: pancreatic ductal adenocarcinoma; PE: phosphatidylethanolamine; PIK3C3/VPS34: phosphatidylinositol 3-kinase catalytic subunit type 3; PtdIns3K: class III phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol 3-phosphate; PROTAC: proteolysis-targeting chimera; SARS-CoV-2: severe acute respiratory syndrome coronavirus 2; SQSTM1/p62: sequestosome 1; ULK1: unc-51 like autophagy activating kinase 1.

FGF7-FGFR2 autocrine signaling increases growth and chemoresistance of fusion-positive rhabdomyosarcomas.

Rhabdomyosarcomas are aggressive pediatric soft-tissue sarcomas and include high-risk PAX3-FOXO1 fusion-gene-positive cases. Fibroblast growth factor receptor 4 (FGFR4) is known to contribute to rhabdomyosarcoma progression; here, we sought to investigate the involvement and potential for therapeutic targeting of other FGFRs in this disease. Cell-based screening of FGFR inhibitors with potential for clinical repurposing (NVP-BGJ398, nintedanib, dovitinib, and ponatinib) revealed greater sensitivity of fusion-gene-positive versus fusion-gene-negative rhabdomyosarcoma cell lines and was shown to be correlated with high expression of FGFR2 and its specific ligand, FGF7. Furthermore, patient samples exhibit higher mRNA levels of FGFR2 and FGF7 in fusion-gene-positive versus fusion-gene-negative rhabdomyosarcomas. Sustained intracellular mitogen-activated protein kinase (MAPK) activity and FGF7 secretion into culture media during serum starvation of PAX3-FOXO1 rhabdomyosarcoma cells together with decreased cell viability after genetic silencing of FGFR2 or FGF7 was in keeping with a novel FGF7-FGFR2 autocrine loop. FGFR inhibition with NVP-BGJ398 reduced viability and was synergistic with SN38, the active metabolite of irinotecan. In vivo, NVP-BGJ398 abrogated xenograft growth and warrants further investigation in combination with irinotecan as a therapeutic strategy for fusion-gene-positive rhabdomyosarcomas.

Modulation of pancreatic cancer cell sensitivity to FOLFIRINOX through microRNA-mediated regulation of DNA damage.

FOLFIRINOX, a combination of chemotherapy drugs (Fluorouracil, Oxaliplatin, Irinotecan -FOI), provides the best clinical benefit in pancreatic ductal adenocarcinoma (PDAC) patients. In this study we explore the role of miRNAs (MIR) as modulators of chemosensitivity to identify potential biomarkers of response. We find that 41 and 84 microRNA inhibitors enhance the sensitivity of Capan1 and MiaPaCa2 PDAC cells respectively. These include a MIR1307-inhibitor that we validate in further PDAC cell lines. Chemotherapy-induced apoptosis and DNA damage accumulation are higher in MIR1307 knock-out (MIR1307KO) versus control PDAC cells, while re-expression of MIR1307 in MIR1307KO cells rescues these effects. We identify binding of MIR1307 to CLIC5 mRNA through covalent ligation of endogenous Argonaute-bound RNAs cross-linking immunoprecipitation assay. We validate these findings in an in vivo model with MIR1307 disruption. In a pilot cohort of PDAC patients undergoing FOLFIRONX chemotherapy, circulating MIR1307 correlates with clinical outcome.

Atg8-Family Proteins-Structural Features and Molecular Interactions in Autophagy and Beyond.

Autophagy is a common name for a number of catabolic processes, which keep the cellular homeostasis by removing damaged and dysfunctional intracellular components. Impairment or misbalance of autophagy can lead to various diseases, such as neurodegeneration, infection diseases, and cancer. A central axis of autophagy is formed along the interactions of autophagy modifiers (Atg8-family proteins) with a variety of their cellular counter partners. Besides autophagy, Atg8-proteins participate in many other pathways, among which membrane trafficking and neuronal signaling are the most known. Despite the fact that autophagy modifiers are well-studied, as the small globular proteins show similarity to ubiquitin on a structural level, the mechanism of their interactions are still not completely understood. A thorough analysis and classification of all known mechanisms of Atg8-protein interactions could shed light on their functioning and connect the pathways involving Atg8-proteins. In this review, we present our views of the key features of the Atg8-proteins and describe the basic principles of their recognition and binding by interaction partners. We discuss affinity and selectivity of their interactions as well as provide perspectives for discovery of new Atg8-interacting proteins and therapeutic approaches to tackle major human diseases.

