Pichai Raman

The Cancer Cell Line Encyclopedia enables predictive modelling of anticancer drug sensitivity.

The systematic translation of cancer genomic data into knowledge of tumour biology and therapeutic possibilities remains challenging. Such efforts should be greatly aided by robust preclinical model systems that reflect the genomic diversity of human cancers and for which detailed genetic and pharmacological annotation is available. Here we describe the Cancer Cell Line Encyclopedia (CCLE): a compilation of gene expression, chromosomal copy number and massively parallel sequencing data from 947 human cancer cell lines. When coupled with pharmacological profiles for 24 anticancer drugs across 479 of the cell lines, this collection allowed identification of genetic, lineage, and gene-expression-based predictors of drug sensitivity. In addition to known predictors, we found that plasma cell lineage correlated with sensitivity to IGF1 receptor inhibitors; AHR expression was associated with MEK inhibitor efficacy in NRAS-mutant lines; and SLFN11 expression predicted sensitivity to topoisomerase inhibitors. Together, our results indicate that large, annotated cell-line collections may help to enable preclinical stratification schemata for anticancer agents. The generation of genetic predictions of drug response in the preclinical setting and their incorporation into cancer clinical trial design could speed the emergence of ‘personalized’ therapeutic regimens.

Activation of a metabolic gene regulatory network downstream of mTOR complex 1.

Aberrant activation of the mammalian target of rapamycin complex 1 (mTORC1) is a common molecular event in a variety of pathological settings, including genetic tumor syndromes, cancer, and obesity. However, the cell-intrinsic consequences of mTORC1 activation remain poorly defined. Through a combination of unbiased genomic, metabolomic, and bioinformatic approaches, we demonstrate that mTORC1 activation is sufficient to stimulate specific metabolic pathways, including glycolysis, the oxidative arm of the pentose phosphate pathway, and de novo lipid biosynthesis. This is achieved through the activation of a transcriptional program affecting metabolic gene targets of hypoxia-inducible factor (HIF1alpha) and sterol regulatory element-binding protein (SREBP1 and SREBP2). We find that SREBP1 and 2 promote proliferation downstream of mTORC1, and the activation of these transcription factors is mediated by S6K1. Therefore, in addition to promoting protein synthesis, mTORC1 activates specific bioenergetic and anabolic cellular processes that are likely to contribute to human physiology and disease.

Convergence of Acquired Mutations and Alternative Splicing of CD19 Enables Resistance to CART-19 Immunotherapy

UNLABELLED The CD19 antigen, expressed on most B-cell acute lymphoblastic leukemias (B-ALL), can be targeted with chimeric antigen receptor-armed T cells (CART-19), but relapses with epitope loss occur in 10% to 20% of pediatric responders. We detected hemizygous deletions spanning the CD19 locus and de novo frameshift and missense mutations in exon 2 of CD19 in some relapse samples. However, we also discovered alternatively spliced CD19 mRNA species, including one lacking exon 2. Pull-down/siRNA experiments identified SRSF3 as a splicing factor involved in exon 2 retention, and its levels were lower in relapsed B-ALL. Using genome editing, we demonstrated that exon 2 skipping bypasses exon 2 mutations in B-ALL cells and allows expression of the N-terminally truncated CD19 variant, which fails to trigger killing by CART-19 but partly rescues defects associated with CD19 loss. Thus, this mechanism of resistance is based on a combination of deleterious mutations and ensuing selection for alternatively spliced RNA isoforms. SIGNIFICANCE CART-19 yield 70% response rates in patients with B-ALL, but also produce escape variants. We discovered that the underlying mechanism is the selection for preexisting alternatively spliced CD19 isoforms with the compromised CART-19 epitope. This mechanism suggests a possibility of targeting alternative CD19 ectodomains, which could improve survival of patients with B-cell neoplasms.

