Pharmaceutical-Grade Rigosertib Is a Microtubule-Destabilizing Agent.

We recently used CRISPRi/a-based chemical-genetic screens and cell biological, biochemical, and structural assays to determine that rigosertib, an anti-cancer agent in phase III clinical trials, kills cancer cells by destabilizing microtubules. Reddy and co-workers (Baker et al., 2020, this issue of Molecular Cell) suggest that a contaminating degradation product in commercial formulations of rigosertib is responsible for the microtubule-destabilizing activity. Here, we demonstrate that cells treated with pharmaceutical-grade rigosertib (>99.9% purity) or commercially obtained rigosertib have qualitatively indistinguishable phenotypes across multiple assays. The two formulations have indistinguishable chemical-genetic interactions with genes that modulate microtubule stability, both destabilize microtubules in cells and in vitro, and expression of a rationally designed tubulin mutant with a mutation in the rigosertib binding site (L240F TUBB) allows cells to proliferate in the presence of either formulation. Importantly, the specificity of the L240F TUBB mutant for microtubule-destabilizing agents has been confirmed independently. Thus, rigosertib kills cancer cells by destabilizing microtubules, in agreement with our original findings.

Combined CRISPRi/a-Based Chemical Genetic Screens Reveal that Rigosertib Is a Microtubule-Destabilizing Agent.

Chemical libraries paired with phenotypic screens can now readily identify compounds with therapeutic potential. A central limitation to exploiting these compounds, however, has been in identifying their relevant cellular targets. Here, we present a two-tiered CRISPR-mediated chemical-genetic strategy for target identification: combined genome-wide knockdown and overexpression screening as well as focused, comparative chemical-genetic profiling. Application of these strategies to rigosertib, a drug in phase 3 clinical trials for high-risk myelodysplastic syndrome whose molecular target had remained controversial, pointed singularly to microtubules as rigosertib's target. We showed that rigosertib indeed directly binds to and destabilizes microtubules using cell biological, in vitro, and structural approaches. Finally, expression of tubulin with a structure-guided mutation in the rigosertib-binding pocket conferred resistance to rigosertib, establishing that rigosertib kills cancer cells by destabilizing microtubules. These results demonstrate the power of our chemical-genetic screening strategies for pinpointing the physiologically relevant targets of chemical agents.

CRISPRi-based genome-scale identification of functional long noncoding RNA loci in human cells.

The human genome produces thousands of long noncoding RNAs (lncRNAs)-transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type-specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs.

A Multiplexed Single-Cell CRISPR Screening Platform Enables Systematic Dissection of the Unfolded Protein Response.

Functional genomics efforts face tradeoffs between number of perturbations examined and complexity of phenotypes measured. We bridge this gap with Perturb-seq, which combines droplet-based single-cell RNA-seq with a strategy for barcoding CRISPR-mediated perturbations, allowing many perturbations to be profiled in pooled format. We applied Perturb-seq to dissect the mammalian unfolded protein response (UPR) using single and combinatorial CRISPR perturbations. Two genome-scale CRISPR interference (CRISPRi) screens identified genes whose repression perturbs ER homeostasis. Subjecting ∼100 hits to Perturb-seq enabled high-precision functional clustering of genes. Single-cell analyses decoupled the three UPR branches, revealed bifurcated UPR branch activation among cells subject to the same perturbation, and uncovered differential activation of the branches across hits, including an isolated feedback loop between the translocon and IRE1α. These studies provide insight into how the three sensors of ER homeostasis monitor distinct types of stress and highlight the ability of Perturb-seq to dissect complex cellular responses.

Paradoxical resistance of multiple myeloma to proteasome inhibitors by decreased levels of 19S proteasomal subunits.

Hallmarks of cancer, including rapid growth and aneuploidy, can result in non-oncogene addiction to the proteostasis network that can be exploited clinically. The defining example is the exquisite sensitivity of multiple myeloma (MM) to 20S proteasome inhibitors, such as carfilzomib. However, MM patients invariably acquire resistance to these drugs. Using a next-generation shRNA platform, we found that proteostasis factors, including chaperones and stress-response regulators, controlled the response to carfilzomib. Paradoxically, 19S proteasome regulator knockdown induced resistance to carfilzomib in MM and non-MM cells. 19S subunit knockdown did not affect the activity of the 20S subunits targeted by carfilzomib nor their inhibition by the drug, suggesting an alternative mechanism, such as the selective accumulation of protective factors. In MM patients, lower 19S levels predicted a diminished response to carfilzomib-based therapies. Together, our findings suggest that an understanding of network rewiring can inform development of new combination therapies to overcome drug resistance.

Efficient ethanol production from brown macroalgae sugars by a synthetic yeast platform.

