Targeting the non-coding genome and temozolomide signature enables CRISPR-mediated glioma oncolysis.

Glioblastoma (GBM) is the most common lethal primary brain cancer in adults. Despite treatment regimens including surgical resection, radiotherapy, and temozolomide (TMZ) chemotherapy, growth of residual tumor leads to therapy resistance and death. At recurrence, a quarter to a third of all gliomas have hypermutated genomes, with mutational burdens orders of magnitude greater than in normal tissue. Here, we quantified the mutational landscape progression in a patient's primary and recurrent GBM, and we uncovered Cas9-targetable repeat elements. We show that CRISPR-mediated targeting of highly repetitive loci enables rapid elimination of GBM cells, an approach we term "genome shredding." Importantly, in the patient's recurrent GBM, we identified unique repeat sequences with TMZ mutational signature and demonstrated that their CRISPR targeting enables cancer-specific cell ablation. "Cancer shredding" leverages the non-coding genome and therapy-induced mutational signatures for targeted GBM cell depletion and provides an innovative paradigm to develop treatments for hypermutated glioma.

Endogenous spacing enables co-processing of microRNAs and efficient combinatorial RNAi.

We present Multi-miR, a microRNA-embedded shRNA system modeled after endogenous microRNA clusters that enables simultaneous expression of up to three or four short hairpin RNAs (shRNAs) from a single promoter without loss of activity, enabling robust combinatorial RNA interference (RNAi). We further developed complementary all-in-one vectors that are over one log-scale more sensitive to doxycycline-mediated activation in vitro than previous methods and resistant to shRNA inactivation in vivo. We demonstrate the utility of this system for intracranial expression of shRNAs in a glioblastoma model. Additionally, we leverage this platform to target the redundant RAF signaling node in a mouse model of KRAS-mutant cancer and show that robust combinatorial synthetic lethality efficiently abolishes tumor growth.

Comprehensive deletion landscape of CRISPR-Cas9 identifies minimal RNA-guided DNA-binding modules.

Proteins evolve through the modular rearrangement of elements known as domains. Extant, multidomain proteins are hypothesized to be the result of domain accretion, but there has been limited experimental validation of this idea. Here, we introduce a technique for genetic minimization by iterative size-exclusion and recombination (MISER) for comprehensively making all possible deletions of a protein. Using MISER, we generate a deletion landscape for the CRISPR protein Cas9. We find that the catalytically-dead Streptococcus pyogenes Cas9 can tolerate large single deletions in the REC2, REC3, HNH, and RuvC domains, while still functioning in vitro and in vivo, and that these deletions can be stacked together to engineer minimal, DNA-binding effector proteins. In total, our results demonstrate that extant proteins retain significant modularity from the accretion process and, as genetic size is a major limitation for viral delivery systems, establish a general technique to improve genome editing and gene therapy-based therapeutics.

PI5P4Ks drive metabolic homeostasis through peroxisome-mitochondria interplay.

PI5P4Ks are a class of phosphoinositide kinases that phosphorylate PI-5-P to PI-4,5-P2. Distinct localization of phosphoinositides is fundamental for a multitude of cellular functions. Here, we identify a role for peroxisomal PI-4,5-P2 generated by the PI5P4Ks in maintaining energy balance. We demonstrate that PI-4,5-P2 regulates peroxisomal fatty acid oxidation by mediating trafficking of lipid droplets to peroxisomes, which is essential for sustaining mitochondrial metabolism. Using fluorescent-tagged lipids and metabolite tracing, we show that loss of the PI5P4Ks significantly impairs lipid uptake and ß-oxidation in the mitochondria. Further, loss of PI5P4Ks results in dramatic alterations in mitochondrial structural and functional integrity, which under nutrient deprivation is further exacerbated, causing cell death. Notably, inhibition of the PI5P4Ks in cancer cells and mouse tumor models leads to decreased cell viability and tumor growth, respectively. Together, these studies reveal an unexplored role for PI5P4Ks in preserving metabolic homeostasis, which is necessary for tumorigenesis.

Cancer-specific loss of TERT activation sensitizes glioblastoma to DNA damage.

