Multi-omic molecular profiling reveals potentially targetable abnormalities shared across multiple histologies of brain metastasis.

The deadly complication of brain metastasis (BM) is largely confined to a relatively narrow cross-section of systemic malignancies, suggesting a fundamental role for biological mechanisms shared across commonly brain metastatic tumor types. To identify and characterize such mechanisms, we performed genomic, transcriptional, and proteomic profiling using whole-exome sequencing, mRNA-seq, and reverse-phase protein array analysis in a cohort of the lung, breast, and renal cell carcinomas consisting of BM and patient-matched primary or extracranial metastatic tissues. While no specific genomic alterations were associated with BM, correlations with impaired cellular immunity, upregulated oxidative phosphorylation (OXPHOS), and canonical oncogenic signaling pathways including phosphoinositide 3-kinase (PI3K) signaling, were apparent across multiple tumor histologies. Multiplexed immunofluorescence analysis confirmed significant T cell depletion in BM, indicative of a fundamentally altered immune microenvironment. Moreover, functional studies using in vitro and in vivo modeling demonstrated heightened oxidative metabolism in BM along with sensitivity to OXPHOS inhibition in murine BM models and brain metastatic derivatives relative to isogenic parentals. These findings demonstrate that pathophysiological rewiring of oncogenic signaling, cellular metabolism, and immune microenvironment broadly characterizes BM. Further clarification of this biology will likely reveal promising targets for therapeutic development against BM arising from a broad variety of systemic cancers.

Genomic characterization of primary central nervous system lymphoma.

Primary central nervous system lymphoma (PCNSL) is a rare malignancy confined to the central nervous system (CNS), and majority of PCNSL is pathologically classified as diffuse large B-cell lymphoma (DLBCL). We have now performed whole-exome sequencing for 41 tumor tissues of DLBCL-type PCNSL and paired normal specimens and also RNA-sequencing for 30 tumors, revealing a very high frequency of nonsynonymous somatic mutations in PIM1 (100 %), BTG2 (92.7 %), and MYD88 (85.4 %). Many genes in the NF-κB pathway are concurrently mutated within the same tumors. Further, focal deletion or somatic mutations in the HLA genes are associated with poor prognosis. Copy number amplification and overexpression of genes at chromosome 7q35 were both found to predict short progression-free survival as well. Oncogenic mutations in GRB2 were also detected, the effects of which in cultured cells were attenuated by inhibitors of the downstream kinases MAP2K1 and MAP2K2. Individuals with tumors positive for MYD88 mutations also harbored the same mutations at a low frequency in peripheral blood mononuclear cells, suggesting that MYD88 mutation-positive precancerous cells originate outside of the CNS and develop into lymphoma after additional genetic hits that confer adaptation to the CNS environment.

Transforming mutations of RAC guanosine triphosphatases in human cancers.

Members of the RAS superfamily of small guanosine triphosphatases (GTPases) transition between GDP-bound, inactive and GTP-bound, active states and thereby function as binary switches in the regulation of various cellular activities. Whereas HRAS, NRAS, and KRAS frequently acquire transforming missense mutations in human cancer, little is known of the oncogenic roles of other small GTPases, including Ras-related C3 botulinum toxin substrate (RAC) proteins. We show that the human sarcoma cell line HT1080 harbors both NRAS(Q61K) and RAC1(N92I) mutant proteins. Whereas both of these mutants were able to transform fibroblasts, knockdown experiments indicated that RAC1(N92I) may be the essential growth driver for this cell line. Screening for RAC1, RAC2, or RAC3 mutations in cell lines and public databases identified several missense mutations for RAC1 and RAC2, with some of the mutant proteins, including RAC1(P29S), RAC1(C157Y), RAC2(P29L), and RAC2(P29Q), being found to be activated and transforming. P29S, N92I, and C157Y mutants of RAC1 were shown to exist preferentially in the GTP-bound state as a result of a rapid transition from the GDP-bound state, rather than as a result of a reduced intrinsic GTPase activity. Activating mutations of RAC GTPases were thus found in a wide variety of human cancers at a low frequency; however, given their marked transforming ability, the mutant proteins are potential targets for the development of new therapeutic agents.

