Distinct resistance mechanisms arise to allosteric vs. ATP-competitive AKT inhibitors.

The AKT kinases have emerged as promising therapeutic targets in oncology and both allosteric and ATP-competitive AKT inhibitors have entered clinical investigation. However, long-term efficacy of such inhibitors will likely be challenged by the development of resistance. We have established prostate cancer models of acquired resistance to the allosteric inhibitor MK-2206 or the ATP-competitive inhibitor ipatasertib following prolonged exposure. While alterations in AKT are associated with acquired resistance to MK-2206, ipatasertib resistance is driven by rewired compensatory activity of parallel signaling pathways. Importantly, MK-2206 resistance can be overcome by treatment with ipatasertib, while ipatasertib resistance can be reversed by co-treatment with inhibitors of pathways including PIM signaling. These findings demonstrate that distinct resistance mechanisms arise to the two classes of AKT inhibitors and that combination approaches may reverse resistance to ATP-competitive inhibition.

Elevated Serum Amino Acids Induce a Subpopulation of Alpha Cells to Initiate Pancreatic Neuroendocrine Tumor Formation.

The cellular origin of sporadic pancreatic neuroendocrine tumors (PNETs) is obscure. Hormone expression suggests that these tumors arise from glucagon-producing alpha cells or insulin-producing ß cells, but instability in hormone expression prevents linage determination. We utilize loss of hepatic glucagon receptor (GCGR) signaling to drive alpha cell hyperproliferation and tumor formation to identify a cell of origin and dissect mechanisms that drive progression. Using a combination of genetically engineered Gcgr knockout mice and GCGR-inhibiting antibodies, we show that elevated plasma amino acids drive the appearance of a proliferative population of SLC38A5+ embryonic progenitor-like alpha cells in mice. Further, we characterize tumors from patients with rare bi-allelic germline GCGR loss-of-function variants and find prominent tumor-cell-associated expression of the SLC38A5 paralog SLC7A8 as well as markers of active mTOR signaling. Thus, progenitor cells arise from adult alpha cells in response to metabolic signals and, when inductive signals are chronically present, drive tumor initiation.

Integrated genomic analysis reveals mutated ELF3 as a potential gallbladder cancer vaccine candidate.

Gallbladder cancer (GBC) is an aggressive gastrointestinal malignancy with no approved targeted therapy. Here, we analyze exomes (n = 160), transcriptomes (n = 115), and low pass whole genomes (n = 146) from 167 gallbladder cancers (GBCs) from patients in Korea, India and Chile. In addition, we also sequence samples from 39 GBC high-risk patients and detect evidence of early cancer-related genomic lesions. Among the several significantly mutated genes not previously linked to GBC are ETS domain genes ELF3 and EHF, CTNNB1, APC, NSD1, KAT8, STK11 and NFE2L2. A majority of ELF3 alterations are frame-shift mutations that result in several cancer-specific neoantigens that activate T-cells indicating that they are cancer vaccine candidates. In addition, we identify recurrent alterations in KEAP1/NFE2L2 and WNT pathway in GBC. Taken together, these define multiple targetable therapeutic interventions opportunities for GBC treatment and management.

S100a4 upregulation in Pik3caH1047R;Trp53R270H;MMTV-Cre-driven mammary tumors promotes metastasis.

BACKGROUND: PIK3CA mutations are frequent in human breast cancer. Pik3caH1047R mutant expression in mouse mammary gland promotes tumorigenesis. TP53 mutations co-occur with PIK3CA mutations in human breast cancers. We previously generated a conditionally activatable Pik3caH1047R;MMTV-Cre mouse model and found a few malignant sarcomatoid (spindle cell) carcinomas that had acquired spontaneous dominant-negative Trp53 mutations. METHODS: A Pik3caH1047R;Trp53R270H;MMTV-Cre double mutant mouse breast cancer model was generated. Tumors were characterized by histology, marker analysis, transcriptional profiling, single-cell RNA-seq, and bioinformatics. Cell lines were developed from mutant tumors and used to identify and confirm genes involved in metastasis. RESULTS: We found Pik3caH1047R and Trp53R270H cooperate in driving oncogenesis in mammary glands leading to a shorter latency than either alone. Double mutant mice develop multiple histologically distinct mammary tumors, including adenocarcinoma and sarcomatoid (spindle cell) carcinoma. We found some tumors to be invasive and a few metastasized to the lung and/or the lymph node. Single-cell RNA-seq analysis of the tumors identified epithelial, stromal, myeloid, and T cell groups. Expression analysis of the metastatic tumors identified S100a4 as a top candidate gene associated with metastasis. Metastatic tumors contained a much higher percentage of epithelial-mesenchymal transition (EMT)-signature positive and S100a4-expressing cells. CRISPR/CAS9-mediated knockout of S100a4 in a metastatic tumor-derived cell line disrupted its metastatic potential indicating a role for S100a4 in metastasis. CONCLUSIONS: Pik3caH1047R;Trp53R270H;MMTV-Cre mouse provides a preclinical model to mimic a subtype of human breast cancers that carry both PIK3CA and TP53 mutations. It also allows for understanding the cooperation between the two mutant genes in tumorigenesis. Our model also provides a system to study metastasis and develop therapeutic strategies for PIK3CA/TP53 double-positive cancers. S100a4 found involved in metastasis in this model can be a potential diagnostic and therapeutic target.