Identification of ovarian high-grade serous carcinoma cell lines that show estrogen-sensitive growth as xenografts in immunocompromised mice.

Ovarian cancer remains a significant challenge in women worldwide. Tumors of the high-grade serous carcinoma (HGSC) type represent the most common form of the disease. Development of new therapies for HGSC has been hampered by a paucity of preclinical models in which new drugs could be tested for target engagement and anti-tumor efficacy. Here, we systematically assessed in vivo growth of ovarian cancer cells, including six validated HGSC cell lines, in highly immunocompromised NSG mice by varying the injection site. We found that, with the exception of OVCAR3, HGSC cell lines COV318, COV362, KURAMOCHI, OVCAR4, and OVSAHO, generally demonstrate poor growth as either subcutaneous or intraperitoneal xenografts. Intrabursal injections performed with KURAMOCHI and COV362 cells did not improve tumor growth in vivo. Additional analysis revealed that OVSAHO and COV362 express moderate levels of estrogen receptor (ERα), which translated into improved growth of xenografts in the presence of 17ß-Estradiol. Surprisingly, we also found that the growth of the widely used non-HGSC ovarian cell line SKOV3 could be significantly improved by estrogen supplementation. By describing successful establishment of estrogen-sensitive HGSC xenograft models, OVSAHO and COV362, this work will enable testing of novel therapies for this aggressive form of ovarian cancer.

Achieving In Vivo Target Depletion through the Discovery and Optimization of Benzimidazolone BCL6 Degraders.

Deregulation of the transcriptional repressor BCL6 enables tumorigenesis of germinal center B-cells, and hence BCL6 has been proposed as a therapeutic target for the treatment of diffuse large B-cell lymphoma (DLBCL). Herein we report the discovery of a series of benzimidazolone inhibitors of the protein-protein interaction between BCL6 and its co-repressors. A subset of these inhibitors were found to cause rapid degradation of BCL6, and optimization of pharmacokinetic properties led to the discovery of 5-((5-chloro-2-((3R,5S)-4,4-difluoro-3,5-dimethylpiperidin-1-yl)pyrimidin-4-yl)amino)-3-(3-hydroxy-3-methylbutyl)-1-methyl-1,3-dihydro-2H-benzo[d]imidazol-2-one (CCT369260), which reduces BCL6 levels in a lymphoma xenograft mouse model following oral dosing.

Combined targeting of MEK and the glucocorticoid receptor for the treatment of RAS-mutant multiple myeloma.

BACKGROUND: Multiple myeloma (MM) remains incurable despite recent therapeutic advances. RAS mutations are frequently associated with relapsed/refractory disease. Efforts to target the mitogen-activated protein kinase (MAPK) pathway with the MEK inhibitor, trametinib (Tra) have been limited by toxicities and the development of resistance. Dexamethasone (Dex) is a corticosteroid commonly used in clinical practice, to enhance efficacy of anti-myeloma therapy. Therefore, we hypothesised that the combination of Tra and Dex would yield synergistic activity in RAS-mutant MM. METHODS: The response of human MM cell lines to drug treatment was analysed using cell proliferation assays, Western blotting, Annexin V and propidium iodide staining by flow cytometry and reverse phase protein arrays. The efficacy of trametinib and dexamethasone treatment in the MM.1S xenograft model was assessed by measuring tumor volume over time. RESULTS: The Tra/Dex combination demonstrated synergistic cytotoxicity in KRASG12A mutant lines MM.1S and RPMI-8226. The induction of apoptosis was associated with decreased MCL-1 expression and increased BIM expression. Reverse phase proteomic arrays revealed suppression of FAK, PYK2, FLT3, NDRG1 and 4EBP1 phosphorylation with the Tra/Dex combination. Notably, NDRG1 expression was associated with the synergistic response to Tra/Dex. MM cells were sensitive to PDK1 inhibition and IGF1-induced signalling partially protected from Tra/Dex treatment, highlighting the importance of this pathway. In the MM.1S tumor xenograft model, only the combination of Tra/Dex resulted in a significant inhibition of tumor growth. CONCLUSIONS: Overall Tra/Dex demonstrates antiproliferative activity in RAS-mutant MM cell lines associated with suppression of pro-survival PDK1 signalling and engagement of apoptotic pathways. Our data support further investigation of this combination in RAS-mutant MM.

Theranostic Attributes of Acoustic Cluster Therapy and Its Use for Enhancing the Effectiveness of Liposomal Doxorubicin Treatment of Human Triple Negative Breast Cancer in Mice.

