Annexin A6 and NPC1 regulate LDL-inducible cell migration and distribution of focal adhesions

Cholesterol is considered indispensable for cell motility, but how physiological cholesterol pools enable cells to move forward remains to be clarified. The majority of cells obtain cholesterol from the uptake of Low-Density lipoproteins (LDL) and here we demonstrate that LDL stimulates A431 squamous epithelial carcinoma and Chinese hamster ovary (CHO) cell migration and invasion. LDL also potentiated epidermal growth factor (EGF) -stimulated A431 cell migration as well as A431 invasion in 3-dimensional environments, using organotypic assays. Blocking cholesterol export from late endosomes (LE), using Niemann Pick Type C1 (NPC1) mutant cells, pharmacological NPC1 inhibition or overexpression of the annexin A6 (AnxA6) scaffold protein, compromised LDL-inducible migration and invasion. Nevertheless, NPC1 mutant cells established focal adhesions (FA) that contain activated focal adhesion kinase (pY397FAK, pY861FAK), vinculin and paxillin. Compared to controls, NPC1 mutants display increased FA numbers throughout the cell body, but lack LDL-inducible FA formation at cell edges. Strikingly, AnxA6 depletion in NPC1 mutant cells, which restores late endosomal cholesterol export in these cells, increases their cell motility and association of the cholesterol biosensor D4H with active FAK at cell edges, indicating that AnxA6-regulated transport routes contribute to cholesterol delivery to FA structures, thereby improving NPC1 mutant cell migratory behaviour.

Synthetic lethal screening identifies existing drugs with selective viability effects on Neurofibromatosis type-1 model systems

Neurofibromatosis type 1 (NF1) is a genetic multi-system disorder. Symptoms include near universal benign neurofibromas, as well as malignant tumours, including generally fatal malignant peripheral nerve sheath tumours. There are limited therapies for any NF1-associated tumours; therefore, there is a clear clinical need to discover new drugs that specifically target NF1-deficient tumour cells. Using a Drosophila NF1-KO cell model, we used synthetic lethal screening to identify candidate drug targets for NF1-deficient tumours and performed statistical enrichment analysis to identify further targets. We then assessed the top 72 candidate synthetic lethal partner genes to NF1 using Variable Dose Analysis, resulting in 15 candidate genes that decreased NF1-KO viability by >10% and were novel druggable targets for NF1. Autophagy inhibitors Chloroquine (CQ) and Bafilomycin A1 resulted in a significant reduction in NF1-KO cell viability, which was conserved in a panel of human NF1 mutant cell lines. AZT and Enzalutamide also selectively reduced NF1 mutant cell viability in human cell lines. Furthermore, the effect of CQ was conserved in a Drosophila NF1-mutant in vivo model. This study highlights two key points: 1) The use of Drosophila cells as a model to screen for drugs specifically targeting NF1 mutant cells was highly successful as candidate interactions were conserved across a panel of human NF1 mutant cells and an in vivo fly NF1 mutant model, and 2) NF1-deficient cells have vulnerability to disruption of the autophagy pathway, telomerase activity, and AR activity. These pathways/drugs represent promising targets for the potential treatment of NF1-associated tumours.

SF3B1 mutant-induced missplicing of MAP3K7 causes anemia in myelodysplastic syndromes

SF3B1 is the most frequently mutated RNA splicing factor in cancer, including in ∼25% of myelodysplastic syndromes (MDS) patients. SF3B1-mutated MDS, which is strongly associated with ringed sideroblast morphology, is characterized by ineffective erythropoiesis, leading to severe, often fatal anemia. However, functional evidence linking SF3B1 mutations to the anemia described in MDS patients harboring this genetic aberration is weak, and the underlying mechanism is completely unknown. Using isogenic SF3B1 WT and mutant cell lines, normal human CD34 cells, and MDS patient cells, we define a previously unrecognized role of the kinase MAP3K7, encoded by a known mutant SF3B1-targeted transcript, in controlling proper terminal erythroid differentiation, and show how MAP3K7 missplicing leads to the anemia characteristic of SF3B1-mutated MDS, although not to ringed sideroblast formation. We found that p38 MAPK is deactivated in SF3B1 mutant isogenic and patient cells and that MAP3K7 is an upstream positive effector of p38 MAPK. We demonstrate that disruption of this MAP3K7-p38 MAPK pathway leads to premature down-regulation of GATA1, a master regulator of erythroid differentiation, and that this is sufficient to trigger accelerated differentiation, erythroid hyperplasia, and ultimately apoptosis. Our findings thus define the mechanism leading to the severe anemia found in MDS patients harboring SF3B1 mutations.

