Andres M. Lebensohn

Toca-1 Mediates Cdc42-Dependent Actin Nucleation by Activating the N-WASP-WIP Complex

An important signaling pathway to the actin cytoskeleton links the Rho family GTPase Cdc42 to the actin-nucleating Arp2/3 complex through N-WASP. Nevertheless, these previously identified components are not sufficient to mediate Cdc42-induced actin polymerization in a physiological context. In this paper, we describe the biochemical purification of Toca-1 (transducer of Cdc42-dependent actin assembly) as an essential component of the Cdc42 pathway. Toca-1 binds both N-WASP and Cdc42 and is a member of the evolutionarily conserved PCH protein family. Toca-1 promotes actin nucleation by activating the N-WASP-WIP/CR16 complex, the predominant form of N-WASP in cells. Thus, the cooperative actions of two distinct Cdc42 effectors, the N-WASP-WIP complex and Toca-1, are required for Cdc42-induced actin assembly. These findings represent a significantly revised view of Cdc42-signaling and shed light on the pathogenesis of Wiskott-Aldrich syndrome.

Activation of the WAVE Complex by Coincident Signals Controls Actin Assembly

WAVE proteins link upstream signals to actin nucleation by activating the Arp2/3 complex and are at the core of regulatory pathways driving membrane protrusion. They are found in heteropentameric complexes whose role in regulating WAVE function is presently unclear. Here we demonstrate that purified native WAVE complexes are basally inactive; previous reports of constitutive activity are artifacts of in vitro manipulation. Further, the native complexes are not activated by Rac alone. Activation of the WAVE2 complex requires simultaneous interactions with prenylated Rac-GTP and acidic phospholipids, as well as a specific state of phosphorylation. Together these signals promote full activation in a highly cooperative process on the membrane surface, by inducing an allosteric change in the complex rather than by simple recruitment or by dissociation of the subunits. These results explain how the WAVE complex can integrate coincident signals to promote localized actin nucleation during cell motility.

Comparative genetic screens in human cells reveal new regulatory mechanisms in WNT signaling

The comprehensive understanding of cellular signaling pathways remains a challenge due to multiple layers of regulation that may become evident only when the pathway is probed at different levels or critical nodes are eliminated. To discover regulatory mechanisms in canonical WNT signaling, we conducted a systematic forward genetic analysis through reporter-based screens in haploid human cells. Comparison of screens for negative, attenuating and positive regulators of WNT signaling, mediators of R-spondin-dependent signaling and suppressors of constitutive signaling induced by loss of the tumor suppressor adenomatous polyposis coli or casein kinase 1α uncovered new regulatory features at most levels of the pathway. These include a requirement for the transcription factor AP-4, a role for the DAX domain of AXIN2 in controlling β-catenin transcriptional activity, a contribution of glycophosphatidylinositol anchor biosynthesis and glypicans to R-spondin-potentiated WNT signaling, and two different mechanisms that regulate signaling when distinct components of the β-catenin destruction complex are lost. The conceptual and methodological framework we describe should enable the comprehensive understanding of other signaling systems. DOI: http://dx.doi.org/10.7554/eLife.21459.001

In vitro reconstitution of cdc42-mediated actin assembly using purified components.

In the accompanying chapter, we describe an in vitro system that uses Xenopus egg extracts to study actin assembly induced by phosphatidylinositol (4,5)bisphosphate (PIP2) and Cdc42. Biochemical fractionation and candidate screening experiments conducted in the extract system have identified the Arp2/3 complex, the N-WASP-WIP (or N-WASP-CR16) complex, and the Cdc42-binding protein Toca-1 as important mediators of PIP2- and Cdc42-actin signaling. Toward our ultimate goal of reconstituting an in vitro system that recapitulates the signaling properties observed in vivo, we then developed a purified actin assembly assay system consisting of the regulatory components that we discovered from extracts. In these assays, the stereotypical sigmoidal kinetics of actin polymerization are monitored by pyrene-actin fluorescence in the presence of defined recombinant or purified proteins, enabling the detailed study of mechanism and protein function. In this chapter, we describe the preparation of the components used in these purified actin assembly reactions, as well as the assay conditions under which we monitor actin polymerization kinetics in vitro.

R-spondins can potentiate WNT signaling without LGRs

The WNT signaling pathway regulates patterning and morphogenesis during development and promotes tissue renewal and regeneration in adults. The R-spondin (RSPO) family of four secreted proteins, RSPO1-4, amplifies target cell sensitivity to WNT ligands by increasing WNT receptor levels. Leucine-rich repeat-containing G-protein coupled receptors (LGRs) 4-6 are considered obligate high-affinity receptors for RSPOs. We discovered that RSPO2 and RSPO3, but not RSPO1 or RSPO4, can potentiate WNT/β-catenin signaling in the absence of all three LGRs. By mapping the domains on RSPO3 that are necessary and sufficient for this activity, we show that the requirement for LGRs is dictated by the interaction between RSPOs and the ZNRF3/RNF43 E3 ubiquitin ligases and that LGR-independent signaling depends on heparan sulfate proteoglycans (HSPGs). We propose that RSPOs can potentiate WNT signals through distinct mechanisms that differ in their use of either LGRs or HSPGs, with implications for understanding their biological functions.

