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Fanconi anemia (FA) is the most common inherited bone marrow failure (BMF) syndrome. Impaired DNA interstrand crosslink (ICL) repair is the underlying mechanism for BMF in FA. FA patients usually develop BMF during the first decade of life, prior to any known exposure to exogenous crosslinking agents. Therefore, endogenous sources of DNA damage are most likely to play an important role in the pathogenesis of FA. Metabolic by-products, such as reactive aldehydes, have been implicated in the acceleration of BMF or leukemia in both humans and mice lacking the ICL repair pathway. However, the potential contribution of other detoxifying metabolic enzymes to genome maintenance has not been systematically investigated. Identification of all sources of endogenous DNA damage will allow us to develop novel strategies to prevent DNA damage from arising, and possibly prevent BMF and leukemia in FA as well as other BMF syndromes. To determine whether detoxifying enzymes other than ALDH2 or ADH5 play a role in the protection of HSPC, we performed a metabolism-focused CRISPR/Cas9 synthetic lethality screen (3000 metabolism genes, 10 sgRNA per gene), using wild-type and FANCD2-/- Jurkat cells. From the screen, we identified ALDH9A1 as the most significantly depleted gene in FANCD2-/- Jurkat cells compared with wild-type. Eight out of ten sgALDH9A1 were significantly depleted in FANCD2-/- Jurkat cells, indicating robust effect of the ALDH9A1 knockout. ADH5, a known synthetic lethal gene with FA, was also depleted in FANCD2-/- Jurkat cells, but to a lesser degree. In vitro fluorescence-based competition assay confirmed synthetic lethal interaction between the two genes, in two independent FANCD2-/- Jurkat clones. To determine whether ALDH9A1 deficiency also caused cell death in FA-deficient human hematopoietic stem progenitor cells (HSPCs), we performed an in vitro validation assay using human umbilical cord blood (UCB). UCB CD34+ cells were edited by ribonucleoprotein delivery of Cas9 and sgRNA, in which either of sgCTRL, sgFANCD2 or sgALDH9A1, or both sgFANCD2 and sgALDH9A1 were used. Edited cells were grown on methylcellulose for 10 to 14 days, after which individual colonies were scored and harvested for sequencing. While CD34+ cells that were targeted by both sgFANCD2 and sgALDH9A1 (double KO) achieved lower editing efficiency for each gene compared with cells targeted by single guides, they produced the fewest hematopoietic colonies and the lowest frequency of GEMM (Granulocyte, Erythrocyte, Macrophage and Megakaryocyte) colonies. We observed fewer colonies targeted for both genes (biallelic double KO; observed to expected ratio 0.33) as compared to either single gene KO. These results suggest that loss of ALDH9A1 is deleterious in FANCD2-deficient HSPC. Lastly, we generated a Fanca-/-Aldh9a1-/- mouse model to examine the in vivo hematopoietic phenotype due to increased endogenous aldehydes. These mice were born at the Mendelian ratio without significant anomalies except rare cases of eye abnormalities. At three months of life, Fanca-/-Aldh9a1-/- mice had lower platelet counts than wild-type, Fanca-/- or Aldh9a1-/- control mice, but total white blood counts and hemoglobin levels were similar between groups. A follow-up result of this mouse model will be presented at the meeting. In conclusion, we identified that cells with ALDH9A1 deficiency require the FA pathway for survival. ALDH9A1 may protect human and mouse HSPC that are deficient in the FA pathway from DNA damage and cell death. Disclosures No relevant conflicts of interest to declare.

Summary Nutrient deprivation is a stimulus shared by both autophagy and the formation of primary cilia. The recently discovered role of primary cilia in nutrient sensing and signaling motivated us to explore the possible functional interactions between this signaling hub and autophagy. Here we show that part of the molecular machinery involved in ciliogenesis also participates in the early steps of the autophagic process. Signaling from the cilia, such as that from the Hedgehog pathway, induces autophagy by acting directly on essential autophagy-related proteins strategically located in the base of the cilium by ciliary trafficking proteins. While abrogation of ciliogenesis partially inhibits autophagy, blockage of autophagy enhances primary cilia growth and cilia-associated signaling during normal nutritional conditions. We propose that basal autophagy regulates ciliary growth through the degradation of proteins required for intraflagellar transport. Compromised ability to activate the autophagic response may underlie the basis of some common ciliopathies.

Chaperone-mediated autophagy (CMA) is a selective mechanism for the degradation of cytosolic proteins in lysosomes that contributes to cellular quality control and becomes an additional source of amino acids when nutrients are scarce. A chaperone complex delivers CMA substrates to a receptor protein at the lysosomal membrane that assembles into multimeric translocation complexes. However, the mechanisms regulating this process remain, for the most part, unknown. In this work, we have identified two regulatory proteins, GFAP and EF1alpha, that mediate a previously unknown inhibitory effect of GTP on CMA. GFAP stabilizes the multimeric translocation complex against chaperone-mediated disassembly, whereas GTP-mediated release of EF1alpha from the lysosomal membrane promotes self-association of GFAP, disassembly of the CMA translocation complex, and the consequent decrease in CMA. The dynamic interactions of these two proteins at the lysosomal membrane unveil now a role for GTP as a negative regulator of CMA.

Corrigendum: Annexin A2 promotes phagophore assembly by enhancing Atg16 + vesicle biogenesis and homotypic fusion

Lipid modifications are essential in cellular sorting and trafficking inside cells. The role of phosphoinositides in trafficking between Golgi and endocytic/lysosomal compartments has been extensively explored and the kinases responsible for these lipid changes have been identified. In contrast, the mechanisms that mediate exit and recycling from lysosomes (Lys), considered for a long time as terminal compartments, are less understood. In this work, we identify a dynamic association of the lipid kinase PI4KIIIβ with Lys and unveil its regulatory function in lysosomal export and retrieval. We have found that absence of PI4KIIIβ leads to abnormal formation of tubular structures from the lysosomal surface and loss of lysosomal constituents through these tubules. We demonstrate that the kinase activity of PI4KIIIβ is necessary to prevent this unwanted lysosomal efflux under normal conditions, and to facilitate proper sorting when recycling of lysosomal material is needed, such as in the physiological context of lysosomal reformation after prolonged starvation.

Autophagy is a process traditionally known to contribute to cellular cleaning through the removal of intracellular components in lysosomes. In recent years, the intensive scrutiny that autophagy has been subjected to at the molecular level, has also contributed to expand our understanding of the physiological role of this pathway. Added to the well-characterized role in quality control, autophagy has proven important in the maintenance of cellular homeostasis and of the energetic balance, in cellular and tissue remodeling and in the cellular defense against extracellular insults and pathogens. It is not a surprise that in light of this growing number of physiological functions, connections between autophagic malfunctioning and human pathologies have also been strengthened. In this review, we focus on several pathological conditions associated to primary or secondary defects in autophagy, and comment on a recurring theme for many of them, that is the fact that autophagy can often exert both beneficial and aggravating effects on the progression of disease. Elucidating the factors that determine the switch between these dual functions of autophagy in disease has become a priority when considering the potential therapeutic implications of the pharmacological modulation of autophagy in many of these pathological conditions.

Chaperone-mediated autophagy (CMA) is an intracellular catabolic pathway that mediates the degradation of a selective subset of cytosolic proteins in lysosomes ([Dice, 2007][1]; [Cuervo, 2010][2]; [Kon and Cuervo, 2010][3]; [Orenstein and Cuervo, 2010][4]). The term autophagy (or self-eating) is