The increasing demands placed on natural resources for fuel and food production require that we explore the use of efficient, sustainable feedstocks such as brown macroalgae. The full potential of brown macroalgae as feedstocks for commercial-scale fuel ethanol production, however, requires extensive re-engineering of the alginate and mannitol catabolic pathways in the standard industrial microbe Saccharomyces cerevisiae. Here we present the discovery of an alginate monomer (4-deoxy-L-erythro-5-hexoseulose uronate, or DEHU) transporter from the alginolytic eukaryote Asteromyces cruciatus. The genomic integration and overexpression of the gene encoding this transporter, together with the necessary bacterial alginate and deregulated native mannitol catabolism genes, conferred the ability of an S. cerevisiae strain to efficiently metabolize DEHU and mannitol. When this platform was further adapted to grow on mannitol and DEHU under anaerobic conditions, it was capable of ethanol fermentation from mannitol and DEHU, achieving titres of 4.6% (v/v) (36.2 g l(-1)) and yields up to 83% of the maximum theoretical yield from consumed sugars. These results show that all major sugars in brown macroalgae can be used as feedstocks for biofuels and value-added renewable chemicals in a manner that is comparable to traditional arable-land-based feedstocks.

A new means to identify type 3 secreted effectors: functionally interchangeable class IB chaperones recognize a conserved sequence.

UNLABELLED: Many Gram-negative bacteria utilize specialized secretion systems to inject proteins (effectors) directly into host cells. Little is known regarding how bacteria ensure that only small subsets of the thousands of proteins they encode are recognized as substrates of the secretion systems, limiting their identification through bioinformatic analyses. Many of these proteins require chaperones to direct their secretion. Here, using the newly described protein interaction platform assay, we demonstrate that type 3 secretion system class IB chaperones from one bacterium directly bind their own effectors as well as those from other species. In addition, we observe that expression of class IB homologs from seven species, including pathogens and endosymbionts, mediate the translocation of effectors from Shigella directly into host cells, demonstrating that class IB chaperones are often functionally interchangeable. Notably, class IB chaperones bind numerous effectors. However, as previously proposed, they are not promiscuous; rather they recognize a defined sequence that we designate the conserved chaperone-binding domain (CCBD) sequence [(LMIF)(1)XXX(IV)(5)XX(IV)(8)X(N)(10)]. This sequence is the first defined amino acid sequence to be identified for any interspecies bacterial secretion system, i.e., a system that delivers proteins directly into eukaryotic cells. This sequence provides a new means to identify substrates of type III secretion systems. Indeed, using a pattern search algorithm for the CCBD sequence, we have identified the first two probable effectors from an endosymbiont, Sodalis glossinidius. IMPORTANCE: Many Gram-negative pathogens utilize type 3 secretion systems to deliver tens of effectors into host cells. In order to understand the diverse ways that these organisms cause disease, it is necessary to identify their effectors, many of which require chaperones to be secreted. Here we establish that class IB chaperones are not promiscuous, as previously proposed, but rather recognize a conserved effector sequence. We demonstrate that pattern search algorithms based on this defined sequence can be used to identify previously unknown effectors. Furthermore, we observe that class IB chaperones from at least seven bacterial species are functionally interchangeable. Not only do they bind and mediate the delivery of their own set of effectors into host cells but they also bind to type 3 substrates from other bacteria, suggesting that inhibitors that block chaperone-effector interactions could provide a novel means to effectively treat infections due to Gram-negative pathogens, including organisms resistant to currently available antibiotics.

Myospryn is a calcineurin-interacting protein that negatively modulates slow-fiber-type transformation and skeletal muscle regeneration.

The calcium-calmodulin-regulated protein phosphatase calcineurin plays an important regulatory role in muscle differentiation, fiber-type determination, hypertrophy, and muscle regeneration. Because calcineurin functions in numerous processes in muscle, multiple mechanisms are likely necessary to ensure that the activity of this phosphatase is appropriately regulated. Here we demonstrate that the muscle-specific scaffolding protein myospryn modulates calcineurin signaling by inhibiting calcineurin-dependent transcriptional activity in C2C12 myoblasts through direct interaction with the enzyme via its noncanonical tripartite motif (TRIM-like). Consistent with these data, transgenic mice overexpressing both the TRIM-like domain of myospryn and constitutively active calcineurin displayed a severe attenuation in the ability of calcineurin to induce a slow-fiber phenotype. Furthermore, transgenic mice overexpressing the TRIM-like domain of myospryn displayed attenuated muscle regeneration after cardiotoxin-induced muscle injury. These results indicate that myospryn functions as a novel inhibitor of the calcineurin signaling pathway in skeletal muscle.