Requirement for an Otopetrin-like protein for acid taste in Drosophila

Receptors for bitter, sugar, and other tastes have been identified in the fruit fly Drosophila melanogaster, while a broadly tuned receptor for the taste of acid has been elusive. Previous work showed that such a receptor was unlikely to be encoded by a gene within one of the two major families of taste receptors in Drosophila, the “gustatory receptors” and “ionotropic receptors.” Here, to identify the acid taste receptor, we tested the contributions of genes encoding proteins distantly related to the mammalian Otopertrin1 (OTOP1) proton channel that functions as a sour receptor in mice. RNA interference (RNAi) knockdown or mutation by CRISPR/Cas9 of one of the genes, Otopetrin-Like A (OtopLA), but not of the others (OtopLB or OtopLC) severely impaired the behavioral rejection to a sweet solution laced with high levels of HCl or carboxylic acids and greatly reduced acid-induced action potentials measured from taste hairs. An isoform of OtopLA that we isolated from the proboscis was sufficient to restore behavioral sensitivity and acid-induced action potential firing in OtopLA mutant flies. At lower concentrations, HCl was attractive to the flies, and this attraction was abolished in the OtopLA mutant. Cell type–specific rescue experiments showed that OtopLA functions in distinct subsets of gustatory receptor neurons for repulsion and attraction to high and low levels of protons, respectively. This work highlights a functional conservation of a sensory receptor in flies and mammals and shows that the same receptor can function in both appetitive and repulsive behaviors.

Clonal dynamics of BRAF-driven drug resistance in EGFR-mutant lung cancer

Activation of MAPK signaling via BRAF mutations may limit the activity of EGFR inhibitors in EGFR-mutant lung cancer patients. However, the impact of BRAF mutations on the selection and fitness of emerging resistant clones during anti-EGFR therapy remains elusive. We tracked the evolution of subclonal mutations by whole-exome sequencing and performed clonal analyses of individual metastases during therapy. Complementary functional analyses of polyclonal EGFR-mutant cell pools showed a dose-dependent enrichment of BRAFV600E and a loss of EGFR inhibitor susceptibility. The clones remain stable and become vulnerable to combined EGFR, RAF, and MEK inhibition. Moreover, only osimertinib/trametinib combination treatment, but not monotherapy with either of these drugs, leads to robust tumor shrinkage in EGFR-driven xenograft models harboring BRAFV600E mutations. These data provide insights into the dynamics of clonal evolution of EGFR-mutant tumors and the therapeutic implications of BRAF co-mutations that may facilitate the development of treatment strategies to improve the prognosis of these patients.

Structural basis for diamide modulation of ryanodine receptor

Diamide insecticides target insect ryanodine receptors (RYRs) and cause dysregulation of calcium signaling in insect muscles and neurons, generating worldwide sales over 2 billion US dollars annually. Several resistance mutations have been reported to reduce the efficacy of the diamides, but the exact binding sites and mechanism of resistance mutations were not clear. Recently, we solved the cryo-electron microscopy (cryo-EM) structure of RYR in complex with the anthranilic diamide chlorantraniliprole (CHL). CHL binds to the pseudo–voltage-sensor domain (pVSD) of RYR, a site in proximity to the previously identified resistance mutations. Mutagenesis studies in silico, in mutant cell lines, and in transgenic Drosophila strains revealed the key residues involved in diamide coordination and the molecular mechanism under species-selectivity and resistance mutations. We also proposed that CHL may alleviate the loss-of-function effects of some central core disease (CCD) mutations by increasing the opening probability (Po) of RYR1. In addition, we solved the crystal structures of several RYR domains from the diamondback moth and the bee, revealing insect-specific structural features which could be potentially targeted by novel insecticides. Interestingly, we found that the phosphorylation of insect RYR is temperature dependent, facilitated by the low thermal stability and dynamic structure of the insect RYR. Our structures provide a foundation for developing novel pesticides to overcome the resistance crisis.