SCINA: A Semi-Supervised Subtyping Algorithm of Single Cells and Bulk Samples.

Advances in single-cell RNA sequencing (scRNA-Seq) have allowed for comprehensive analyses of single cell data. However, current analyses of scRNA-Seq data usually start from unsupervised clustering or visualization. These methods ignore prior knowledge of transcriptomes and the probable structures of the data. Moreover, cell identification heavily relies on subjective and possibly inaccurate human inspection afterwards. To address these analytical challenges, we developed SCINA (Semi-supervised Category Identification and Assignment), a semi-supervised model that exploits previously established gene signatures using an expectation-maximization (EM) algorithm. SCINA is applicable to scRNA-Seq and flow cytometry/CyTOF data, as well as other data of similar format. We applied SCINA to a wide range of datasets, and showed its accuracy, stability and efficiency, which exceeded most popular unsupervised approaches. SCINA discovered an intermediate stage of oligodendrocytes from mouse brain scRNA-Seq data. SCINA also detected immune cell population changes in cytometry data in a genetically-engineered mouse model. Furthermore, SCINA performed well with bulk gene expression data. Specifically, we identified a new kidney tumor clade with similarity to FH-deficient tumors (FHD), which we refer to as FHD-like tumors (FHDL). Overall, SCINA provides both methodological advances and biological insights from perspectives different from traditional analytical methods.

The tumor suppressor BAP1 cooperates with BRAFV600E to promote tumor formation in cutaneous melanoma.

The deubiquitinating enzyme BAP1 is mutated in a hereditary cancer syndrome with a high risk of mesothelioma and melanocytic tumors. Here, we show that Bap1 deletion in melanocytes cooperates with the constitutively active, oncogenic form of BRAF (BRAFV600E ) and UV to cause melanoma in mice, albeit at very low frequency. In addition, Bap1-null melanoma cells derived from mouse tumors are more aggressive and colonize and grow at distant sites more than their wild-type counterparts. Molecularly, Bap1-null melanoma cell lines have increased DNA damage measured by γH2aX and hyperubiquitination of histone H2a. Therapeutically, these Bap1-null tumors are completely responsive to BRAF- and MEK-targeted therapies. Therefore, BAP1 functions as a tumor suppressor and limits tumor progression in melanoma.

Actionable Activating Oncogenic ERBB2/HER2 Transmembrane and Juxtamembrane Domain Mutations.

Deregulated HER2 is a target of many approved cancer drugs. We analyzed 111,176 patient tumors and identified recurrent mutations in HER2 transmembrane domain (TMD) and juxtamembrane domain (JMD) that include G660D, R678Q, E693K, and Q709L. Using a saturation mutagenesis screen and testing of patient-derived mutations we found several activating TMD and JMD mutations. Structural modeling and analysis showed that the TMD/JMD mutations function by improving the active dimer interface or stabilizing an activating conformation. Further, we found that HER2 G660D employed asymmetric kinase dimerization for activation and signaling. Importantly, anti-HER2 antibodies and small-molecule kinase inhibitors blocked the activity of TMD/JMD mutants. Consistent with this, a G660D germline mutant lung cancer patient showed remarkable clinical response to HER2 blockade.

ERK Mutations and Amplification Confer Resistance to ERK-Inhibitor Therapy.