Most glioblastomas (GBMs) achieve cellular immortality by acquiring a mutation in the telomerase reverse transcriptase (TERT) promoter. TERT promoter mutations create a binding site for a GA binding protein (GABP) transcription factor complex, whose assembly at the promoter is associated with TERT reactivation and telomere maintenance. Here, we demonstrate increased binding of a specific GABPB1L-isoform-containing complex to the mutant TERT promoter. Furthermore, we find that TERT promoter mutant GBM cells, unlike wild-type cells, exhibit a critical near-term dependence on GABPB1L for proliferation, notably also posttumor establishment in vivo. Up-regulation of the protein paralogue GABPB2, which is normally expressed at very low levels, can rescue this dependence. More importantly, when combined with frontline temozolomide (TMZ) chemotherapy, inducible GABPB1L knockdown and the associated TERT reduction led to an impaired DNA damage response that resulted in profoundly reduced growth of intracranial GBM tumors. Together, these findings provide insights into the mechanism of cancer-specific TERT regulation, uncover rapid effects of GABPB1L-mediated TERT suppression in GBM maintenance, and establish GABPB1L inhibition in combination with chemotherapy as a therapeutic strategy for TERT promoter mutant GBM.

Site-Specific Bioconjugation through Enzyme-Catalyzed Tyrosine-Cysteine Bond Formation.

The synthesis of protein-protein and protein-peptide conjugates is an important capability for producing vaccines, immunotherapeutics, and targeted delivery agents. Herein we show that the enzyme tyrosinase is capable of oxidizing exposed tyrosine residues into o-quinones that react rapidly with cysteine residues on target proteins. This coupling reaction occurs under mild aerobic conditions and has the rare ability to join full-size proteins in under 2 h. The utility of the approach is demonstrated for the attachment of cationic peptides to enhance the cellular delivery of CRISPR-Cas9 20-fold and for the coupling of reporter proteins to a cancer-targeting antibody fragment without loss of its cell-specific binding ability. The broad applicability of this technique provides a new building block approach for the synthesis of protein chimeras.

MAP kinase and autophagy pathways cooperate to maintain RAS mutant cancer cell survival.

Oncogenic mutations in the small GTPase KRAS are frequently found in human cancers, and, currently, there are no effective targeted therapies for these tumors. Using a combinatorial siRNA approach, we analyzed a panel of KRAS mutant colorectal and pancreatic cancer cell lines for their dependency on 28 gene nodes that represent canonical RAS effector pathways and selected stress response pathways. We found that RAF node knockdown best differentiated KRAS mutant and KRAS WT cancer cells, suggesting RAF kinases are key oncoeffectors for KRAS addiction. By analyzing all 376 pairwise combination of these gene nodes, we found that cotargeting the RAF, RAC, and autophagy pathways can improve the capture of KRAS dependency better than targeting RAF alone. In particular, codepletion of the oncoeffector kinases BRAF and CRAF, together with the autophagy E1 ligase ATG7, gives the best therapeutic window between KRAS mutant cells and normal, untransformed cells. Distinct patterns of RAS effector dependency were observed across KRAS mutant cell lines, indicative of heterogeneous utilization of effector and stress response pathways in supporting KRAS addiction. Our findings revealed previously unappreciated complexity in the signaling network downstream of the KRAS oncogene and suggest rational target combinations for more effective therapeutic intervention.

Disruption of the ß1L Isoform of GABP Reverses Glioblastoma Replicative Immortality in a TERT Promoter Mutation-Dependent Manner.

TERT promoter mutations reactivate telomerase, allowing for indefinite telomere maintenance and enabling cellular immortalization. These mutations specifically recruit the multimeric ETS factor GABP, which can form two functionally independent transcription factor species: a dimer or a tetramer. We show that genetic disruption of GABPß1L (ß1L), a tetramer-forming isoform of GABP that is dispensable for normal development, results in TERT silencing in a TERT promoter mutation-dependent manner. Reducing TERT expression by disrupting ß1L culminates in telomere loss and cell death exclusively in TERT promoter mutant cells. Orthotopic xenografting of ß1L-reduced, TERT promoter mutant glioblastoma cells rendered lower tumor burden and longer overall survival in mice. These results highlight the critical role of GABPß1L in enabling immortality in TERT promoter mutant glioblastoma.