Chromatin-Remodeling Complex SWI/SNF Controls Multidrug Resistance by Transcriptionally Regulating the Drug Efflux Pump ABCB1.

Anthracyclines are among the most effective yet most toxic drugs used in the oncology clinic. The nucleosome-remodeling SWI/SNF complex, a potent tumor suppressor, is thought to promote sensitivity to anthracyclines by recruiting topoisomerase IIa (TOP2A) to DNA and increasing double-strand breaks. In this study, we discovered a novel mechanism through which SWI/SNF influences resistance to the widely used anthracycline doxorubicin based on the use of a forward genetic screen in haploid human cells, followed by a rigorous single and double-mutant epistasis analysis using CRISPR/Cas9-mediated engineering. Doxorubicin resistance conferred by loss of the SMARCB1 subunit of the SWI/SNF complex was caused by transcriptional upregulation of a single gene, encoding the multidrug resistance pump ABCB1. Remarkably, both ABCB1 upregulation and doxorubicin resistance caused by SMARCB1 loss were dependent on the function of SMARCA4, a catalytic subunit of the SWI/SNF complex. We propose that residual SWI/SNF complexes lacking SMARCB1 are vital determinants of drug sensitivity, not just to TOP2A-targeted agents, but to the much broader range of cancer drugs effluxed by ABCB1. Cancer Res; 76(19); 5810-21. ©2016 AACR.

Discovery of gene regulatory elements through a new bioinformatics analysis of haploid genetic screens

The systematic identification of regulatory elements that control gene expression remains a challenge. Genetic screens that use untargeted mutagenesis have the potential to identify protein-coding genes, non-coding RNAs and regulatory elements, but their analysis has mainly focused on identifying the former two. To identify regulatory elements, we conducted a new bioinformatics analysis of insertional mutagenesis screens interrogating WNT signaling in haploid human cells. We searched for specific patterns of retroviral gene trap integrations (used as mutagens in haploid screens) in short genomic intervals overlapping with introns and regions upstream of genes. We uncovered atypical patterns of gene trap insertions that were not predicted to disrupt coding sequences, but caused changes in the expression of two key regulators of WNT signaling, suggesting the presence of cis-regulatory elements. Our methodology extends the scope of haploid genetic screens by enabling the identification of regulatory elements that control gene expression.

R-spondins can potentiate WNT signaling without LGR receptors

The WNT signaling pathway regulates patterning and morphogenesis during embryonic development and promotes tissue renewal and regeneration in adults. Some WNT responses in vertebrates depend on a second signal provided by the R-spondin family of four secreted proteins (RSPO1-4) that drive the renewal of stem cells in many tissues. RSPOs markedly amplify target cell sensitivity to WNT ligands by neutralizing two transmembrane E3 ligases, ZNRF3 and RNF43, which reduce cell-surface levels of WNT receptors. Chromosomal translocations that increase RSPO expression or that inactivate ZNRF3/RNF43 can drive human cancers. RSPOs contain tandem furin-like repeats (FU1 and FU2), a thrombospondin type I (TSP) domain, and a basic region (BR). RSPOs simultaneously engage ZNRF3/RNF43 through their FU1 domain and one of three leucine-rich repeat-containing G-protein coupled receptors (LGR4-6) through their FU2 domain, triggering the clearance of ZNRF3/RNF43 and the consequent rise in WNT receptor levels. LGRs are selectively expressed in various tissue stem cells and are considered the primary high-affinity receptors for RSPOs. Using purified mutant and chimeric RSPOs and cell lines lacking various receptors, we show that RSPO2 and RSPO3, but not RSPO1 and RSPO4, can potentiate WNT/β-catenin signaling in the absence of all three LGRs. The ZNRF3/RNF43-interacting FU1 domain was necessary for LGR-independent signaling, while the LGR-interacting FU2 domain was dispensable. The FU1 domain of RSPO3 was also sufficient to confer LGR-independence when transplanted to RSPO1, demonstrating that its interaction with ZNRF3/RNF43 dictates LGR-independent signaling. The enigmatic TSP/BR domains of RSPOs and their interaction with heparan sulfate proteoglycans (HSPGs), previously considered dispensable for WNT/β-catenin signaling, became essential in the absence of LGRs. These results define two alternative modes of RSPO-mediated signaling that share a common dependence on ZNRF3/RNF43, but differ in their use of either LGRs or HSPGs, with implications for understanding their mechanism of action, biological functions and evolutionary origins.