Cancer-associated missense mutations of caspase-8 activate nuclear factor-κB signaling.

Head and neck squamous cell carcinoma (HNSCC) is an aggressive cancer with a 5-year survival rate of ~50%. With the use of a custom cDNA-capture system coupled with massively parallel sequencing, we have now investigated transforming mechanisms for this malignancy. The cDNAs of cancer-related genes (n = 906) were purified from a human HNSCC cell line (T3M-1 Cl-10) and subjected to high-throughput resequencing, and the clinical relevance of non-synonymous mutations thus identified was evaluated with luciferase-based reporter assays. A CASP8 (procaspase-8) cDNA with a novel G-to-C point mutation that results in the substitution of alanine for glycine at codon 325 was identified, and the mutant protein, CASP8 (G325A), was found to activate nuclear factor-κB (NF-κB) signaling to an extent far greater than that achieved with the wild-type protein. Moreover, forced expression of wild-type CASP8 suppressed the growth of T3M-1 Cl-10 cells without notable effects on apoptosis. We further found that most CASP8 mutations previously detected in various epithelial tumors also increase the ability of the protein to activate NF-κB signaling. Such NF-κB activation was shown to be mediated through the COOH-terminal region of the second death effector domain of CASP8. Although CASP8 mutations associated with cancer have been thought to promote tumorigenesis as a result of attenuation of the proapoptotic function of the protein, our results now show that most such mutations, including the novel G325A identified here, separately confer a gain of function with regard to activation of NF-κB signaling, indicating another role of CASP8 in the transformation of human malignancies including HNSCC.

Delineating genotypes and phenotypes of individual cells from long-read single cell transcriptomes.

Single-cell nanopore sequencing of full-length mRNAs (scNanoRNAseq) is transforming singlecell multi-omics studies. However, challenges include computational complexity and dependence on short-read curation. To address this, we developed a comprehensive toolkit, scNanoGPS to calculate same-cell genotypes-phenotypes without short-read guidance. We applied scNanoGPS onto 23,587 long-read transcriptomes from 4 tumors and 2 cell lines. Standalone, scNanoGPS accurately deconvoluted error-prone long-reads into single-cells and single-molecules. Further, scNanoGPS simultaneously accessed both phenotypes (expressions/isoforms) and genotypes (mutations) of individual cells. Our analyses revealed that tumor and stroma/immune cells often expressed significantly distinct combinations of isoforms (DCIs). In a kidney tumor, we identified 924 genes with DCIs involved in cell-type-specific functions such as PDE10A in tumor cells and CCL3 in lymphocytes. Moreover, transcriptome-wide mutation analyses identified many cell-type-specific mutations including VEGFA mutations in tumor cells and HLA-A mutations in immune cells, highlighting critical roles of different populations in tumors. Together, scNanoGPS facilitates applications of single-cell long-read sequencing.

High throughput single cell long-read sequencing analyses of same-cell genotypes and phenotypes in human tumors.

Single-cell nanopore sequencing of full-length mRNAs transforms single-cell multi-omics studies. However, challenges include high sequencing errors and dependence on short-reads and/or barcode whitelists. To address these, we develop scNanoGPS to calculate same-cell genotypes (mutations) and phenotypes (gene/isoform expressions) without short-read nor whitelist guidance. We apply scNanoGPS onto 23,587 long-read transcriptomes from 4 tumors and 2 cell-lines. Standalone, scNanoGPS deconvolutes error-prone long-reads into single-cells and single-molecules, and simultaneously accesses both phenotypes and genotypes of individual cells. Our analyses reveal that tumor and stroma/immune cells express distinct combination of isoforms (DCIs). In a kidney tumor, we identify 924 DCI genes involved in cell-type-specific functions such as PDE10A in tumor cells and CCL3 in lymphocytes. Transcriptome-wide mutation analyses identify many cell-type-specific mutations including VEGFA mutations in tumor cells and HLA-A mutations in immune cells, highlighting the critical roles of different mutant populations in tumors. Together, scNanoGPS facilitates applications of single-cell long-read sequencing technologies.