Extension of the crRNA enhances Cpf1 gene editing in vitro and in vivo.

Engineering of the Cpf1 crRNA has the potential to enhance its gene editing efficiency and non-viral delivery to cells. Here, we demonstrate that extending the length of its crRNA at the 5' end can enhance the gene editing efficiency of Cpf1 both in cells and in vivo. Extending the 5' end of the crRNA enhances the gene editing efficiency of the Cpf1 RNP to induce non-homologous end-joining and homology-directed repair using electroporation in cells. Additionally, chemical modifications on the extended 5' end of the crRNA result in enhanced serum stability. Also, extending the 5' end of the crRNA by 59 nucleotides increases the delivery efficiency of Cpf1 RNP in cells and in vivo cationic delivery vehicles including polymer nanoparticle. Thus, 5' extension and chemical modification of the Cpf1 crRNA is an effective method for enhancing the gene editing efficiency of Cpf1 and its delivery in vivo.

Differential Effector Engagement by Oncogenic KRAS.

KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention.

Profiling of engineering hotspots identifies an allosteric CRISPR-Cas9 switch.

The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated protein Cas9 from Streptococcus pyogenes is an RNA-guided DNA endonuclease with widespread utility for genome modification. However, the structural constraints limiting the engineering of Cas9 have not been determined. Here we experimentally profile Cas9 using randomized insertional mutagenesis and delineate hotspots in the structure capable of tolerating insertions of a PDZ domain without disruption of the enzyme's binding and cleavage functions. Orthogonal domains or combinations of domains can be inserted into the identified sites with minimal functional consequence. To illustrate the utility of the identified sites, we construct an allosterically regulated Cas9 by insertion of the estrogen receptor-α ligand-binding domain. This protein showed robust, ligand-dependent activation in prokaryotic and eukaryotic cells, establishing a versatile one-component system for inducible and reversible Cas9 activation. Thus, domain insertion profiling facilitates the rapid generation of new Cas9 functionalities and provides useful data for future engineering of Cas9.

MYC Drives Pten/Trp53-Deficient Proliferation and Metastasis due to IL6 Secretion and AKT Suppression via PHLPP2.

UNLABELLED: We have recently recapitulated metastasis of human PTEN/TP53-mutant prostate cancer in the mouse using the RapidCaP system. Surprisingly, we found that this metastasis is driven by MYC, and not AKT, activation. Here, we show that cell-cell communication by IL6 drives the AKT-MYC switch through activation of the AKT-suppressing phosphatase PHLPP2, when PTEN and p53 are lost together, but not separately. IL6 then communicates a downstream program of STAT3-mediated MYC activation, which drives cell proliferation. Similarly, in tissues, peak proliferation in Pten/Trp53-mutant primary and metastatic prostate cancer does not correlate with activated AKT, but with STAT3/MYC activation instead. Mechanistically, MYC strongly activates the AKT phosphatase PHLPP2 in primary cells and prostate cancer metastasis. We show genetically that Phlpp2 is essential for dictating the proliferation of MYC-mediated AKT suppression. Collectively, our data reveal competition between two proto-oncogenes, MYC and AKT, which ensnarls the Phlpp2 gene to facilitate MYC-driven prostate cancer metastasis after loss of Pten and Trp53. SIGNIFICANCE: Our data identify IL6 detection as a potential causal biomarker for MYC-driven metastasis after loss of PTEN and p53. Second, our finding that MYC then must supersede AKT to drive cell proliferation points to MYC inhibition as a critical part of PI3K pathway therapy in lethal prostate cancer.

A computational algorithm to predict shRNA potency.

The strength of conclusions drawn from RNAi-based studies is heavily influenced by the quality of tools used to elicit knockdown. Prior studies have developed algorithms to design siRNAs. However, to date, no established method has emerged to identify effective shRNAs, which have lower intracellular abundance than transfected siRNAs and undergo additional processing steps. We recently developed a multiplexed assay for identifying potent shRNAs and used this method to generate ∼250,000 shRNA efficacy data points. Using these data, we developed shERWOOD, an algorithm capable of predicting, for any shRNA, the likelihood that it will elicit potent target knockdown. Combined with additional shRNA design strategies, shERWOOD allows the ab initio identification of potent shRNAs that specifically target the majority of each gene's multiple transcripts. We validated the performance of our shRNA designs using several orthogonal strategies and constructed genome-wide collections of shRNAs for humans and mice based on our approach.

