Anthony B. Mak

A Lentiviral Functional Proteomics Approach Identifies Chromatin Remodeling Complexes Important for the Induction of Pluripotency

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.e. multiple N- and C-terminal tags and multiple promoters), is compatible with GatewayTM cloning, and incorporates a reference peptide. Its major advantage is that it permits efficient and stable delivery of affinity-tagged open reading frames into most mammalian cell types. We benchmarked MAPLE with a number of human protein complexes involved in transcription, including the RNA polymerase II-associated factor, negative elongation factor, positive transcription elongation factor b, SWI/SNF, and mixed lineage leukemia complexes. In addition, MAPLE was used to identify an interaction between the reprogramming factor Klf4 and the Swi/Snf chromatin remodeling complex in mouse embryonic stem cells. We show that the SWI/SNF catalytic subunit Smarca2/Brm is up-regulated during the process of induced pluripotency and demonstrate a role for the catalytic subunits of the SWI/SNF complex during somatic cell reprogramming. Our data suggest that the transcription factor Klf4 facilitates chromatin remodeling during reprogramming.

CD133 Protein N-Glycosylation Processing Contributes to Cell Surface Recognition of the Primitive Cell Marker AC133 Epitope

Background: Cell surface recognition of the AC133 epitope on CD133 marks many stem cell and cancer stem cell types. Results: A large scale RNA interference screen identifies genes involved in N-glycosylation that regulate cell surface AC133 recognition. Conclusion: CD133 N-glycosylation and its processing contribute to cell surface AC133 recognition. Significance: Glycobiological differences between primitive and differentiated cells may be responsible for regulating cell surface AC133. The AC133 epitope expressed on the CD133 glycoprotein has been widely used as a cell surface marker of numerous stem cell and cancer stem cell types. It has been recently proposed that posttranslational modification and regulation of CD133 may govern cell surface AC133 recognition. Therefore, we performed a large scale pooled RNA interference (RNAi) screen to identify genes involved in cell surface AC133 expression. Gene hits could be validated at a rate of 70.5% in a secondary assay using an orthogonal RNAi system, demonstrating that our primary RNAi screen served as a powerful genetic screening approach. Within the list of hits from the primary screen, genes involved in N-glycan biosynthesis were significantly enriched as determined by Ingenuity Canonical Pathway analyses. Indeed, inhibiting biosynthesis of the N-glycan precursor using the small molecule tunicamycin or inhibiting its transfer to CD133 by generating N-glycan-deficient CD133 mutants resulted in undetectable cell surface AC133. Among the screen hits involved in N-glycosylation were genes involved in complex N-glycan processing, including the poorly characterized MGAT4C, which we demonstrate to be a positive regulator of cell surface AC133 expression. Our study identifies a set of genes involved in CD133 N-glycosylation as a direct contributing factor to cell surface AC133 recognition and provides biochemical evidence for the function and structure of CD133 N-glycans.

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large‐scale sequencing efforts. Using genome‐scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co‐culture competition assays to generate a high‐confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non‐isogenic cancer cell lines. For example, the PTEN−/− DiE genes reveal a signature that can preferentially classify PTEN‐dependent genotypes across a series of non‐isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.

The mixed lineage leukemia (MLL)-fusion associated gene AF4 promotes CD133 transcription

The AC133 epitope has been used as a marker for both normal and cancer stem cells from multiple tissue lineages. To identify transcription factors that regulate CD133 expression, we conducted parallel large-scale RNA interference screens in Caco-2 cancer cells that endogenously express CD133 and in engineered HEK293 cells that express CD133 from a heterologous promoter. The transcription factor AF4 was identified following a comparative analysis between the two screens. We then showed that AF4 is a promoter of CD133 transcription in multiple cancer cell lines. Knockdown of AF4 resulted in a dramatic reduction in CD133 transcript levels. Importantly, a subset of pediatric acute lymphoblastic leukemias (ALL) harbor a fusion oncogene results from a chromosomal translocation that juxtaposes the mixed-lineage leukemia (MLL) gene and the AF4 gene. An investigation of the functional role of CD133 in the MLL-AF4-dependent ALL cells revealed that CD133 was required for leukemia cell survival. Together, our findings show AF4-dependent regulation of CD133 expression, which is required for the growth of ALL cells. CD133 may therefore represent a therapeutic target in a subset of cancers.

