Search

B-cell acute lymphoblastic leukemia (B-ALL), a genetically heterogeneous disease, is classified into different molecular subtypes that are defined by recurrent gene rearrangements, gross chromosomal abnormalities, or specific gene mutations. Cells with these genetic alterations acquire a leukemia-initiating ability and show unique expression profiles. The distribution of B-ALL molecular subtypes is greatly dependent on age, which also affects treatment responsiveness and long-term survival, partly accounting for the inferior outcome in adolescents and young adults (AYA) and (older) adults with B-ALL. Recent advances in sequencing technology, especially RNA sequencing and the application of these technologies in large B-ALL cohorts have uncovered B-ALL molecular subtypes prevalent in AYA and adults. These new insights supply more precise estimations of prognoses and targeted therapies informed by sequencing results, as well as a deeper understanding of the genetic basis of AYA/adult B-ALL. This article provides an account of these technological advances and an overview of the recent major findings of B-ALL molecular subtypes in adults.

The cellular context that integrates gene expression, signaling, and metabolism dictates the oncogenic behavior and shapes the treatment responses in distinct cancer types. Although chimeric fusion proteins involving transcription factors (TF) are hallmarks of many types of acute lymphoblastic leukemia (ALL), therapeutically targeting the fusion proteins is a challenge. In this work, we characterize the core regulatory circuitry (CRC; interconnected autoregulatory loops of TFs) of B-ALL involving MEF2D-fusions and identify MEF2D-fusion and SREBF1 TFs as crucial CRC components. By gene silencing and pharmacologic perturbation, we reveal that the CRC integrates the pre-B-cell receptor (BCR) and lipid metabolism to maintain itself and govern malignant phenotypes. Small-molecule inhibitors of pre-BCR signaling and lipid biosynthesis disrupt the CRC and silence the MEF2D fusion in cell culture and show therapeutic efficacy in xenografted mice. Therefore, pharmacologic disruption of CRC presents a potential therapeutic strategy to target fusion protein–driven leukemia.Significance:Cancer type–specific gene expression is governed by transcription factors involved in a highly interconnected autoregulatory loop called CRC. Here, we characterized fusion protein–driven CRC and identified its pharmacologic vulnerabilities, opening therapeutic avenues to indirectly target fusion-driven leukemia by disrupting its CRC.See related commentary by Sadras and Müschen, p. 18.This article is highlighted in the In This Issue feature, p. 5

SupplementaryFigures

Expression level of ELF4 in stably transduced T3M-1 Cl-10 cells

Caspase activities in T3M-1 Cl-10 cells infected with recombinant retroviruses for the indicated proteins.

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.

CEBPA-IGH, a fusion gene of the immunoglobulin heavy-chain locus (IGH) and the CCAAT enhancer-binding protein α (C/EBPα) gene, is recurrently found in B-ALL cases and causes aberrant expression of C/EBPα, a master regulator of granulocyte differentiation, in B cells. Forced expression of C/EBPα in B cells was reported to cause loss of B-cell identity due to the inhibition of Pax5, a master regulator of B-cell differentiation; however, it is not known whether the same mechanism is applicable for B-ALL development by CEBPA-IGH. It is known that a full-length isoform of C/EBPα, p42, promotes myeloid differentiation, whereas its N-terminal truncated isoform, p30, inhibits myeloid differentiation through the inhibition of p42; however, the differential role between p42 and p30 in ALL development has not been clarified. In the present study, we examined the effect of the expression of p42 and p30 in B cells by performing RNA-seq of mRNA from LCL stably transfected with p42 or p30. Unexpectedly, suppression of PAX5 target genes was barely observed. Instead, both isoforms suppressed the target genes of MEF2 family members (MEF2s), other regulators of B-cell differentiation. Similarly, MEF2s target genes rather than PAX5 target genes were suppressed in CEBP-IGH-positive ALL (n = 8) compared with other B-ALL (n = 315). Furthermore, binding of both isoforms to MEF2s target genes and the reduction of surrounding histone acetylation were observed in ChIP-qPCR. Our data suggest that the inhibition of MEF2s by C/EBPα plays a role in the development of CEBPA-IGH-positive ALL and that both isoforms work co-operatively to achieve it.