Functional analysis of N-acetylglucosaminyltransferase-I knockdown in 2D and 3D neuroblastoma cell cultures

Tumor development can be promoted/suppressed by certain N-glycans attached to proteins at the cell surface. Here we examined aberrant neuronal properties in 2D and 3D rat neuroblastoma (NB) cell cultures with different N-glycan populations. Lectin binding studies revealed that the engineered N-glycosylation mutant cell line, NB_1(-Mgat1), expressed solely oligomannose N-glycans, and verified that the parental cell line, NB_1, and a previous engineered N-glycosylation mutant, NB_1(-Mgat2), expressed significant levels of higher order N-glycans, complex and hybrid N-glycans, respectively. NB_1 grew faster than mutant cell lines in monolayer and spheroid cell cultures. A 2-fold difference in growth between NB_1 and mutants occurred much sooner in 2D cultures relative to that observed in 3D cultures. Neurites and spheroid cell sizes were reduced in mutant NB cells of 2D and 3D cultures, respectively. Cell invasiveness was highest in 2D cultures of NB_1 cells compared to that of NB_1(-Mgat1). In contrast, NB_1 spheroid cells were much less invasive relative to NB_1(-Mgat1) spheroid cells while they were more invasive than NB_1(-Mgat2). Gelatinase activities supported the ranking of cell invasiveness in various cell lines. Both palladin and HK2 were more abundant in 3D than 2D cultures. Levels of palladin, vimentin and EGFR were modified in a different manner under 2D and 3D cultures. Thus, our results support variations in the N-glycosylation pathway and in cell culturing to more resemble in vivo tumor environments can impact the aberrant cellular properties, particularly cell invasiveness, of NB.

TP53 Null AML Line Does Not Selectively Expand Under Lenalidomide Treatment

Lenalidomide is an immunomodulatory therapeutic (IMiD) with known anti-proliferative and anti-angiogenic effects, and is an approved therapy for both 5q- Myelodysplastic Syndromes and Multiple Myeloma (MM). This thalidomide analog is known to retarget the E3 ubiquitin ligase cereblon to differential ubiquitination, and therefore degradation, of specific substrates. While lenalidomide maintenance is known to promote progression-free survival, increased rates of therapy-related myeloid neoplasms (TMNs) have been observed in MM patients on IMiD maintenance therapy following autologous stem cell transplant. Of note, TP53 is the most commonly mutated gene in TMN arising after lenalidomide therapy (Mouhieddine et al. 2020). TP53 is also recurrently mutated in TMNs following chemotherapy, suggesting that genotoxic stress results in the selective expansion of hematopoietic stem and progenitor cells (HSPCs) carrying mutations in TP53 (TN Wong et al. 2018). Based on these observations, we hypothesize that lenalidomide may contribute to the development of TMN by providing a fitness advantage to HSPCs carrying TP53 mutations. To test this hypothesis, we generated TP53 -/- deficient MOLM13 AML cells using CRISPR-Cas9 gene editing. We confirmed guide editing efficiency by next generation sequencing of a bulk edited pool of cells and generated isogenic clones via outgrowth of singly sorted cells, in an accepted approach for the field (Boettcher et al. 2019). We validated clones via sequencing and western blotting, and identified no differences in basal proliferation characteristics. Next, we further compared TP53 -/- deficient clones' with TP53 +/+ clones' response to cisplatin treatment (0.05-10µM) and observed a 2.8 fold increase in IC50 for TP53 -/- deficient clones. Satisfied that TP53 -/- isogenic clones successfully recapitulated this known resistance phenotype, we examined the response to lenalidomide by mixing TP53 -/- and TP53 +/+(AAVS1 edited control) clones and measuring expansion under lenalidomide treatment. However, we observed substantial inter-clone heterogeneity, with widely varying cellular proliferation characteristics following clone mixing, despite our prior validation. Therefore, we shifted to using bulk edited MOLM-13 cells in order to mitigate the potential impact of off-target effects and strong selective pressure of singly sorted clones. We edited a pool of cells at either TP53 or the safe harbor locus AAVS1 , allowed cells to recover, and mixed the two edited populations before treating with PBS, vehicle (DMSO), 1µm or 10µM lenalidomide for up to 28 days. We sequenced both AAVS1 and TP53 loci to measure cellular expansion by tracking the fraction of unedited, safe harbor edited, and TP53-/- genomic DNA at 0, 3, 7, and 28 days. Contrary to our hypothesis, TP53 -/- cells did not expand over time in response to lenalidomide treatment, and we did not detect a difference in TP53 -/- cell expansion with lenalidomide treatment compared to vehicle control (Figure 1). While we did not detect a cell intrinsic effect of TP53 mutations on lenalidomide response, it is possible that there are cell extrinsic effects, such as alterations in the bone marrow microenvironment, that contribute to the development of TP53-mutant TMN. It is also possible that a more prolonged exposure of lenalidomide is needed to see a significant effect of TP53-mutant cell expansion. This is relevant, since patients may be maintained on lenalidomide therapy for years. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Model Identifies Genetic Predisposition of Alzheimer’s Disease as Key Decider in Cell Susceptibility to Stress