Translation initiation factor eIF4F modifies the dexamethasone response in multiple myeloma.

Enhanced protein synthesis capacity is associated with increased tumor cell survival, proliferation, and resistance to chemotherapy. Cancers like multiple myeloma (MM), which display elevated activity in key translation regulatory nodes, such as the PI3K/mammalian target of rapamycin and MYC-eukaryotic initiation factor (eIF) 4E pathways, are predicted to be particularly sensitive to therapeutic strategies that target this process. To identify novel vulnerabilities in MM, we undertook a focused RNAi screen in which components of the translation apparatus were targeted. Our screen was designed to identify synthetic lethal relationships between translation factors or regulators and dexamethasone (DEX), a corticosteroid used as frontline therapy in this disease. We find that suppression of all three subunits of the eIF4F cap-binding complex synergizes with DEX in MM to induce cell death. Using a suite of small molecules that target various activities of eIF4F, we observed that cell survival and DEX resistance are attenuated upon eIF4F inhibition in MM cell lines and primary human samples. Levels of MYC and myeloid cell leukemia 1, two known eIF4F-responsive transcripts and key survival factors in MM, were reduced upon eIF4F inhibition, and their independent suppression also synergized with DEX. Inhibition of eIF4F in MM exerts pleotropic effects unraveling a unique therapeutic opportunity.

Development of siRNA payloads to target KRAS-mutant cancer.

UNLABELLED: RNAi is a powerful tool for target identification and can lead to novel therapies for pharmacologically intractable targets such as KRAS. RNAi therapy must combine potent siRNA payloads with reliable in vivo delivery for efficient target inhibition. We used a functional "Sensor" assay to establish a library of potent siRNAs against RAS pathway genes and to show that they efficiently suppress their targets at low dose. This reduces off-target effects and enables combination gene knockdown. We administered Sensor siRNAs in vitro and in vivo and validated the delivery of KRAS siRNA alone and siRNA targeting the complete RAF effector node (A/B/CRAF) as promising strategies to treat KRAS-mutant colorectal cancer. We further demonstrate that improved therapeutic efficacy is achieved by formulating siRNA payloads that combine both single-gene siRNA and node-targeted siRNAs (KRAS + PIK3CA/B). The customizable nature of Sensor siRNA payloads offers a universal platform for the combination target identification and development of RNAi therapeutics. SIGNIFICANCE: To advance RNAi therapy for KRAS-mutant cancer, we developed a validated siRNA library against RAS pathway genes that enables combination gene silencing. Using an in vivo model for real-time siRNA delivery tracking, we show that siRNA-mediated inhibition of KRAS as well as RAF or PI3K combinations can impair KRAS-mutant colorectal cancer in xenograft models.

Disruption of CRAF-mediated MEK activation is required for effective MEK inhibition in KRAS mutant tumors.

MEK inhibitors are clinically active in BRAF(V600E) melanomas but only marginally so in KRAS mutant tumors. Here, we found that MEK inhibitors suppress ERK signaling more potently in BRAF(V600E), than in KRAS mutant tumors. To understand this, we performed an RNAi screen in a KRAS mutant model and found that CRAF knockdown enhanced MEK inhibition. MEK activated by CRAF was less susceptible to MEK inhibitors than when activated by BRAF(V600E). MEK inhibitors induced RAF-MEK complexes in KRAS mutant models, and disrupting such complexes enhanced inhibition of CRAF-dependent ERK signaling. Newer MEK inhibitors target MEK catalytic activity and also impair its reactivation by CRAF, either by disrupting RAF-MEK complexes or by interacting with Ser 222 to prevent MEK phosphorylation by RAF.

An optimized microRNA backbone for effective single-copy RNAi.

