Subtractive CRISPR screen identifies the ATG16L1/vacuolar ATPase axis as required for non-canonical LC3 lipidation.

Although commonly associated with autophagosomes, LC3 can also be recruited to membranes by covalent lipidation in a variety of non-canonical contexts. These include responses to ionophores such as the M2 proton channel of influenza A virus. We report a subtractive CRISPR screen that identifies factors required for non-canonical LC3 lipidation. As well as the enzyme complexes directly responsible for LC3 lipidation in all contexts, we show the RALGAP complex is important for M2-induced, but not ionophore drug-induced, LC3 lipidation. In contrast, ATG4D is responsible for LC3 recycling in M2-induced and basal LC3 lipidation. Identification of a vacuolar ATPase subunit in the screen suggests a common mechanism for non-canonical LC3 recruitment. Influenza-induced and ionophore drug-induced LC3 lipidation lead to association of the vacuolar ATPase and ATG16L1 and can be antagonized by Salmonella SopF. LC3 recruitment to erroneously neutral compartments may therefore represent a response to damage caused by diverse invasive pathogens.

The Cellular Origins of Breast Implant-Associated Anaplastic Large Cell Lymphoma (BIA-ALCL): Implications for Immunogenesis.

The exact cellular origins of most malignancies are unknown, largely because of the complex nature of malignancies, and because the potential vast number of pathways towards transformation are difficult to discern from established growths. This is compounded by the fact that cancer cells have evolved rather than being the consequence of a design process, with most data collected from (sometimes epidemiological) studies of large numbers of related malignancies. In the case of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL), the relative rarity of this disease, coupled with limited insight into its biological basis, have hampered progress. The known facts that are holding up as our knowledge increases with rising incidences are that most cases have been reported in the context of textured breast implants, although not all women with these implants develop BIA-ALCL, and cure for early-stage disease (accounting for the majority of patients) can be achieved via complete capsulectomy and implant removal. However, some theories can be gleaned from the limited biological studies conducted to date whereby a T-helper cell derivation is implicated, with its specific and apparent subset of origin dependent on, and shaped by, a number of factors, including the inflammatory microenvironment (the presence of other inflammatory cell types), the driving antigen (bacterial and/or synthetic), the acquisition of driving oncogenic events, and the inherent genetics/health status of the patient.

Aberrant anaplastic lymphoma kinase activity induces a p53 and Rb-dependent senescence-like arrest in the absence of detectable p53 stabilization.

Anaplastic Lymphoma Kinase (ALK) is a receptor tyrosine kinase aberrantly expressed in a variety of tumor types, most notably in Anaplastic Large Cell Lymphoma (ALCL) where a chromosomal translocation generates the oncogenic fusion protein, Nucleophosmin-ALK (NPM-ALK). Whilst much is known regarding the mechanism of transformation by NPM-ALK, the existence of cellular defence pathways to prevent this pathological process has not been investigated. Employing the highly tractable primary murine embryonic fibroblast (MEF) system we show that cellular transformation is not an inevitable consequence of NPM-ALK activity but is combated by p53 and Rb. Activation of p53 and/or Rb by NPM-ALK triggers a potent proliferative block with features reminiscent of senescence. While loss of p53 alone is sufficient to circumvent NPM-ALK-induced senescence and permit cellular transformation, sole loss of Rb permits continued proliferation but not transformation due to p53-imposed restraints. Furthermore, NPM-ALK attenuates p53 activity in an Rb and MDM2 dependent manner but this activity is not sufficient to bypass senescence. These data indicate that senescence may constitute an effective barrier to ALK-induced malignancies that ultimately must be overcome for tumor development.

NPM-ALK inhibits the p53 tumor suppressor pathway in an MDM2 and JNK-dependent manner.

Anaplastic large cell lymphoma (ALCL) is characterized by the presence of the t(2;5)(p23;q35) generating the nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion protein, a hyperactive kinase with transforming properties. Among these properties is the ability to regulate activity of the p53 tumor suppressor protein. In many human cancers, p53 is inactivated by mutation or other means, in some cases as a result of up-regulation of the negative regulator MDM2. However, the majority of ALK-expressing ALCL carry wild-type p53 and do not over express MDM2. We demonstrate a novel p53-dependent pathogenetic mechanism in ALK-expressing lymphoma. We confirm previously published reports of NPM-ALK-induced activation of the phosphoinositide (PI) 3-kinase and Jun N-terminal kinase (JNK) stress-activated protein (SAP) kinase proteins, but in this study demonstrate a role for these in the regulation of p53 activity in an intricate signaling system. Specifically, constitutive ALK signaling leads to the functional inactivation and/or degradation of p53 in JNK and MDM2 dependent manners. We also show nuclear exclusion of p53 in a PI 3-kinase-dependent manner. Furthermore, we demonstrate that reactivation of p53 in ALK-expressing cells as a result of pharmacologic inhibition of JNK, PI 3-kinase, and/or MDM2 activities results in the induction of apoptosis suggesting a novel therapeutic modality.