Oncogenic MAP2K1 mutations in human epithelial tumors.

The scirrhous subtype of gastric cancer is a highly infiltrative tumor with a poor outcome. To identify a transforming gene in this intractable disorder, we constructed a retroviral complementary DNA (cDNA) expression library from a cell line (OCUM-1) of scirrhous gastric cancer. A focus formation assay with the library and mouse 3T3 fibroblasts led to the discovery of a transforming cDNA, encoding for MAP2K1 with a glutamine-to-proline substitution at amino acid position 56. Interestingly, treatment with a MAP2K1-specific inhibitor clearly induced cell death of OCUM-1 but not of other two cells lines of scirrhous gastric cancer that do not carry MAP2K1 mutations, revealing the essential role of MAP2K1(Q56P) in the transformation mechanism of OCUM-1 cells. By using a next-generation sequencer, we further conducted deep sequencing of the MAP2K1 cDNA among 171 human cancer specimens or cell lines, resulting in the identification of one known (D67N) and four novel (R47Q, R49L, I204T and P306H) mutations within MAP2K1. The latter four changes were further shown to confer transforming potential to MAP2K1. In our experiments, a total of six (3.5%) activating mutations in MAP2K1 were thus identified among 172 of specimens or cell lines for human epithelial tumors. Given the addiction of cancer cells to the elevated MAP2K1 activity for proliferation, human cancers with such MAP2K1 mutations are suitable targets for the treatment with MAP2K1 inhibitors.

High-throughput resequencing of target-captured cDNA in cancer cells.

The recent advent of whole exon (exome)-capture technology, coupled with second-generation sequencers, has made it possible to readily detect genomic alterations that affect encoded proteins in cancer cells. Such target resequencing of the cancer genome, however, fails to detect most clinically-relevant gene fusions, given that such oncogenic fusion genes are often generated through intron-to-intron ligation. To develop a resequencing platform that simultaneously captures point mutations, insertions-deletions (indels), and gene fusions in the cancer genome, we chose cDNA as the input for target capture and extensive resequencing, and we describe the versatility of such a cDNA-capture system. As a test case, we constructed a custom target-capture system for 913 cancer-related genes, and we purified cDNA fragments for the target gene set from five cell lines of CML. Our target gene set included Abelson murine leukemia viral oncogene homolog 1 (ABL1), but it did not include breakpoint cluster region (BCR); however, the sequence output faithfully detected reads spanning the fusion points of these two genes in all cell lines, confirming the ability of cDNA capture to detect gene fusions. Furthermore, computational analysis of the sequence dataset successfully identified non-synonymous mutations and indels, including those of tumor protein p53 (TP53). Our data might thus support the feasibility of a cDNA-capture system coupled with massively parallel sequencing as a simple platform for the detection of a variety of anomalies in protein-coding genes among hundreds of cancer specimens.

Blocking immunosuppressive neutrophils deters pY696-EZH2-driven brain metastases.

The functions of immune cells in brain metastases are unclear because the brain has traditionally been considered "immune privileged." However, we found that a subgroup of immunosuppressive neutrophils is recruited into the brain, enabling brain metastasis development. In brain metastatic cells, enhancer of zeste homolog 2 (EZH2) is highly expressed and phosphorylated at tyrosine-696 (pY696)-EZH2 by nuclear-localized Src tyrosine kinase. Phosphorylation of EZH2 at Y696 changes its binding preference from histone H3 to RNA polymerase II, which consequently switches EZH2's function from a methyltransferase to a transcription factor that increases c-JUN expression. c-Jun up-regulates protumorigenic inflammatory cytokines, including granulocyte colony-stimulating factor (G-CSF), which recruits Arg1+- and PD-L1+ immunosuppressive neutrophils into the brain to drive metastasis outgrowth. G-CSF-blocking antibodies or immune checkpoint blockade therapies combined with Src inhibitors impeded brain metastasis in multiple mouse models. These findings indicate that pY696-EZH2 can function as a methyltransferase-independent transcription factor to facilitate the brain infiltration of immunosuppressive neutrophils, which could be clinically targeted for brain metastasis treatment.