Short hairpin RNA (shRNA) technology enables stable and regulated gene repression. For establishing experimentally versatile RNAi tools and minimizing toxicities, synthetic shRNAs can be embedded into endogenous microRNA contexts. However, due to our incomplete understanding of microRNA biogenesis, such "shRNAmirs" often fail to trigger potent knockdown, especially when expressed from a single genomic copy. Following recent advances in design of synthetic shRNAmir stems, here we take a systematic approach to optimize the experimental miR-30 backbone. Among several favorable features, we identify a conserved element 3' of the basal stem as critically required for optimal shRNAmir processing and implement it in an optimized backbone termed "miR-E", which strongly increases mature shRNA levels and knockdown efficacy. Existing miR-30 reagents can be easily converted to miR-E, and its combination with up-to-date design rules establishes a validated and accessible platform for generating effective single-copy shRNA libraries that will facilitate the functional annotation of the genome.

Tiling genomes of pathogenic viruses identifies potent antiviral shRNAs and reveals a role for secondary structure in shRNA efficacy.

shRNAs can trigger effective silencing of gene expression in mammalian cells, thereby providing powerful tools for genetic studies, as well as potential therapeutic strategies. Specific shRNAs can interfere with the replication of pathogenic viruses and are currently being tested as antiviral therapies in clinical trials. However, this effort is hindered by our inability to systematically and accurately identify potent shRNAs for viral genomes. Here we apply a recently developed highly parallel sensor assay to identify potent shRNAs for HIV, hepatitis C virus (HCV), and influenza. We observe known and previously unknown sequence features that dictate shRNAs efficiency. Validation using HIV and HCV cell culture models demonstrates very high potency of the top-scoring shRNAs. Comparing our data with the secondary structure of HIV shows that shRNA efficacy is strongly affected by the secondary structure at the target RNA site. Artificially introducing secondary structure to the target site markedly reduces shRNA silencing. In addition, we observe that HCV has distinct sequence features that bias HCV-targeting shRNAs toward lower efficacy. Our results facilitate further development of shRNA based antiviral therapies and improve our understanding and ability to predict efficient shRNAs.

An integrated approach to dissecting oncogene addiction implicates a Myb-coordinated self-renewal program as essential for leukemia maintenance.

Although human cancers have complex genotypes and are genomically unstable, they often remain dependent on the continued presence of single-driver mutations-a phenomenon dubbed "oncogene addiction." Such dependencies have been demonstrated in mouse models, where conditional expression systems have revealed that oncogenes able to initiate cancer are often required for tumor maintenance and progression, thus validating the pathways they control as therapeutic targets. Here, we implement an integrative approach that combines genetically defined mouse models, transcriptional profiling, and a novel inducible RNAi platform to characterize cellular programs that underlie addiction to MLL-AF9-a fusion oncoprotein involved in aggressive forms of acute myeloid leukemia (AML). We show that MLL-AF9 contributes to leukemia maintenance by enforcing a Myb-coordinated program of aberrant self-renewal involving genes linked to leukemia stem cell potential and poor prognosis in human AML. Accordingly, partial and transient Myb suppression precisely phenocopies MLL-AF9 withdrawal and eradicates aggressive AML in vivo without preventing normal myelopoiesis, indicating that strategies to inhibit Myb-dependent aberrant self-renewal programs hold promise as effective and cancer-specific therapeutics. Together, our results identify Myb as a critical mediator of oncogene addiction in AML, delineate relevant Myb target genes that are amenable to pharmacologic inhibition, and establish a general approach for dissecting oncogene addiction in vivo.

Toolkit for evaluating genes required for proliferation and survival using tetracycline-regulated RNAi.

Short hairpin RNAs (shRNAs) are versatile tools for analyzing loss-of-function phenotypes in vitro and in vivo. However, their use for studying genes involved in proliferation and survival, which are potential therapeutic targets in cancer and other diseases, is confounded by the strong selective advantage of cells in which shRNA expression is inefficient. We therefore developed a toolkit that combines Tet-regulated miR30-shRNA technology, robust transactivator expression and two fluorescent reporters to track and isolate cells with potent target knockdown. We demonstrated that this system improves the study of essential genes and was sufficiently robust to eradicate aggressive cancer in mice by suppressing a single gene. Further, we applied this system for in vivo negative-selection screening with pooled shRNAs and propose a streamlined, inexpensive workflow that will facilitate the use of RNA interference (RNAi) for the identification and evaluation of essential therapeutic targets.