Purpose: MAPK pathway inhibitors targeting BRAF and MEK have shown clinical efficacy in patients with RAF- and/or RAS-mutated tumors. However, acquired resistance to these agents has been an impediment to improved long-term survival in the clinic. In such cases, targeting ERK downstream of BRAF/MEK has been proposed as a potential strategy for overcoming acquired resistance. Preclinical studies suggest that ERK inhibitors are effective at inhibiting BRAF/RAS-mutated tumor growth and overcome BRAF or/and MEK inhibitor resistance. However, as observed with other MAPK pathway inhibitors, treatment with ERK inhibitors is likely to cause resistance in the clinic. Here, we aimed to model the mechanism of resistance to ERK inhibitors.Experimental Design: We tested five structurally different ATP-competitive ERK inhibitors representing three different scaffolds on BRAF/RAS-mutant cancer cell lines of different tissue types to generate resistant lines. We have used in vitro modeling, structural biology, and genomic analysis to understand the development of resistance to ERK inhibitors and the mechanisms leading to it.Results: We have identified mutations in ERK1/2, amplification and overexpression of ERK2, and overexpression of EGFR/ERBB2 as mechanisms of acquired resistance. Structural analysis of ERK showed that specific compounds that induced on-target ERK mutations were impaired in their ability to bind mutant ERK. We show that in addition to MEK inhibitors, ERBB receptor and PI3K/mTOR pathway inhibitors are effective in overcoming ERK-inhibitor resistance.Conclusions: These findings suggest that combination therapy with MEK or ERBB receptor or PI3K/mTOR and ERK inhibitors may be an effective strategy for managing the emergence of resistance in the clinic. Clin Cancer Res; 24(16); 4044-55. ©2018 AACR.

Comprehensive genomic analysis identifies pathogenic variants in maturity-onset diabetes of the young (MODY) patients in South India.

BACKGROUND: Maturity-onset diabetes of the young (MODY) is an early-onset, autosomal dominant form of non-insulin dependent diabetes. Genetic diagnosis of MODY can transform patient management. Earlier data on the genetic predisposition to MODY have come primarily from familial studies in populations of European origin. METHODS: In this study, we carried out a comprehensive genomic analysis of 289 individuals from India that included 152 clinically diagnosed MODY cases to identify variants in known MODY genes. Further, we have analyzed exome data to identify putative MODY relevant variants in genes previously not implicated in MODY. Functional validation of MODY relevant variants was also performed. RESULTS: We found MODY 3 (HNF1A; 7.2%) to be most frequently mutated followed by MODY 12 (ABCC8; 3.3%). They together account for ~ 11% of the cases. In addition to known MODY genes, we report the identification of variants in RFX6, WFS1, AKT2, NKX6-1 that may contribute to development of MODY. Functional assessment of the NKX6-1 variants showed that they are functionally impaired. CONCLUSIONS: Our findings showed HNF1A and ABCC8 to be the most frequently mutated MODY genes in south India. Further we provide evidence for additional MODY relevant genes, such as NKX6-1, and these require further validation.

Comprehensive genomic analysis of malignant pleural mesothelioma identifies recurrent mutations, gene fusions and splicing alterations.

We analyzed transcriptomes (n = 211), whole exomes (n = 99) and targeted exomes (n = 103) from 216 malignant pleural mesothelioma (MPM) tumors. Using RNA-seq data, we identified four distinct molecular subtypes: sarcomatoid, epithelioid, biphasic-epithelioid (biphasic-E) and biphasic-sarcomatoid (biphasic-S). Through exome analysis, we found BAP1, NF2, TP53, SETD2, DDX3X, ULK2, RYR2, CFAP45, SETDB1 and DDX51 to be significantly mutated (q-score ≥ 0.8) in MPMs. We identified recurrent mutations in several genes, including SF3B1 (∼2%; 4/216) and TRAF7 (∼2%; 5/216). SF3B1-mutant samples showed a splicing profile distinct from that of wild-type tumors. TRAF7 alterations occurred primarily in the WD40 domain and were, except in one case, mutually exclusive with NF2 alterations. We found recurrent gene fusions and splice alterations to be frequent mechanisms for inactivation of NF2, BAP1 and SETD2. Through integrated analyses, we identified alterations in Hippo, mTOR, histone methylation, RNA helicase and p53 signaling pathways in MPMs.

Targeting PTPRK-RSPO3 colon tumours promotes differentiation and loss of stem-cell function.

Colorectal cancer remains a major unmet medical need, prompting large-scale genomics efforts in the field to identify molecular drivers for which targeted therapies might be developed. We previously reported the identification of recurrent translocations in R-spondin genes present in a subset of colorectal tumours. Here we show that targeting RSPO3 in PTPRK-RSPO3-fusion-positive human tumour xenografts inhibits tumour growth and promotes differentiation. Notably, genes expressed in the stem-cell compartment of the intestine were among those most sensitive to anti-RSPO3 treatment. This observation, combined with functional assays, suggests that a stem-cell compartment drives PTPRK-RSPO3 colorectal tumour growth and indicates that the therapeutic targeting of stem-cell properties within tumours may be a clinically relevant approach for the treatment of colorectal tumours.