Multiomics profiling of primary lung cancers and distant metastases reveals immunosuppression as a common characteristic of tumor cells with metastatic plasticity.

BACKGROUND: Metastasis is the primary cause of cancer mortality accounting for 90% of cancer deaths. Our understanding of the molecular mechanisms driving metastasis is rudimentary. RESULTS: We perform whole exome sequencing (WES), RNA sequencing, methylation microarray, and immunohistochemistry (IHC) on 8 pairs of non-small cell lung cancer (NSCLC) primary tumors and matched distant metastases. Furthermore, we analyze published WES data from 35 primary NSCLC and metastasis pairs, and transcriptomic data from 4 autopsy cases with metastatic NSCLC and one metastatic lung cancer mouse model. The majority of somatic mutations are shared between primary tumors and paired distant metastases although mutational signatures suggest different mutagenesis processes in play before and after metastatic spread. Subclonal analysis reveals evidence of monoclonal seeding in 41 of 42 patients. Pathway analysis of transcriptomic data reveals that downregulated pathways in metastases are mainly immune-related. Further deconvolution analysis reveals significantly lower infiltration of various immune cell types in metastases with the exception of CD4+ T cells and M2 macrophages. These results are in line with lower densities of immune cells and higher CD4/CD8 ratios in metastases shown by IHC. Analysis of transcriptomic data from autopsy cases and animal models confirms that immunosuppression is also present in extracranial metastases. Significantly higher somatic copy number aberration and allelic imbalance burdens are identified in metastases. CONCLUSIONS: Metastasis is a molecularly late event, and immunosuppression driven by different molecular events, including somatic copy number aberration, may be a common characteristic of tumors with metastatic plasticity.

Spatial Distance Correlates With Genetic Distance in Diffuse Glioma.

Background: Treatment effectiveness and overall prognosis for glioma patients depend heavily on the genetic and epigenetic factors in each individual tumor. However, intra-tumoral genetic heterogeneity is known to exist and needs to be managed. Currently, evidence for genetic changes varying spatially within the tumor is qualitative, and quantitative data is lacking. We hypothesized that a greater genetic diversity or "genetic distance" would be observed for distinct tumor samples taken with larger physical distances between them. Methods: Stereotactic biopsies were obtained from untreated primary glioma patients as part of a clinical trial between 2011 and 2016, with at least one biopsy pair collected in each case. The physical (Euclidean) distance between biopsy sites was determined using coordinates from imaging studies. The tissue samples underwent whole exome DNA sequencing and epigenetic methylation profiling and genomic distances were defined in three separate ways derived from differences in number of genes, copy number variations (CNV), and methylation profiles. Results: Of the 31 patients recruited to the trial, 23 were included in DNA methylation analysis, for a total of 71 tissue samples (14 female, 9 male patients, age range 21-80). Samples from an 8 patient subset of the 23 evaluated patients were further included in whole exome and copy number variation analysis. Physical and genomic distances were found to be independently and positively correlated for each of the three genomic distance measures. The correlation coefficients were 0.63, 0.65, and 0.35, respectively for (a) gene level mutations, (b) copy number variation, and (c) methylation status. We also derived quantitative linear relationships between physical and genomic distances. Conclusion: Primary brain tumors are genetically heterogeneous, and the physical distance within a given glioma correlates to genomic distance using multiple orthogonal genomic assessments. These data should be helpful in the clinical diagnostic and therapeutic management of glioma, for example by: managing sampling error, and estimating genetic heterogeneity using simple imaging inputs.

Elucidating the molecular pathogenesis of glioma: integrated germline and somatic profiling of a familial glioma case series.