INTRODUCTION: Acoustic cluster therapy (ACT) comprises co-administration of a formulation containing microbubble/microdroplet clusters (PS101), together with a regular medicinal drug (e.g., a chemotherapeutic) and local ultrasound (US) insonation of the targeted pathological tissue (e.g., the tumor). PS101 is confined to the vascular compartment and, when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase-shift to produce bubbles with a median diameter of 22 µm when unconstrained by the capillary wall. In vivo these bubbles transiently lodge in the tumor's microvasculature. Low frequency ultrasound (300 kHz) at a low mechanical index (MI = 0.15) is then applied to drive oscillations of the deposited ACT bubbles to induce a range of biomechanical effects that locally enhance extravasation, distribution, and uptake of the co-administered drug, significantly increasing its therapeutic efficacy. METHODS: In this study we investigated the therapeutic efficacy of ACT with liposomal doxorubicin for the treatment of triple negative breast cancer using orthotopic human tumor xenografts (MDA-MB-231-H.luc) in athymic mice (ICR-NCr-Foxn1nu). Doxil® (6 mg/kg, i.v.) was administered at days 0 and 21, each time immediately followed by three sequential ACT (20 ml/kg PS101) treatment procedures (n = 7-10). B-mode and nonlinear ultrasound images acquired during the activation phase were correlated to the therapeutic efficacy. RESULTS: Results show that combination with ACT induces a strong increase in the therapeutic efficacy of Doxil®, with 63% of animals in complete, stable remission at end of study, vs. 10% for Doxil® alone (p < 0.02). A significant positive correlation (p < 0.004) was found between B-mode contrast enhancement during ACT activation and therapy response. These observations indicate that ACT may also be used as a theranostic agent and that ultrasound contrast enhancement during or before ACT treatment may be employed as a biomarker of therapeutic response during clinical use.

Pharmacologic Inhibitor of DNA-PK, M3814, Potentiates Radiotherapy and Regresses Human Tumors in Mouse Models.

Physical and chemical DNA-damaging agents are used widely in the treatment of cancer. Double-strand break (DSB) lesions in DNA are the most deleterious form of damage and, if left unrepaired, can effectively kill cancer cells. DNA-dependent protein kinase (DNA-PK) is a critical component of nonhomologous end joining (NHEJ), one of the two major pathways for DSB repair. Although DNA-PK has been considered an attractive target for cancer therapy, the development of pharmacologic DNA-PK inhibitors for clinical use has been lagging. Here, we report the discovery and characterization of a potent, selective, and orally bioavailable DNA-PK inhibitor, M3814 (peposertib), and provide in vivo proof of principle for DNA-PK inhibition as a novel approach to combination radiotherapy. M3814 potently inhibits DNA-PK catalytic activity and sensitizes multiple cancer cell lines to ionizing radiation (IR) and DSB-inducing agents. Inhibition of DNA-PK autophosphorylation in cancer cells or xenograft tumors led to an increased number of persistent DSBs. Oral administration of M3814 to two xenograft models of human cancer, using a clinically established 6-week fractionated radiation schedule, strongly potentiated the antitumor activity of IR and led to complete tumor regression at nontoxic doses. Our results strongly support DNA-PK inhibition as a novel approach for the combination radiotherapy of cancer. M3814 is currently under investigation in combination with radiotherapy in clinical trials.

De novo phosphatidylcholine synthesis is required for autophagosome membrane formation and maintenance during autophagy.