Spectrum of diverse genomic alterations define non-clear cell renal carcinoma subtypes.

To further understand the molecular distinctions between kidney cancer subtypes, we analyzed exome, transcriptome and copy number alteration data from 167 primary human tumors that included renal oncocytomas and non-clear cell renal cell carcinomas (nccRCCs), consisting of papillary (pRCC), chromophobe (chRCC) and translocation (tRCC) subtypes. We identified ten significantly mutated genes in pRCC, including MET, NF2, SLC5A3, PNKD and CPQ. MET mutations occurred in 15% (10/65) of pRCC samples and included previously unreported recurrent activating mutations. In chRCC, we found TP53, PTEN, FAAH2, PDHB, PDXDC1 and ZNF765 to be significantly mutated. Gene expression analysis identified a five-gene set that enabled the molecular classification of chRCC, renal oncocytoma and pRCC. Using RNA sequencing, we identified previously unreported gene fusions, including ACTG1-MITF fusion. Ectopic expression of the ACTG1-MITF fusion led to cellular transformation and induced the expression of downstream target genes. Finally, we observed upregulation of the anti-apoptotic factor BIRC7 in MiTF-high RCC tumors, suggesting a potential therapeutic role for BIRC7 inhibitors.

A comprehensive transcriptional portrait of human cancer cell lines.

Tumor-derived cell lines have served as vital models to advance our understanding of oncogene function and therapeutic responses. Although substantial effort has been made to define the genomic constitution of cancer cell line panels, the transcriptome remains understudied. Here we describe RNA sequencing and single-nucleotide polymorphism (SNP) array analysis of 675 human cancer cell lines. We report comprehensive analyses of transcriptome features including gene expression, mutations, gene fusions and expression of non-human sequences. Of the 2,200 gene fusions catalogued, 1,435 consist of genes not previously found in fusions, providing many leads for further investigation. We combine multiple genome and transcriptome features in a pathway-based approach to enhance prediction of response to targeted therapeutics. Our results provide a valuable resource for studies that use cancer cell lines.

Diverse modes of genomic alteration in hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is a heterogeneous disease with high mortality rate. Recent genomic studies have identified TP53, AXIN1, and CTNNB1 as the most frequently mutated genes. Lower frequency mutations have been reported in ARID1A, ARID2 and JAK1. In addition, hepatitis B virus (HBV) integrations into the human genome have been associated with HCC. RESULTS: Here, we deep-sequence 42 HCC patients with a combination of whole genome, exome and transcriptome sequencing to identify the mutational landscape of HCC using a reasonably large discovery cohort. We find frequent mutations in TP53, CTNNB1 and AXIN1, and rare but likely functional mutations in BAP1 and IDH1. Besides frequent hepatitis B virus integrations at TERT, we identify translocations at the boundaries of TERT. A novel deletion is identified in CTNNB1 in a region that is heavily mutated in multiple cancers. We also find multiple high-allelic frequency mutations in the extracellular matrix protein LAMA2. Lower expression levels of LAMA2 correlate with a proliferative signature, and predict poor survival and higher chance of cancer recurrence in HCC patients, suggesting an important role of the extracellular matrix and cell adhesion in tumor progression of a subgroup of HCC patients. CONCLUSIONS: The heterogeneous disease of HCC features diverse modes of genomic alteration. In addition to common point mutations, structural variations and methylation changes, there are several virus-associated changes, including gene disruption or activation, formation of chimeric viral-human transcripts, and DNA copy number changes. Such a multitude of genomic events likely contributes to the heterogeneous nature of HCC.

Integrated exome and transcriptome sequencing reveals ZAK isoform usage in gastric cancer.

