Prioritization of cancer therapeutic targets using CRISPR-Cas9 screens.

Functional genomics approaches can overcome limitations-such as the lack of identification of robust targets and poor clinical efficacy-that hamper cancer drug development. Here we performed genome-scale CRISPR-Cas9 screens in 324 human cancer cell lines from 30 cancer types and developed a data-driven framework to prioritize candidates for cancer therapeutics. We integrated cell fitness effects with genomic biomarkers and target tractability for drug development to systematically prioritize new targets in defined tissues and genotypes. We verified one of our most promising dependencies, the Werner syndrome ATP-dependent helicase, as a synthetic lethal target in tumours from multiple cancer types with microsatellite instability. Our analysis provides a resource of cancer dependencies, generates a framework to prioritize cancer drug targets and suggests specific new targets. The principles described in this study can inform the initial stages of drug development by contributing to a new, diverse and more effective portfolio of cancer drug targets.

Dichotomous parvalbumin interneuron populations in dorsolateral and dorsomedial striatum.

KEY POINTS: There are two electrophysiological dichotomous populations of parvalbumin (PV) interneurons located in the dorsal striatum. Striatal PV interneurons in medial and lateral regions differ significantly in their intrinsic excitability. Parvalbumin interneurons in the dorsomedial striatum, but not in the dorsolateral striatum, receive afferent glutamatergic input from cingulate cortex. ABSTRACT: Dorsomedial striatum circuitry is involved in goal-directed actions or movements that become habits upon repetition, as encoded by the dorsolateral striatum. An inability to shift from habits can compromise action-control and prevent behavioural adaptation. Although these regions appear to be clearly behaviourally distinct, little is known about their distinct physiology. Parvalbumin (PV) interneurons are a major source of striatal inhibition and are usually considered as a homogeneous population in the entire dorsal striatum. In the present study, we recorded PV interneurons in dorsal striatum slices from wild-type male mice and suggest the existence of two electrophysiological dichotomous populations. We found that PV interneurons located at the dorsomedial striatum region have increased intrinsic excitability compared to PV interneurons in dorsolateral region. We also found that PV interneurons in the dorsomedial region, but not in the dorsolateral striatum region, receive short-latency excitatory inputs from cingulate cortex. Therefore, the results of the present study demonstrate the importance of considering region specific parvalbumin interneuron populations when studying dorsal striatal function.

Rab11-FIP1A regulates early trafficking into the recycling endosomes.

The Rab11 family of small GTPases, along with the Rab11-family interacting proteins (Rab11-FIPs), are critical regulators of intracellular vesicle trafficking and recycling. We have identified a point mutation of Threonine-197 site to an Alanine in Rab11-FIP1A, which causes a dramatic dominant negative phenotype when expressed in HeLa cells. The normally perinuclear distribution of GFP-Rab11-FIP1A was condensed into a membranous cisternum with almost no GFP-Rab11-FIP1A(T197A) remaining outside of this central locus. Also, this condensed GFP-FIP1A(T197A) altered the distribution of proteins in the Rab11a recycling pathway including endogenous Rab11a, Rab11-FIP1C, and transferrin receptor (CD71). Furthermore, this condensed GFP-FIP1A(T197A)-containing structure exhibited little movement in live HeLa cells. Expression of GFP-FIP1A(T197A) caused a strong blockade of transferrin recycling. Treatment of cells expressing GFP-FIP1A(T197A) with nocodazole did not disperse the Rab11a-containing recycling system. We also found that Rab5 and EEA1 were accumulated in membranes by GFP-Rab11-FIP1A but Rab4 was unaffected, suggesting that a direct pathway may exist from early endosomes into the Rab11a-containing recycling system. Our study of a potent inhibitory trafficking mutation in Rab11-FIP1A shows that Rab11-FIP1A associates with and regulates trafficking at an early step in the process of membrane recycling.

Rab11-FIP2 interaction with MYO5B regulates movement of Rab11a-containing recycling vesicles.

A tripartite association of Rab11a with both Rab11-FIP2 and MYO5B regulates recycling endosome trafficking. We sought to define the intermolecular interactions required between Rab11-FIP2 and MYO5B. Using a random mutagenesis strategy, we identified point mutations at S229P or G233E in Rab11-FIP2 that caused loss of interaction with MYO5B in yeast two-hybrid assays as well as loss of interaction of Rab11-FIP2(129-356) with MYO5B tail when expressed in HeLa cells. Single mutations or the double S229P/G233E mutation failed to alter the association of full-length Rab11-FIP2 with MYO5B tail in HeLa cells. While EGFP-Rab11-FIP2 wild type colocalized with endogenous MYO5B staining in MDCK cells, EGFP-Rab11-FIP2(S229P/G233E) showed a significant decrease in localization with endogenous MYO5B. Analysis of Rab11a-containing vesicle movement in live HeLa cells demonstrated that when the MYO5B/Rab11-FIP2 association is perturbed by mutation or by Rab11-FIP2 knockdown, vesicle movement is increased in both speed and track length, consistent with an impairment of MYO5B tethering at the cytoskeleton. These results support a critical role for the interaction of MYO5B with Rab11-FIP2 in stabilizing the functional complex with Rab11a, which regulates dynamic movements of membrane recycling vesicles.

Rab11-FIP1 phosphorylation by MARK2 regulates polarity in MDCK cells.

MARK2/Par1b/EMK1, a serine/threonine kinase, is required for correct apical/basolateral membrane polarization in epithelial cells. However, the specific substrates mediating MARK2 action are less well understood. We have now found that MARK2 phosphorylates Rab11-FIP1B/C at serine 234 in a consensus site similar to that previously identified in Rab11-FIP2. In MDCK cells undergoing repolarization after a calcium switch, antibodies specific for pS234-Rab11-FIP1 or pS227-Rab11-FIP2 demonstrate that the spatial and temporal activation of Rab11-FIP1 phosphorylation is distinct from that for Rab11-FIP2. Phosphorylation of Rab11-FIP1 persists through calcium switch and remains high after polarity has been reestablished whereas FIP2 phosphorylation is highest early in reestablishment of polarity but significantly reduced once polarity has been re-established. MARK2 colocalized with FIP1B/C/D and p(S234)-FIP1 in vivo. Overexpression of GFP-Rab11-FIP1C wildtype or non-phosphorylatable GFP-Rab11-FIP1C(S234A) induced two significant phenotypes following calcium switch. Overexpression of FIP1C wildtype and FIP1C(S234A) caused a psuedo-stratification of cells in early time points following calcium switch. At later time points most prominently observed in cells expressing FIP1C(S234A) a significant lateral lumen phenotype was observed, where F-actin-rich lateral lumens appeared demarcated by a ring of ZO1 and also containing ezrin, syntaxin 3 and podocalyxin. In contrast, p120 and E-Cadherin were excluded from the new apical surface at the lateral lumens and now localized to the new lateral surface oriented toward the media. GFP-FIP1C(S234A) localized to membranes deep to the lateral lumens, and immunostaining demonstrated the reorientation of the centrosome and the Golgi apparatus toward the lateral lumen. These results suggest that both Rab11-FIP1B/C and Rab11-FIP2 serve as critical substrates mediating aspects of MARK2 regulation of epithelial polarity.