Macroautophagy/autophagy can enable cancer cells to withstand cellular stress and maintain bioenergetic homeostasis by sequestering cellular components into newly formed double-membrane vesicles destined for lysosomal degradation, potentially affecting the efficacy of anti-cancer treatments. Using 13C-labeled choline and 13C-magnetic resonance spectroscopy and western blotting, we show increased de novo choline phospholipid (ChoPL) production and activation of PCYT1A (phosphate cytidylyltransferase 1, choline, alpha), the rate-limiting enzyme of phosphatidylcholine (PtdCho) synthesis, during autophagy. We also discovered that the loss of PCYT1A activity results in compromised autophagosome formation and maintenance in autophagic cells. Direct tracing of ChoPLs with fluorescence and immunogold labeling imaging revealed the incorporation of newly synthesized ChoPLs into autophagosomal membranes, endoplasmic reticulum (ER) and mitochondria during anticancer drug-induced autophagy. Significant increase in the colocalization of fluorescence signals from the newly synthesized ChoPLs and mCherry-MAP1LC3/LC3 (microtubule-associated protein 1 light chain 3) was also found on autophagosomes accumulating in cells treated with autophagy-modulating compounds. Interestingly, cells undergoing active autophagy had an altered ChoPL profile, with longer and more unsaturated fatty acid/alcohol chains detected. Our data suggest that de novo synthesis may be required to increase autophagosomal ChoPL content and alter its composition, together with replacing phospholipids consumed from other organelles during autophagosome formation and turnover. This addiction to de novo ChoPL synthesis and the critical role of PCYT1A may lead to development of agents targeting autophagy-induced drug resistance. In addition, fluorescence imaging of choline phospholipids could provide a useful way to visualize autophagosomes in cells and tissues. ABBREVIATIONS: AKT: AKT serine/threonine kinase; BAX: BCL2 associated X, apoptosis regulator; BECN1: beclin 1; ChoPL: choline phospholipid; CHKA: choline kinase alpha; CHPT1: choline phosphotransferase 1; CTCF: corrected total cell fluorescence; CTP: cytidine-5'-triphosphate; DCA: dichloroacetate; DMEM: dulbeccos modified Eagles medium; DMSO: dimethyl sulfoxide; EDTA: ethylenediaminetetraacetic acid; ER: endoplasmic reticulum; GDPD5: glycerophosphodiester phosphodiesterase domain containing 5; GFP: green fluorescent protein; GPC: glycerophosphorylcholine; HBSS: hanks balances salt solution; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LPCAT1: lysophosphatidylcholine acyltransferase 1; LysoPtdCho: lysophosphatidylcholine; MRS: magnetic resonance spectroscopy; MTORC1: mechanistic target of rapamycin kinase complex 1; PCho: phosphocholine; PCYT: choline phosphate cytidylyltransferase; PLA2: phospholipase A2; PLB: phospholipase B; PLC: phospholipase C; PLD: phospholipase D; PCYT1A: phosphate cytidylyltransferase 1, choline, alpha; PI3K: phosphoinositide-3-kinase; pMAFs: pancreatic mouse adult fibroblasts; PNPLA6: patatin like phospholipase domain containing 6; Pro-Cho: propargylcholine; Pro-ChoPLs: propargylcholine phospholipids; PtdCho: phosphatidylcholine; PtdEth: phosphatidylethanolamine; PtdIns3P: phosphatidylinositol-3-phosphate; RPS6: ribosomal protein S6; SCD: stearoyl-CoA desaturase; SEM: standard error of the mean; SM: sphingomyelin; SMPD1/SMase: sphingomyelin phosphodiesterase 1, acid lysosomal; SGMS: sphingomyelin synthase; WT: wild-type.

An atypical LIR motif within UBA5 (ubiquitin like modifier activating enzyme 5) interacts with GABARAP proteins and mediates membrane localization of UBA5.

Short linear motifs, known as LC3-interacting regions (LIRs), interact with mactoautophagy/autophagy modifiers (Atg8/LC3/GABARAP proteins) via a conserved universal mechanism. Typically, this includes the occupancy of 2 hydrophobic pockets on the surface of Atg8-family proteins by 2 specific aromatic and hydrophobic residues within the LIR motifs. Here, we describe an alternative mechanism of Atg8-family protein interaction with the non-canonical UBA5 LIR, an E1-like enzyme of the ufmylation pathway that preferentially interacts with GABARAP but not LC3 proteins. By solving the structures of both GABARAP and GABARAPL2 in complex with the UBA5 LIR, we show that in addition to the binding to the 2 canonical hydrophobic pockets (HP1 and HP2), a conserved tryptophan residue N-terminal of the LIR core sequence binds into a novel hydrophobic pocket on the surface of GABARAP proteins, which we term HP0. This mode of action is unique for UBA5 and accompanied by large rearrangements of key residues including the side chains of the gate-keeping K46 and the adjacent K/R47 in GABARAP proteins. Swapping mutations in LC3B and GABARAPL2 revealed that K/R47 is the key residue in the specific binding of GABARAP proteins to UBA5, with synergetic contributions of the composition and dynamics of the loop L3. Finally, we elucidate the physiological relevance of the interaction and show that GABARAP proteins regulate the localization and function of UBA5 on the endoplasmic reticulum membrane in a lipidation-independent manner.Abbreviations: ATG: AuTophaGy-related; EGFP: enhanced green fluorescent protein; GABARAP: GABA-type A receptor-associated protein; ITC: isothermal titration calorimetry; KO: knockout; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NMR: nuclear magnetic resonance; RMSD: root-mean-square deviation of atomic positions; TKO: triple knockout; UBA5: ubiquitin like modifier activating enzyme 5.