Calcium-activated chloride channel ANO1 promotes breast cancer progression by activating EGFR and CAMK signaling

The calcium-activated chloride channel anoctamin 1 (ANO1) is located within the 11q13 amplicon, one of the most frequently amplified chromosomal regions in human cancer, but its functional role in tumorigenesis has remained unclear. The 11q13 region is amplified in ∼15% of breast cancers. Whether ANO1 is amplified in breast tumors, the extent to which gene amplification contributes to ANO1 overexpression, and whether overexpression of ANO1 is important for tumor maintenance have remained unknown. We have found that ANO1 is amplified and highly expressed in breast cancer cell lines and primary tumors. Amplification of ANO1 correlated with disease grade and poor prognosis. Knockdown of ANO1 in ANO1-amplified breast cancer cell lines and other cancers bearing 11q13 amplification inhibited proliferation, induced apoptosis, and reduced tumor growth in established cancer xenografts. Moreover, ANO1 chloride channel activity was important for cell viability. Mechanistically, ANO1 knockdown or pharmacological inhibition of its chloride-channel activity reduced EGF receptor (EGFR) and calmodulin-dependent protein kinase II (CAMKII) signaling, which subsequently attenuated AKT, v-src sarcoma viral oncogene homolog (SRC), and extracellular signal-regulated kinase (ERK) activation in vitro and in vivo. Our results highlight the involvement of the ANO1 chloride channel in tumor progression and provide insights into oncogenic signaling in human cancers with 11q13 amplification, thereby establishing ANO1 as a promising target for therapy in these highly prevalent tumor types.

CASC15-S Is a Tumor Suppressor lncRNA at the 6p22 Neuroblastoma Susceptibility Locus.

Chromosome 6p22 was identified recently as a neuroblastoma susceptibility locus, but its mechanistic contributions to tumorigenesis are as yet undefined. Here we report that the most highly significant single-nucleotide polymorphism (SNP) associations reside within CASC15, a long noncoding RNA that we define as a tumor suppressor at 6p22. Low-level expression of a short CASC15 isoform (CASC15-S) associated highly with advanced neuroblastoma and poor patient survival. In human neuroblastoma cells, attenuating CASC15-S increased cellular growth and migratory capacity. Gene expression analysis revealed downregulation of neuroblastoma-specific markers in cells with attenuated CASC15-S, with concomitant increases in cell adhesion and extracellular matrix transcripts. Altogether, our results point to CASC15-S as a mediator of neural growth and differentiation, which impacts neuroblastoma initiation and progression.

A macrocyclic HCV NS3/4A protease inhibitor interacts with protease and helicase residues in the complex with its full-length target

Hepatitis C virus (HCV) infection is a global health burden with over 170 million people infected worldwide. In a significant portion of patients chronic hepatitis C infection leads to serious liver diseases, including fibrosis, cirrhosis, and hepatocellular carcinoma. The HCV NS3 protein is essential for viral polyprotein processing and RNA replication and hence viral replication. It is composed of an N-terminal serine protease domain and a C-terminal helicase/NTPase domain. For full activity, the protease requires the NS4A protein as a cofactor. HCV NS3/4A protease is a prime target for developing direct-acting antiviral agents. First-generation NS3/4A protease inhibitors have recently been introduced into clinical practice, markedly changing HCV treatment options. To date, crystal structures of HCV NS3/4A protease inhibitors have only been reported in complex with the protease domain alone. Here, we present a unique structure of an inhibitor bound to the full-length, bifunctional protease-helicase NS3/4A and show that parts of the P4 capping and P2 moieties of the inhibitor interact with both protease and helicase residues. The structure sheds light on inhibitor binding to the more physiologically relevant form of the enzyme and supports exploring inhibitor-helicase interactions in the design of the next generation of HCV NS3/4A protease inhibitors. In addition, small angle X-ray scattering confirmed the observed protease-helicase domain assembly in solution.

Englerin A Agonizes the TRPC4/C5 Cation Channels to Inhibit Tumor Cell Line Proliferation.