Abstract PR05: Systematic forward genetic screens in haploid human cells reveal new players and regulatory mechanisms in Wnt signaling

Wnt signaling is at the core of animal development and regeneration. It controls cell proliferation, differentiation and survival. In humans, defective Wnt signaling leads to several forms of cancer, most notably colorectal cancer (CRC). A convergence of methodological advances enabled us to do a systematic forward genetic analysis of canonical Wnt signaling in human cells, revealing new players and regulatory mechanisms. Using HAP1 cells, a haploid human cell line, we conducted a set of genome-wide screens to interrogate the Wnt pathway under normal and pathological conditions. We constructed a HAP1 line harboring a Wnt-responsive GFP reporter, mutagenized the cells by insertion of a gene trap retrovirus throughout the haploid genome, and sorted for cells exhibiting phenotypes of interest using FACS. By setting appropriate gates we enriched for negative and positive regulators of the pathway. These screens yielded many known players as well as a new transcription factor, TFAP4, required for signaling downstream of β-catenin. Unexpectedly AXIN2, a scaffold in the β-catenin destruction complex, was a prominent hit in the screen for positive regulators. However, the distribution of gene trap insertions in AXIN2 suggested that truncation of the last one or two exons encompassing the DAX domain, as opposed to disruption of the entire AXIN2 gene, was responsible for the observed phenotype. Follow-up analysis of cells in which AXIN1 was eliminated and AXIN2 C-terminal truncations were generated at the single endogenous locus confirmed that this domain is dispensable for destruction complex function. Furthermore, in these cells responsiveness to Wnt was only partially diminished, suggesting that the DAX domain is not essential for transduction of the Wnt signal from the receptor. Instead we show that the C-terminus is involved in regulating Axin2 protein levels. Using CRISPR/CAS9 we then generated haploid cell lines lacking adenomatous polyposis coli (APC), a scaffold in the β-catenin destruction complex, or casein kinase α (CK1α), the priming kinase for β-catenin degradation. These lines have constitutive Wnt signaling activity, and the former recapitulates the APC mutations found in CRC patients. We conducted synthetic genome-wide screens looking for modifiers of these mutations in an otherwise isogenic background. We found that in APC-null cells, deletion of the RNA binding protein SERBP1 reduces β-catenin protein levels significantly, restoring normal levels of Wnt-stimulated signaling. In CK1α-null cells, deletion of the E3 ubiquitin ligase HUWE1 decreases Wnt signaling by over 85% with only a minor reduction of β-catenin protein level. This defect is specific to the CK1α genetic background, since deletion of HUWE1 in APC-null cells has no effect on signaling. These results suggest a destruction complex independent role of CK1α in Wnt signaling, mediated by HUWE1. Our findings are supported by experiments in model organisms. The experimental approaches used in this study are generally applicable to other signaling pathways and more broadly to any cellular process in which a phenotypic readout can be used to enrich for mutant cells. The combination of forward genetics in haploid cells and CRISPR/CAS9-based genome engineering brings to bear on cultured human cells the immense power of genetics traditionally limited to model organisms such as yeast. This abstract is also presented as Poster B11. Citation Format: Andres M. Lebensohn, Ramin Dubey, Leif R. Neitzel, Ofelia Tacchelly, Caleb D. Marceau, Zahra Bahrami, Amanda G. Hansen, Yashi Ahmed, Ethan Lee, Jan Carette, Rajat Rohatgi. Systematic forward genetic screens in haploid human cells reveal new players and regulatory mechanisms in Wnt signaling. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR05.

Abstract PR03: Genome-wide screens for Wnt signaling in human haploid cells

Wnt signaling is at the core of human development and stem cell biology. Defective Wnt signaling leads to several forms of cancer, most notably colorectal cancer. Using HAP1 cells, a human haploid cell line, we conducted a set of genome-wide forward genetic screens to identify new Wnt pathway components. We constructed a HAP1 line harboring a GFP reporter driven by the Wnt-responsive TCF promoter, we mutagenized the cells by insertion of a gene-trap retrovirus throughout the haploid genome, and we sorted the mutagenized population for cells exhibiting phenotypes of interest using FACS. In order to find 1. negative regulators, 2. positive regulators and 3. feedback modulators of the pathway, we sorted for 1. cells expressing GFP in the absence of Wnt, 2. cells with reduced GFP expression after strong Wnt stimulation and 3. cells with enhanced GFP expression after mild Wnt stimulation, respectively. Deep sequencing of amplified genomic DNA flanking the retroviral insertion sites in the sorted cell populations revealed genes whose disruption may have led to the phenotype of interest. Among the most frequently disrupted genes (some with over 150 unique retroviral insertions) we found many of the known components of the canonical Wnt pathway, including LRP6, casein kinase I, APC, β-catenin, DOT1-L, TCF4 and the recently discovered negative feedback regulator ZNRF3. Unexpectedly, the screen for positive regulators revealed retroviral insertions in AXIN2 (a scaffold protein found in the β-catenin destruction complex) predicted to yield a C-terminally truncated form that appears to act as a dominant negative. We are currently assessing hits not previously known to be involved in Wnt signaling. This abstract is also presented as Poster A40. Citation Format: Andres Lebensohn, Casey Hughes, Caleb Marceau, Rajat Rohatgi, Jan Carette. Genome-wide screens for Wnt signaling in human haploid cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Synthetic Lethal Approaches to Cancer Vulnerabilities; May 17-20, 2013; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(5 Suppl):Abstract nr PR03.