History of the Selective Autophagy Research: How Did It Begin and Where Does It Stand Today?

Autophagy, self-eating, is a pivotal catabolic mechanism that ensures homeostasis and survival of the cell in the face of stressors as different as starvation, infection, or protein misfolding. The importance of the research in this field was recognized by the general public after the Nobel Prize for Physiology or Medicine was awarded in 2016 to Yoshinori Ohsumi for discoveries of the mechanisms of autophagy using yeast as a model organism. One of the seminal findings of Ohsumi was on the role ubiquitin-like proteins (UBLs)-Atg5, Atg12, and Atg8-play in the formation of the double-membrane vesicle autophagosome, which is the functional unit of autophagy. Subsequent work by several groups demonstrated that, like the founding member of the UBL family ubiquitin, these small but versatile protein and lipid modifiers interact with a plethora of proteins, which either directly regulate autophagosome formation, for example, components of the Atg1/ULK1 complex, or are involved in cargo recognition, for example, Atg19 and p62/SQSTM1. By tethering the cargo to the UBLs present on the forming autophagosome, the latter proteins were proposed to effectively act as selective autophagy receptors. The discovery of the selective autophagy receptors brought a breakthrough in the autophagy field, supplying the mechanistic underpinning for the formation of an autophagosome selectively around the cytosolic cargo, that is, a protein aggregate, a mitochondrion, or a cytosolic bacterium. In this historical overview, I highlight key steps that the research into selective autophagy has been taking over the past 20 years. I comment on their significance and discuss current challenges in developing more detailed knowledge of the mechanisms of selective autophagy. I will conclude by introducing the new directions that this dynamic research field is taking into its third decade.

Selective Autophagy Receptors in Neuronal Health and Disease.

Neurons are electrically excitable, postmitotic cells that perform sensory, relaying, and motor functions. Because of their unique morphological and functional specialization, cells of this type are sensitive to the stress caused by accumulation of misfolded proteins or damaged organelles. Autophagy is the fundamental mechanism that ensures sequestration of cytosolic material and its subsequent degradation in lysosomes of eukaryotic cells, thereby providing cell-autonomous nutrients and removing harmful cargos. Strikingly, mice and flies lacking functional autophagy develop early onset progressive neurodegeneration. Like in human neurodegenerative diseases (NDDs)-Alzheimer's disease, Parkinson's disease, frontotemporal dementia, Huntington's disease, and amyotrophic lateral sclerosis-characteristic protein aggregates observed in autophagy-deficient neurons in the animal models are indicators of the ongoing neuronal pathology. A number of selective autophagy receptors (SARs) have been characterized that interact both with the cargo and components of the autophagic machinery, thus providing the molecular basis for selective degradation of sizable cytosolic components. Interference with autophagy in experimental models, but also during the pathological vagaries in neurons, will thus have far-reaching consequences for a range of selective autophagy pathways critical for the normal functioning of the nervous system. Here, we review the key principles behind the selective autophagy and discuss how the SARs may be involved in the pathogenesis of NDDs. Using recently published examples, we also examine the emerging role of less well studied selective autophagy pathways in neuronal health and disease. We conclude by discussing targeting selective autophagy as an emerging therapeutic modality in NDDs.

Modulation of Biliary Cancer Chemo-Resistance Through MicroRNA-Mediated Rewiring of the Expansion of CD133+ Cells.