Accumulation of unfolded/misfolded proteins in neuronal cells perturbs endoplasmic reticulum homeostasis, triggering a stress cascade called unfolded protein response (UPR), markers of which are upregulated in Alzheimer’s disease (AD) brain specimens. We measured the UPR dynamic response in three human neuroblastoma cell lines overexpressing the wild-type and two familial AD (FAD)-associated mutant forms of amyloid precursor protein (APP), the Swedish and Swedish-Indiana mutations, using gene expression analysis. The results reveal a differential response to subsequent environmental stress depending on the genetic background, with cells overexpressing the Swedish variant of APP exhibiting the highest global response. We further developed a dynamic mathematical model of the UPR that describes the activation of the three branches of this stress response in response to unfolded protein accumulation. Model-based analysis of the experimental data suggests that the mutant cell lines experienced a higher protein load and subsequent magnitude of transcriptional activation compared to the cells overexpressing wild-type APP, pointing to higher susceptibility of mutation-carrying cells to stress. The model was then used to understand the effect of therapeutic agents salubrinal, lithium, and valproate on signalling through different UPR branches. This study proposes a novel integrated platform to support the development of therapeutics for AD.

Genomic Landscape in Neoplasm-Like Stroma Reveals Distinct Prognostic Subtypes of Pancreatic Ductal Adenocarcinoma

As a main component of the tumor microenvironment, the stroma is critical in development, progression, and metastasis of pancreatic ductal adenocarcinoma (PDAC). The genomic status and its relationship of neoplastic and stromal components remain unclear in PDAC. We performed targeted sequencing for 1,021 cancer-suspected genes on parallel microdissected stromal and neoplastic components from 50 operable PDAC patients. Clonality analysis of mutations was conducted to reconstruct the evolutionary trajectory, and then molecular subtypes were established. Multi-lineage differentiation potential and mesenchymal transformation of KRAS-mutant cell line Panc1 were evaluated using RT-PCR and immunofluorescence staining. In this study, 39 (78.0%) were genomically altered in stroma, with KRAS (71.8%), TP53 (61.5%), and CDKN2A (23.1%) as the most commonly mutated genes. The majority of stromal mutations (89.8%) were detected in matched neoplastic components. Patients with KRAS/TP53-mut stroma demonstrated a higher tumor cell fraction (TCF) than did those with wild-type (WT) stroma (p = 0.0371, p = 0.0014). In both components, mutants KRAS and TP53 often occurred as clonal events, and the allele frequencies presented linear correlation in the same specimen. All neoplasm-like stroma (characterized with all or initial neoplastic clones and driver events in stroma) harbored KRAS or TP53 mutations. Neoplasm-like and KRAS-mutant stroma was associated with shorter disease-free survival. It is a new finding for the existence of driver gene mutations in PDAC stroma. These data suggest that genomic features of stromal components may serve as prognostic biomarkers in resectable PDAC and might help to guide a more precise treatment paradigm in therapeutic options.