Englerin A is a structurally unique natural product reported to selectively inhibit growth of renal cell carcinoma cell lines. A large scale phenotypic cell profiling experiment (CLiP) of englerin A on ¬over 500 well characterized cancer cell lines showed that englerin A inhibits growth of a subset of tumor cell lines from many lineages, not just renal cell carcinomas. Expression of the TRPC4 cation channel was the cell line feature that best correlated with sensitivity to englerin A, suggesting the hypothesis that TRPC4 is the efficacy target for englerin A. Genetic experiments demonstrate that TRPC4 expression is both necessary and sufficient for englerin A induced growth inhibition. Englerin A induces calcium influx and membrane depolarization in cells expressing high levels of TRPC4 or its close ortholog TRPC5. Electrophysiology experiments confirmed that englerin A is a TRPC4 agonist. Both the englerin A induced current and the englerin A induced growth inhibition can be blocked by the TRPC4/C5 inhibitor ML204. These experiments confirm that activation of TRPC4/C5 channels inhibits tumor cell line proliferation and confirms the TRPC4 target hypothesis generated by the cell line profiling. In selectivity assays englerin A weakly inhibits TRPA1, TRPV3/V4, and TRPM8 which suggests that englerin A may bind a common feature of TRP ion channels. In vivo experiments show that englerin A is lethal in rodents near doses needed to activate the TRPC4 channel. This toxicity suggests that englerin A itself is probably unsuitable for further drug development. However, since englerin A can be synthesized in the laboratory, it may be a useful chemical starting point to identify novel modulators of other TRP family channels.

Integrated Molecular Meta-Analysis of 1,000 Pediatric High-Grade and Diffuse Intrinsic Pontine Glioma

Summary We collated data from 157 unpublished cases of pediatric high-grade glioma and diffuse intrinsic pontine glioma and 20 publicly available datasets in an integrated analysis of >1,000 cases. We identified co-segregating mutations in histone-mutant subgroups including loss of FBXW7 in H3.3G34R/V, TOP3A rearrangements in H3.3K27M, and BCOR mutations in H3.1K27M. Histone wild-type subgroups are refined by the presence of key oncogenic events or methylation profiles more closely resembling lower-grade tumors. Genomic aberrations increase with age, highlighting the infant population as biologically and clinically distinct. Uncommon pathway dysregulation is seen in small subsets of tumors, further defining the molecular diversity of the disease, opening up avenues for biological study and providing a basis for functionally defined future treatment stratification.

A LIN28B-RAN-AURKA Signaling Network Promotes Neuroblastoma Tumorigenesis.

A more complete understanding of aberrant oncogenic signaling in neuroblastoma, a malignancy of the developing sympathetic nervous system, is paramount to improving patient outcomes. Recently, we identified LIN28B as an oncogenic driver in high-risk neuroblastoma. Here, we identify the oncogene RAN as a LIN28B target and show regional gain of chromosome 12q24 as an additional somatic alteration resulting in increased RAN expression. We show that LIN28B influences RAN expression by promoting RAN Binding Protein 2 expression and by directly binding RAN mRNA. Further, we demonstrate a convergence of LIN28B and RAN signaling on Aurora kinase A activity. Collectively, these findings demonstrate that LIN28B-RAN-AURKA signaling drives neuroblastoma oncogenesis, suggesting that this pathway may be amenable to therapeutic targeting.

The Myc-miR-17-92 axis amplifies B-cell receptor signaling via inhibition of ITIM proteins: a novel lymphomagenic feed-forward loop

The c-Myc oncoprotein regulates >15% of the human transcriptome and a limited number of microRNAs (miRNAs). Here, we establish that in a human B-lymphoid cell line, Myc-repressed, but not Myc-stimulated, genes are significantly enriched for predicted binding sites of Myc-regulated miRNAs, primarily those comprising the Myc-activated miR-17~92 cluster. Notably, gene set enrichment analysis demonstrates that miR-17∼92 is a major regulator of B-cell receptor (BCR) pathway components. Many of them are immunoreceptor tyrosine inhibitory motif (ITIM)-containing proteins, and ITIM proteins CD22 and FCGR2B were found to be direct targets of miR-17∼92. Consistent with the propensity of ITIM proteins to recruit phosphatases, either MYC or miR-17~92 expression was necessary to sustain phosphorylation of spleen tyrosine kinase (SYK) and the B-cell linker protein (BLNK) upon ligation of the BCR. Further downstream, stimulation of the BCR response by miR-17-92 resulted in the enhanced calcium flux and elevated levels of Myc itself. Notably, inhibition of the miR-17~92 cluster in diffuse large B-cell lymphoma (DLBCL) cell lines diminished the BCR response as measured by SYK and BLNK phosphorylation. Conversely, human DLBCLs of the BCR subtype express higher Myc and mir17hg transcript levels than other subtypes. Hence, the Myc-miR-17-92-BCR axis, frequently affected by genomic rearrangements, constitutes a novel lymphomagenic feed-forward loop.