Kazutaka Fukumura

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.

Leukemic evolution of donor-derived cells harboring IDH2 and DNMT3A mutations after allogeneic stem cell transplantation.

Leukemic evolution of donor-derived cells harboring IDH2 and DNMT3A mutations after allogeneic stem cell transplantation

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.

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. (Cancer Sci 2012; 103: 131–135)

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.

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.

Genome-wide DNA methylation profiling identifies primary central nervous system lymphoma as a distinct entity different from systemic diffuse large B-cell lymphoma

Taishi Nakamura1,2 · Satoshi Yamashita3 · Kazutaka Fukumura4 · Jun Nakabayashi5 · Kazuhiro Tanaka6 · Kaoru Tamura7 · Kensuke Tateishi2 · Manabu Kinoshita8 · Shintaro Fukushima1 · Hirokazu Takami1 · Kohei Fukuoka1 · Kai Yamazaki1 · Yuko Matsushita9 · Makoto Ohno9 · Yasuji Miyakita9 · Soichiro Shibui10 · Atsuhiko Kubo11 · Takashi Shuto12 · Sylvia Kocialkowski13 · Shoji Yamanaka14 · Akitake Mukasa15 · Takashi Sasayama6 · Kazuhiko Mishima16 · Taketoshi Maehara7 · Nobutaka Kawahara2 · Motoo Nagane17 · Yoshitaka Narita9 · Hiroyuki Mano4 · Toshikazu Ushijima3 · Koichi Ichimura2

Abstract 3893: Whole-exome sequencing analysis of primary central nervous system lymphoma reveals recurrent MYD88 and PIM1 mutations

Primary central nervous system lymphoma (PCNSL) is defined as diffuse large B-cell lymphoma (DLBCL) confined to the central nervous system. Although PCNSL cannot be histologically distinguished from extracerebral DLBCL, its prognosis is quite poor. To gain insights into the transforming mechanism of PCNSL, we conducted whole-exome sequencing for 44 PCNSL specimens. Genomic DNA was extracted from tumors and their matched normal samples. Exome fragments were captured by using SureSelectXT Human All ExonV5+lncRNA, and subjected to deep sequencing with the HiSeq 2000 system. There were 8 cases with germinal center B-cell-like (GCB) subtype and 34 cases with non-GCB subtype. Whole-exome sequencing of tumor and paired normal yielded sequencing data with a mean coverage of 156X and 80X, with 98% and 99% of the targeted bases covered by ≥20 independent reads, respectively. Of 17,832 somatic mutations identified in the PCNSL specimens, 11,611 (65%) were nonsynonymous single nucleotide variations. Predominant nucleotide substitution was a C>T transition (57%) especially in the context of the sequence GCG. We also identified 798 insertions/deletions. Interestingly, MYD88 mutations were detected in 35 of 44 patients (79.5%), and PIM1 mutations were detected in 42 patients (95.5%). Also, a number of genetic aberrations were shown to activate the NF-κB pathway. These results suggest that MYD88 and PIM1 mutations have important roles in the development of PCNSL and could be the therapeutic targets of this intractable disorder. Citation Format: Motoo Nagane, Kazutaka Fukumura, Toshihide Ueno, Jeunghun Lee, Yukiko Shishido-Hara, Mitsuaki Shirahata, Kazuhiko Mishima, Koichi Ichimura, Akitake Mukasa, Yoshitaka Narita, Ryo Nishikawa, Hiroyuki Mano. Whole-exome sequencing analysis of primary central nervous system lymphoma reveals recurrent MYD88 and PIM1 mutations. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3893. doi:10.1158/1538-7445.AM2015-3893