Background: The genomic characterization of sporadically arising gliomas has delineated molecularly and clinically distinct subclasses of disease. However, less is known about the molecular nature of gliomas that are familial in origin. We performed molecular subtyping of 163 tumor specimens from individuals with a family history of glioma and integrated germline and somatic genomic data to characterize the pathogenesis of 20 tumors in additional detail. Methods: Immunohistochemical analyses were performed on formalin-fixed, paraffin-embedded tumor sections to determine molecular subtypes of glioma. For 20 cases, tumor DNA was exome sequenced on an Illumina HiSeq 2000 platform and copy number profiling was performed on the Illumina HumanOmniExpress BeadChip. Genotypes at glioma risk polymorphisms were determined from germline DNA profiled on the Illumina Infinium OncoArray and deleterious germline mutations were identified from germline sequencing data. Results: All 3 molecular subtypes of sporadic glioma were represented in the overall case series, including molecular glioblastoma (n = 102), oligodendroglioma (n = 21), and astrocytoma (n = 20). Detailed profiling of 20 of these cases showed characteristic subtype-specific alterations at frequencies comparable to sporadic glioma cases. All 20 cases had alterations in genes regulating telomere length. Frequencies of common glioma risk alleles were similar to those among sporadic cases, and correlations between risk alleles and same-gene somatic mutations were not observed. Conclusions: This study illustrates that the molecular characteristics of familial tumors profiled largely recapitulate what is known about sporadic glioma and that both germline and somatic molecular features target common core pathways involved in gliomagenesis. Key Points: 1. Familial and sporadic gliomas display highly comparable molecular landscapes. 2. Germline and somatic molecular events target common core pathways involved in gliomagenesis. 3. Carriage of germline glioma risk variants is not associated with somatic events in the same gene.

Recurrent CCND3 mutations in MLL-rearranged acute myeloid leukemia.

In acute myeloid leukemia (AML), MLL (KMT2A) rearrangements are among the most frequent chromosomal abnormalities; however, knowledge of the genetic landscape of MLL-rearranged AML is limited. In this study, we performed whole-exome sequencing (n = 9) and targeted sequencing (n = 56) of samples from pediatric MLL-rearranged AML patients enrolled in the Japanese Pediatric Leukemia/Lymphoma Study Group AML-05 study. Additionally, we analyzed 105 pediatric t(8;21) AML samples and 30 adult MLL-rearranged AML samples. RNA-sequencing data from 31 patients published in a previous study were also reanalyzed. As a result, we identified 115 mutations in pediatric MLL-rearranged AML patients (2.1 mutations/patient), with mutations in signaling pathway genes being the most frequently detected (60.7%). Mutations in genes associated with epigenetic regulation (21.4%), transcription factors (16.1%), and the cohesin complex (8.9%) were also commonly detected. Novel CCND3 mutations were identified in 5 pediatric MLL-rearranged AML patients (8.9%) and 2 adult MLL-rearranged AML patients (3.3%). Recurrent mutations of CCND1 (n = 3, 2.9%) and CCND2 (n = 8, 7.6%) were found in pediatric t(8;21) AML patients, whereas no CCND3 mutations were found, suggesting that D-type cyclins exhibit a subtype-specific mutation pattern in AML. Treatment of MLL-rearranged AML cell lines with CDK4/6 inhibitors (abemaciclib and palbociclib) blocked G1 to S phase cell-cycle progression and impaired proliferation. Pediatric MLL-MLLT3-rearranged AML patients with coexisting mutations (n = 16) had significantly reduced relapse-free survival and overall survival compared with those without coexisting mutations (n = 9) (P = .048 and .046, respectively). These data provide insights into the genetics of MLL-rearranged AML and suggest therapeutic strategies.

Mutational Landscape and Antiproliferative Functions of ELF Transcription Factors in Human Cancer.