Gastric cancer is the second leading cause of worldwide cancer mortality, yet the underlying genomic alterations remain poorly understood. Here we perform exome and transcriptome sequencing and SNP array assays to characterize 51 primary gastric tumours and 32 cell lines. Meta-analysis of exome data and previously published data sets reveals 24 significantly mutated genes in microsatellite stable (MSS) tumours and 16 in microsatellite instable (MSI) tumours. Over half the patients in our collection could potentially benefit from targeted therapies. We identify 55 splice site mutations accompanied by aberrant splicing products, in addition to mutation-independent differential isoform usage in tumours. ZAK kinase isoform TV1 is preferentially upregulated in gastric tumours and cell lines relative to normal samples. This pattern is also observed in colorectal, bladder and breast cancers. Overexpression of this particular isoform activates multiple cancer-related transcription factor reporters, while depletion of ZAK in gastric cell lines inhibits proliferation. These results reveal the spectrum of genomic and transcriptomic alterations in gastric cancer, and identify isoform-specific oncogenic properties of ZAK.

The emerging mutational landscape of G proteins and G-protein-coupled receptors in cancer.

Aberrant expression and activity of G proteins and G-protein-coupled receptors (GPCRs) are frequently associated with tumorigenesis. Deep sequencing studies show that 4.2% of tumours carry activating mutations in GNAS (encoding Gαs), and that oncogenic activating mutations in genes encoding Gαq family members (GNAQ or GNA11) are present in ~66% and ~6% of melanomas arising in the eye and skin, respectively. Furthermore, nearly 20% of human tumours harbour mutations in GPCRs. Many human cancer-associated viruses also express constitutively active viral GPCRs. These studies indicate that G proteins, GPCRs and their linked signalling circuitry represent novel therapeutic targets for cancer prevention and treatment.

Oncogenic ERBB3 mutations in human cancers.

The human epidermal growth factor receptor (HER) family of tyrosine kinases is deregulated in multiple cancers either through amplification, overexpression, or mutation. ERBB3/HER3, the only member with an impaired kinase domain, although amplified or overexpressed in some cancers, has not been reported to carry oncogenic mutations. Here, we report the identification of ERBB3 somatic mutations in ~11% of colon and gastric cancers. We found that the ERBB3 mutants transformed colonic and breast epithelial cells in a ligand-independent manner. However, the mutant ERBB3 oncogenic activity was dependent on kinase-active ERBB2. Furthermore, we found that anti-ERBB antibodies and small molecule inhibitors effectively blocked mutant ERBB3-mediated oncogenic signaling and disease progression in vivo.

Comprehensive genomic analysis identifies SOX2 as a frequently amplified gene in small-cell lung cancer.

Small-cell lung cancer (SCLC) is an exceptionally aggressive disease with poor prognosis. Here, we obtained exome, transcriptome and copy-number alteration data from approximately 53 samples consisting of 36 primary human SCLC and normal tissue pairs and 17 matched SCLC and lymphoblastoid cell lines. We also obtained data for 4 primary tumors and 23 SCLC cell lines. We identified 22 significantly mutated genes in SCLC, including genes encoding kinases, G protein-coupled receptors and chromatin-modifying proteins. We found that several members of the SOX family of genes were mutated in SCLC. We also found SOX2 amplification in ∼27% of the samples. Suppression of SOX2 using shRNAs blocked proliferation of SOX2-amplified SCLC lines. RNA sequencing identified multiple fusion transcripts and a recurrent RLF-MYCL1 fusion. Silencing of MYCL1 in SCLC cell lines that had the RLF-MYCL1 fusion decreased cell proliferation. These data provide an in-depth view of the spectrum of genomic alterations in SCLC and identify several potential targets for therapeutic intervention.

Recurrent R-spondin fusions in colon cancer.

Identifying and understanding changes in cancer genomes is essential for the development of targeted therapeutics. Here we analyse systematically more than 70 pairs of primary human colon tumours by applying next-generation sequencing to characterize their exomes, transcriptomes and copy-number alterations. We have identified 36,303 protein-altering somatic changes that include several new recurrent mutations in the Wnt pathway gene TCF7L2, chromatin-remodelling genes such as TET2 and TET3 and receptor tyrosine kinases including ERBB3. Our analysis for significantly mutated cancer genes identified 23 candidates, including the cell cycle checkpoint kinase ATM. Copy-number and RNA-seq data analysis identified amplifications and corresponding overexpression of IGF2 in a subset of colon tumours. Furthermore, using RNA-seq data we identified multiple fusion transcripts including recurrent gene fusions involving R-spondin family members RSPO2 and RSPO3 that together occur in 10% of colon tumours. The RSPO fusions were mutually exclusive with APC mutations, indicating that they probably have a role in the activation of Wnt signalling and tumorigenesis. Consistent with this we show that the RSPO fusion proteins were capable of potentiating Wnt signalling. The R-spondin gene fusions and several other gene mutations identified in this study provide new potential opportunities for therapeutic intervention in colon cancer.