Genetic analysis of cancer drivers reveals cohesin and CTCF as suppressors of PD-L1.

Immune evasion is a significant contributor to tumor evolution, and the immunoinhibitory axis PD-1/PD-L1 is a frequent mechanism employed to escape tumor immune surveillance. To identify cancer drivers involved in immune evasion, we performed a CRISPR-Cas9 screen of tumor suppressor genes regulating the basal and interferon (IFN)-inducible cell surface levels of PD-L1. Multiple regulators of PD-L1 were identified, including IRF2, ARID2, KMT2D, and AAMP. We also identified CTCF and the cohesin complex proteins, known regulators of chromatin architecture and transcription, among the most potent negative regulators of PD-L1 cell surface expression. Additionally, loss of the cohesin subunit RAD21 was shown to up-regulate PD-L2 and MHC-I surface expression. PD-L1 and MHC-I suppression by cohesin were shown to be conserved in mammary epithelial and myeloid cells. Comprehensive examination of the transcriptional effect of STAG2 deficiency in epithelial and myeloid cells revealed an activation of strong IFN and NF-κB expression signatures. Inhibition of JAK-STAT or NF-κB pathways did not result in rescue of PD-L1 up-regulation in RAD21-deficient cells, suggesting more complex or combinatorial mechanisms at play. Discovery of the PD-L1 and IFN up-regulation in cohesin-mutant cells expands our understanding of the biology of cohesin-deficient cells as well as molecular regulation of the PD-L1 molecule.

Identification of FUBP1 as a Long Tail Cancer Driver and Widespread Regulator of Tumor Suppressor and Oncogene Alternative Splicing.

Comprehensive sequencing approaches have allowed for the identification of the most frequent contributors to cancer, known as drivers. They have also revealed a class of mutations in understudied, infrequently altered genes, referred to as "long tail" (LT) drivers. A key challenge has been to find clinically relevant LT drivers and to understand how they cooperate to drive disease. Here, we identified far upstream binding protein 1 (FUBP1) as an LT driver using an in vivo CRISPR screen. FUBP1 cooperates with other tumor suppressor genes to transform mammary epithelial cells by disrupting cellular differentiation and tissue architecture. Mechanistically, FUBP1 participates in regulating N6-methyladenosine (m6A) RNA methylation, and its loss leads to global changes in RNA splicing and widespread expression of aberrant driver isoforms. These findings suggest that somatic alteration of a single gene involved in RNA splicing and m6A methylation can produce the necessary panoply of contributors for neoplastic transformation.

Genetic Screens Reveal FEN1 and APEX2 as BRCA2 Synthetic Lethal Targets.

BRCA1 or BRCA2 inactivation drives breast and ovarian cancer but also creates vulnerability to poly(ADP-ribose) polymerase (PARP) inhibitors. To search for additional targets whose inhibition is synthetically lethal in BRCA2-deficient backgrounds, we screened two pairs of BRCA2 isogenic cell lines with DNA-repair-focused small hairpin RNA (shRNA) and CRISPR (clustered regularly interspaced short palindromic repeats)-based libraries. We found that BRCA2-deficient cells are selectively dependent on multiple pathways including base excision repair, ATR signaling, and splicing. We identified APEX2 and FEN1 as synthetic lethal genes with both BRCA1 and BRCA2 loss of function. BRCA2-deficient cells require the apurinic endonuclease activity and the PCNA-binding domain of Ape2 (APEX2), but not Ape1 (APEX1). Furthermore, BRCA2-deficient cells require the 5' flap endonuclease but not the 5'-3' exonuclease activity of Fen1, and chemically inhibiting Fen1 selectively targets BRCA-deficient cells. Finally, we developed a microhomology-mediated end-joining (MMEJ) reporter and showed that Fen1 participates in MMEJ, underscoring the importance of MMEJ as a collateral repair pathway in the context of homologous recombination (HR) deficiency.

Functional genomics reveals that tumors with activating phosphoinositide 3-kinase mutations are dependent on accelerated protein turnover.

Activating mutations in the phosphoinositide 3-kinase (PI3K) signaling pathway are frequently identified in cancer. To identify pathways that support PI3K oncogenesis, we performed a genome-wide RNAi screen in isogenic cell lines harboring wild-type or mutant PIK3CA to search for PI3K synthetic-lethal (SL) genes. A combined analysis of these results with a meta-analysis of two other large-scale RNAi screening data sets in PI3K mutant cancer cell lines converged on ribosomal protein translation and proteasomal protein degradation as critical nononcogene dependencies for PI3K-driven tumors. Genetic or pharmacologic inhibition of either pathway alone, but not together, selectively killed PI3K mutant tumor cells in an mTOR-dependent manner. The expression of ribosomal and proteasomal components was significantly up-regulated in primary human colorectal tumors harboring PI3K pathway activation. Importantly, a PI3K SL gene signature containing the top hits of the SL genes identified in our meta-analysis robustly predicted overall patient survival in colorectal cancer, especially among patients with tumors with an activated PI3K pathway. These results suggest that disruption of protein turnover homeostasis via ribosome or proteasome inhibition may be a novel treatment strategy for PI3K mutant human tumors.