ELF4 (also known as MEF) is a member of the ETS family of transcription factors. An oncogenic role for ELF4 has been demonstrated in hematopoietic malignancies, but its function in epithelial tumors remains unclear. Here, we show that ELF4 can function as a tumor suppressor and is somatically inactivated in a wide range of human tumors. We identified a missense mutation affecting the transactivation potential of ELF4 in oral squamous cell carcinoma cells. Restoration of the transactivation activity through introduction of wild-type ELF4 significantly inhibited cell proliferation in vitro and tumor xenograft growth. Furthermore, we found that ELF1 and ELF2, closely related transcription factors to ELF4, also exerted antiproliferative effects in multiple cancer cell lines. Mutations in ELF1 and ELF2, as in ELF4, were widespread across human cancers, but were almost all mutually exclusive. Moreover, chromatin immunoprecipitation coupled with high-throughput sequencing revealed ELF4-binding sites in genomic regions adjacent to genes related to cell-cycle regulation and apoptosis. Finally, we provide mechanistic evidence that the antiproliferative effects of ELF4 were mediated through the induction of HRK, an activator of apoptosis, and DLX3, an inhibitor of cell growth. Collectively, our findings reveal a novel subtype of human cancer characterized by inactivating mutations in the ELF subfamily of proteins, and warrant further investigation of the specific settings where ELF restoration may be therapeutically beneficial. Cancer Res; 76(7); 1814-24. ©2016 AACR.

STK10 missense mutations associated with anti-apoptotic function.

Peripheral T-cell lymphoma (PTCL) is an aggressive lymphoma with a 5-year overall survival rate of <30%. To identify carcinogenesis-related genes in PTCL, we conducted high-throughput resequencing of target-captured cDNA in a PTCL specimen, revealing a total of 19 missense mutations among 18 independent genes. One of such substitutions, c.2201G>A in STK10 cDNA, replaces an arginine residue to a histidine (R634H) in the encoded protein. Of note, while wild-type STK10 suppresses NF-κB activity and potentiates dexamethasone-induced apoptosis, the R634H change significantly decreases such pro-apoptotic activity. This c.2201G>A change of STK10 was also identified in another PTCL specimen, but now registered as a single nucleotide polymorphism in the latest dbSNP database. Furthermore, other somatic mutations of STK10 have been reported, and we now reveal that some of them (L85P and K277E) have more profound anti-apoptotic effects compared to R634H. These results suggest that STK10 functions as a tumor suppressor gene, and that dysfunction of STK10 activity either through polymorphism or somatic mutations may confer anti-apoptotic effects contributing to carcinogenesis.

Diarylheptanoids derived from Alpinia officinarum induce apoptosis, S-phase arrest and differentiation in human neuroblastoma cells.

BACKGROUND: We have previously screened many natural products for their cytotoxic effects on neuroblastoma cell lines, and two diarylheptanoids derived from Alpinia officinarum, 7-(4''-hydroxy-3''-methoxyphenyl)-1-phenyl-4E-hepten-3-one (Compound 1) and (5R)-5-methoxy-7-(4''-hydroxy-3''-methoxyphenyl)-1-phenyl-3-heptanone (Compound 2), were shown to have potent cytotoxicity. On this basis, a detailed study of the antitumor activities of these two diarylheptanoids in neuroblastoma cell lines was performed. MATERIALS AND METHODS: Cytotoxicity was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. To measure apoptosis, nuclear shrinkage was monitored by Hoechst 33342 staining, and activation of caspases 3 and 9 was monitored by Western blotting. Cell cycle status was evaluated by flow cytometry with propidium iodide staining. Differentiated cells were photographed and counted in a randomized fashion. RESULTS: Both diarylheptanoids showed significant cytotoxicity against neuroblastoma cell lines (IMR-32, SK-N-SH, NB-39). The compounds induced nuclear shrinkage and fragmentation, and activated caspase-3 and caspase-9. Flow cytometric analysis revealed induction of S-phase cell cycle arrest concurrently with an increased sub-G(1) cell population. Moreover, a low concentration (10(-8) M) of Compound 1 induced significant neurite branching in the NB-39 cell line. CONCLUSION: Diarylheptanoids derived from A. officinarum have marked activity against neuroblastoma cells, acting through multiple mechanisms. Our results suggest that the two compounds studied here may be useful for the treatment of patients with neuroblastoma.