A genome scale overexpression screen to reveal drug activity in human cells

Target identification is a critical step in the lengthy and expensive process of drug development. Here, we describe a genome-wide screening platform that uses systematic overexpression of pooled human ORFs to understand drug mode-of-action and resistance mechanisms. We first calibrated our screen with the well-characterized drug methotrexate. We then identified new genes involved in the bioactivity of diverse drugs including antineoplastic agents and biologically active molecules. Finally, we focused on the transcription factor RHOXF2 whose overexpression conferred resistance to DNA damaging agents. This approach represents an orthogonal method for functional screening and, to our knowledge, has never been reported before.

CD133-Targeted Niche-Dependent Therapy in Cancer A Multipronged Approach

Cancer treatment continues to be challenged by the development of therapeutic resistances and relapses in the clinical setting, which are largely attributed to tumor heterogeneity, particularly the existence of cancer stem cells (CSCs). Thus, targeting the CSC subpopulation may represent an effective therapeutic strategy. However, despite advances in identifying and characterizing CD133(+) CSCs in various human cancers, efforts to translate these experimental findings to clinical modalities have been slow in the making, especially in light of the growing awareness of CSC plasticity and the foreseeable pitfall of therapeutically targeting CSC base sorely on a surface marker. We, and others, have demonstrated that the CD133(+) CSCs reside in complex vascular niches, where reciprocal signaling between the CD133(+) CSCs and their microenvironment may govern niche morphogenesis and homeostasis. Herein, we discuss the multifaceted functional role of the CD133(+) cells in the context of their niche, and the potential of targeting CD133 as a niche-dependent approach in effective therapy.

Post-Translational Regulation of CD133 by ATase1/ATase2-Mediated Lysine Acetylation

The CD133 cell-surface protein expresses the AC133 epitope that is associated with cancer progenitor cells and cancer resistance to traditional anticancer therapies. We report that the endoplasmic reticulum Golgi intermediate compartment residing acetyltransferases, ATase1 (NAT8B) and ATase2 (NAT8), can physically interact with CD133 to acetylate the protein on three lysine residues predicted to reside on the first extracellular loop of CD133. Site-directed mutagenesis of these residues mimicking a loss of acetylation and downregulation or inhibition of ATase1/ATase2 resulted in near-complete abolishment of CD133 protein expression. We also demonstrate that targeting ATase1/ATase2 results in apoptosis of CD133 expressing acute lymphoblastic leukemia cells. Taken together, we suggest that lysine acetylation on predicted extracellular residues plays a key role in expression and trafficking of CD133 protein to the cell surface and can be targeted to disrupt CD133 regulation and function.

A versatile lentiviral expression system to identify mammalian protein–protein interactions

Protein-protein interactions (PPIs) are central to our understanding of protein function, biological processes and signaling pathways. Affinity purification coupled with mass spectrometry (AP-MS) is a powerful approach for detecting PPIs and protein complexes and relies on the purification of bait proteins using bait-specific binding reagents. These binding reagents may recognize bait proteins directly or affinity tags that are fused to bait proteins. A limitation of the latter approach is that expression of affinity tagged baits is largely constrained to engineered or unnatural cell lines, which results in the AP-MS identification of PPIs that may not accurately reflect those seen in nature. Therefore, generating cell lines stably expressing affinity tagged bait proteins in a broad range of cell types and cell lines is important for identifying PPIs that are dependent on different contexts. To facilitate the identification of PPIs across many mammalian cell types, we developed the mammalian affinity purification and lentiviral expression (MAPLE) system. MAPLE uses recombinant lentiviral technology to stably and efficiently express affinity tagged complementary DNA (cDNA) in mammalian cells, including cells that are difficult to transfect and non-dividing cells. The MAPLE vectors contain a versatile affinity (VA) tag for multi-step protein purification schemes and subcellular localization studies. In this methods article, we present a step-by-step overview of the MAPLE system workflow.