BACKGROUND AND AIMS: Changes in single microRNA (miRNA) expression have been associated with chemo-resistance in biliary tract cancers (BTCs). However, a global assessment of the dynamic role of the microRNome has never been performed to identify potential therapeutic targets that are functionally relevant in the BTC cell response to chemotherapy. APPROACH AND RESULTS: High-throughput screening (HTS) of 997 locked nucleic acid miRNA inhibitors was performed in six cholangiocarcinoma cell lines treated with cisplatin and gemcitabine (CG) seeking changes in cell viability. Validation experiments were performed with mirVana probes. MicroRNA and gene expression was assessed by TaqMan assay, RNA-sequencing, and in situ hybridization in four independent cohorts of human BTCs. Knockout of microRNA was achieved by CRISPR-CAS9 in CCLP cells (MIR1249KO) and tested for effects on chemotherapy sensitivity in vitro and in vivo. HTS revealed that MIR1249 inhibition enhanced chemotherapy sensitivity across all cell lines. MIR1249 expression was increased in 41% of cases in human BTCs. In validation experiments, MIR1249 inhibition did not alter cell viability in untreated or dimethyl sulfoxide-treated cells; however, it did increase the CG effect. MIR1249 expression was increased in CD133+ biliary cancer cells freshly isolated from the stem cell niche of human BTCs as well as in CD133+ chemo-resistant CCLP cells. MIR1249 modulated the chemotherapy-induced enrichment of CD133+ cells by controlling their clonal expansion through the Wnt-regulator FZD8. MIR1249KO cells had impaired expansion of the CD133+ subclone and its enrichment after chemotherapy, reduced expression of cancer stem cell markers, and increased chemosensitivity. MIR1249KO xenograft BTC models showed tumor shrinkage after exposure to weekly CG, whereas wild-type models showed only stable disease over treatment. CONCLUSIONS: MIR1249 mediates resistance to CG in BTCs and may be tested as a target for therapeutics.

Therapeutic Dose Response of Acoustic Cluster Therapy in Combination With Irinotecan for the Treatment of Human Colon Cancer in Mice.

Introduction: Acoustic Cluster Therapy (ACT) comprises coadministration of a formulation containing microbubble-microdroplet clusters (PS101) together with a regular medicinal drug and local ultrasound (US) insonation of the targeted pathological tissue. PS101 is confined to the vascular compartment and when the clusters are exposed to regular diagnostic imaging US fields, the microdroplets undergo a phase shift to produce bubbles with a median diameter of 22 µm. Low frequency, low mechanical index US is then applied to drive oscillations of the deposited ACT bubbles to induce biomechanical effects that locally enhance extravasation, distribution, and uptake of the coadministered drug, significantly increasing its therapeutic efficacy. Methods: The therapeutic efficacy of ACT with irinotecan (60 mg/kg i.p.) was investigated using three treatment sessions given on day 0, 7, and 14 on subcutaneous human colorectal adenocarcinoma xenografts in mice. Treatment was performed with three back-to-back PS101+US administrations per session with PS101 doses ranging from 0.40-2.00 ml PS101/kg body weight (n = 8-15). To induce the phase shift, 45 s of US at 8 MHz at an MI of 0.30 was applied using a diagnostic US system; low frequency exposure consisted of 1 or 5 min at 500 kHz with an MI of 0.20. Results: ACT with irinotecan induced a strong, dose dependent increase in the therapeutic effect (R2 = 0.95). When compared to irinotecan alone, at the highest dose investigated, combination treatment induced a reduction in average normalized tumour volume from 14.6 (irinotecan), to 5.4 (ACT with irinotecan, p = 0.002) on day 27. Median survival increased from 34 days (irinotecan) to 54 (ACT with irinotecan, p = 0.002). Additionally, ACT with irinotecan induced an increase in the fraction of complete responders; from 7% to 26%. There was no significant difference in the therapeutic efficacy whether the low frequency US lasted 1 or 5 min. Furthermore, there was no significant difference between the enhancement observed in the efficacy of ACT with irinotecan when PS101+US was administered before or after irinotecan. An increase in early dropouts was observed at higher PS101 doses. Both mean tumour volume (on day 27) and median survival indicate that the PS101 dose response was linear in the range investigated.

A Diversity of Selective Autophagy Receptors Determines the Specificity of the Autophagy Pathway.

The clearance of surplus, broken, or dangerous components is key for maintaining cellular homeostasis. The failure to remove protein aggregates, damaged organelles, or intracellular pathogens leads to diseases, including neurodegeneration, cancer, and infectious diseases. Autophagy is the evolutionarily conserved pathway that sequesters cytoplasmic components in specialized vesicles, autophagosomes, which transport the cargo to the degradative compartments (vacuoles or lysosomes). Research during the past few decades has elucidated how autophagosomes engulf their substrates selectively. This type of autophagy involves a growing number of selective autophagy receptors (SARs) (e.g., Atg19 in yeasts, p62/SQSTM1 in mammals), which bind to the cargo and simultaneously engage components of the core autophagic machinery via direct interaction with the ubiquitin-like proteins (UBLs) of the Atg8/LC3/GABARAP family and adaptors, Atg11 (in yeasts) or FIP200 (in mammals). In this Review, we critically discuss the biology of the SARs with special emphasis on their interactions with UBLs.