Cumulative haploinsufficiency and triplosensitivity drive aneuploidy patterns and shape the cancer genome.

Aneuploidy has been recognized as a hallmark of cancer for more than 100 years, yet no general theory to explain the recurring patterns of aneuploidy in cancer has emerged. Here, we develop Tumor Suppressor and Oncogene (TUSON) Explorer, a computational method that analyzes the patterns of mutational signatures in tumors and predicts the likelihood that any individual gene functions as a tumor suppressor (TSG) or oncogene (OG). By analyzing >8,200 tumor-normal pairs, we provide statistical evidence suggesting that many more genes possess cancer driver properties than anticipated, forming a continuum of oncogenic potential. Integrating our driver predictions with information on somatic copy number alterations, we find that the distribution and potency of TSGs (STOP genes), OGs, and essential genes (GO genes) on chromosomes can predict the complex patterns of aneuploidy and copy number variation characteristic of cancer genomes. We propose that the cancer genome is shaped through a process of cumulative haploinsufficiency and triplosensitivity.

LPS ligand and culture additives improve production of monomeric MD-1 and 2 in Pichia pastoris by decreasing aggregation and intermolecular disulfide bonding.

Myeloid differentiation proteins MD-1 and MD-2 have both been shown to form a heterogeneous collection of oligomers when expressed in absence of their respective receptor, RP105 and TLR4. The biological relevance of these oligomers is not clear. Only monomeric proteins have been found to be active and able to trigger an immune response to endotoxin by modulating the TLR4 pathway. In this study, we produced variants of MD-1 and MD-2 in Pichia pastoris. To minimize the time and expense of initial expression tests, small-scale cultures have been set up to allow the rapid identification of the highest expressing clone and the optimal expression conditions. The expression vectors used, the site of linearization and the locus of integration affected the yield of transformation. Next we screened culture additives and found that they significantly increased the fraction of monomeric proteins secreted in the culture medium (up to 15% of the total MD protein produced). We confirmed their presence by size-exclusion chromatography. Optimal anti-aggregation agents were protein-dependent except for LPS that presented stabilizing effects for all MD proteins. Contrary to previous reports, this study suggests that MD-1 can bind to LPS.

The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP.

Lung cancers caused by activating mutations in the epidermal growth factor receptor (EGFR) are initially responsive to small molecule tyrosine kinase inhibitors (TKIs), but the efficacy of these agents is often limited because of the emergence of drug resistance conferred by a second mutation, T790M. Threonine 790 is the "gatekeeper" residue, an important determinant of inhibitor specificity in the ATP binding pocket. The T790M mutation has been thought to cause resistance by sterically blocking binding of TKIs such as gefitinib and erlotinib, but this explanation is difficult to reconcile with the fact that it remains sensitive to structurally similar irreversible inhibitors. Here, we show by using a direct binding assay that T790M mutants retain low-nanomolar affinity for gefitinib. Furthermore, we show that the T790M mutation activates WT EGFR and that introduction of the T790M mutation increases the ATP affinity of the oncogenic L858R mutant by more than an order of magnitude. The increased ATP affinity is the primary mechanism by which the T790M mutation confers drug resistance. Crystallographic analysis of the T790M mutant shows how it can adapt to accommodate tight binding of diverse inhibitors, including the irreversible inhibitor HKI-272, and also suggests a structural mechanism for catalytic activation. We conclude that the T790M mutation is a "generic" resistance mutation that will reduce the potency of any ATP-competitive kinase inhibitor and that irreversible inhibitors overcome this resistance simply through covalent binding, not as a result of an alternative binding mode.

Hirudin binding reveals key determinants of thrombin allostery.

Thrombin exists in two allosteric forms, slow (S) and fast (F), that recognize natural substrates and inhibitors with significantly different affinities. Because under physiologic conditions the two forms are almost equally populated, investigation of thrombin function must address the contribution from the S and F forms and the molecular origin of their differential recognition of ligands. Using a panel of 79 Ala mutants, we have mapped for the first time the epitopes of thrombin recognizing a macromolecular ligand, hirudin, in the S and F forms. Hirudin binding is a relevant model for the interaction of thrombin with fibrinogen and PAR1 and is likewise influenced by the allosteric S-->F transition. The epitopes are nearly identical and encompass two hot spots, one in exosite I and the other in the Na+ site at the opposite end of the protein. The higher affinity of the F form is due to the preferential interaction of hirudin with Lys-36, Leu-65, Thr-74, and Arg-75 in exosite I; Gly-193 in the oxyanion hole; and Asp-221 and Asp-222 in the Na+ site. Remarkably, no correlation is found between the energetic and structural involvements of thrombin residues in hirudin recognition, which invites caution in the analysis of protein-protein interactions in general.