RNA interference screens to uncover membrane protein biology.

In this review, we discuss the use of RNA interference screens to identify genes involved in the regulation and function of membrane proteins. Briefly, cells expressing the membrane protein of interest can be transduced with a pooled lentiviral short-hairpin RNA (shRNA) library containing tens of thousands of unique shRNAs. Transduced cells are then selected or fractionated based on specific critera, such as membrane protein expression or function. shRNAs from selected cell populations are then deconvoluted and quantified using microarray analyses or high-throughput sequencing technologies. This allows individual shRNAs to be scored and cutoffs can be made to generate a list of shRNA hits. Bioinformatic analyses of gene targets of shRNA hits can be used to identify pathways and processes associated with membrane protein biology. To illustrate this functional genomics approach, we discuss pooled lentiviral shRNA screens that were performed to identify genes that regulate the transcription and cell-surface expression of the cancer stem cell marker CD133. This approach can be adapted to study other membrane proteins, as well as specific aspects of membrane proteins, such as their function or downstream signaling effects.

Abstract A11: Regulation of CD133 by the tubulin deacetylase HDAC6 can alter cancer cell state

The CD133 antigen AC133/1 is a putative marker for enriching certain stem cells and cancer progenitor cells. Despite its wide application, the function and regulation of its expression remains unclear. Given the presence of its transcript in more differentiated cells, epigenetic regulation of CD133 transcripts alone cannot vouch for its utility as a marker. Therefore, understanding the regulation of CD133 protein expression is important for further defining the cellular state of tumor-initiating cells. We used the MAPLE system (Mak et al, Mol Cell Proteomics, 2010) to identify bona fide protein interaction partners of CD133. We present the microtubule-associated histone deacetylase 6 (HDAC6) as a physical interaction partner of CD133. This interaction was validated by co-immunoprecipitation/Western Blot analyses and by a membrane yeast two-hybrid (MYTH) assay. In order to understand the functional consequence of the HDAC6 and CD133 interaction, we used lentiviral short-hairpin RNAs to knockdown HDAC6 in multiple cell types, including the human epithelial colorectal adenocarcinoma lines Caco- 2 and HT-29 and in the retinoblastoma cell line Weri-Rb-1. Interestingly, we observed a significant reduction of CD133 protein expression. This reduction was shown to be dependent on HDAC6 deacetylase activity using a deacetlyase deficient HDAC6 mutant as well as when using the small molecule inhibitor of HDAC6, tubacin. HDAC6 promotes de-acetylation of alpha-tubulin to influence re-cycling of cell surface proteins. Furthermore, when we performed a CD133 MYTH screen against a cDNA library isolated from an adult human brain, we identified the late endosomal- and lysosomal-associated marker CD63 as an interaction partner of CD133. In fact, treatment of Caco-2 cells with tubacin resulted in trafficking of CD133 into endosomes for lysosomal degradation as determined by increased co-localization with CD63. In an attempt to study the affect of HDAC6 knockdown on cell viability, we noticed that CD133 knockdown significantly impacted cell proliferation and clonogenicity of Caco-2 cells. We determined that the cause of this is likely due to increased differentiation as determined by the level of alkaline phosphatase activity and by quantitative realtime PCR for markers of differentiated colon cells. To assess the tumorigenic potential of Caco-2 cells with reduced CD133 expression in vitro we performed soft agar colony-forming assay, which resulted in the loss of anchorage independent growth, a well-known hallmark of cancer. Together, these results suggest that CD133 can exercise a function in establishing and maintaining a primitive cancer cell state and loss or reduction of its expression marks more differentiated cancer cells that may be nontumorigenic. Citation Information: Clin Cancer Res 2010;16(14 Suppl):A11.