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy of T cell progenitors, known to be a heterogeneous disease in pediatric and adult patients. Here we attempted to better understand the disease at the molecular level based on the transcriptomic landscape of 707 T-ALL patients (510 pediatric, 190 adult patients, and 7 with unknown age; 599 from published cohorts and 108 newly investigated). Leveraging the information of gene expression enabled us to identify 10 subtypes (G1–G10), including the previously undescribed one characterized by GATA3 mutations, with GATA3R276Q capable of affecting lymphocyte development in zebrafish. Through associating with T cell differentiation stages, we found that high expression of LYL1/LMO2/SPI1/HOXA (G1–G6) might represent the early T cell progenitor, pro/precortical/cortical stage with a relatively high age of disease onset, and lymphoblasts with TLX3/TLX1 high expression (G7–G8) could be blocked at the cortical/postcortical stage, while those with high expression of NKX2-1/TAL1/LMO1 (G9–G10) might correspond to cortical/postcortical/mature stages of T cell development. Notably, adult patients harbored more cooperative mutations among epigenetic regulators, and genes involved in JAK-STAT and RAS signaling pathways, with 44% of patients aged 40 y or above in G1 bearing DNMT3A/IDH2 mutations usually seen in acute myeloid leukemia, suggesting the nature of mixed phenotype acute leukemia.

Although next‐generation sequencing‐based panel testing is well practiced in the field of cancer medicine for the identification of target molecules in solid tumors, the clinical utility and clinical issues surrounding panel testing in hematological malignancies have yet to be fully evaluated. We conducted a multicenter prospective clinical sequencing study to verify the feasibility of a panel test for hematological tumors, including acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, and diffuse large B‐cell lymphoma. Out of 96 eligible patients, 79 patients (82%) showed potentially actionable findings, based on the clinical sequencing assays. We identified that genetic alterations with a strong clinical significance were found at a higher frequency in terms of diagnosis (n = 60; 63%) and prognosis (n = 61; 64%) than in terms of therapy (n = 8; 8%). Three patients who harbored a germline mutation in either DDX41 (n = 2) or BRCA2 (n = 1) were provided with genetic counseling. At 6 mo after sequencing, clinical actions based on the diagnostic (n = 5) or prognostic (n = 3) findings were reported, but no patients were enrolled in a clinical trial or received targeted therapies based on the sequencing results. These results suggest that panel testing for hematological malignancies would be feasible given the availability of useful diagnostic and prognostic information. This study is registered with the UMIN Clinical Trial Registry (UMIN000029879, multiple myeloma; UMIN000031343, adult acute myeloid leukemia; UMIN000033144, diffuse large B‐cell lymphoma; and UMIN000034243, childhood leukemia)., This multicenter prospective study investigated feasibility of target capture‐based panel testing, focusing on hematological malignancies. Our results suggest that panel testing for hematological malignancies is feasible given the availability of useful diagnostic and prognostic information.

Previous genomic studies have revealed the genomic landscape of myeloma cells. Although some of the genomic abnormalities shown are believed to be correlated to the molecular pathogenesis of multiple myeloma and/or clinical outcome, these correlations are not fully understood. The aim of this study is to elucidate the correlation between genomic abnormalities and clinical characteristics by targeted capture sequencing in the Japanese multiple myeloma cohort. We analysed 154 patients with newly diagnosed multiple myeloma. The analysis revealed that the study cohort consisted of a less frequent hyperdiploid subtype (37·0%) with relatively high frequencies of KRAS mutation (36·4%) and IGH-CCND1 translocation (26·6%) compared with previous reports. Moreover, our targeted capture sequencing strategy was able to detect rare IGH-associated chromosomal translocations, such as IGH-CCND2 and IGH-MAFA. Interestingly, all 10 patients harboured MAX mutations accompanied by 14q23 deletion. The patients with del(17p) exhibited an unfavourable clinical outcome, and the presence of KRAS mutation was associated with shorter survival in patients with multiple myeloma, harbouring IGH-CCND1. Thus, our study provides a detailed landscape of genomic abnormalities, which may have potential clinical application for patients with multiple myeloma.

Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is intractable and mostly harbors genetic alterations that activate JAK or ABL signaling. The commonest subtype of Ph-like ALL exhibits the CRLF2 gene arrangement that brings about JAK1/2-STAT5 pathway activation. However, JAK1/2 inhibition alone is insufficient for treatment, necessitating combinatorial therapies targeting multiple signals. To better understand the mechanisms underlying the insufficient efficacy of JAK inhibition, this study explored gene expression changes upon treatment with a JAK1/2 inhibitor (ruxolitinib) and revealed BCL6 elevation as one such mechanism. Upregulated BCL6 suppressed TP53 expression along with its downstream cell cycle inhibitor p21 (CDKN2A) and proapoptotic molecules, such as FAS, TNFRSF10B, BID, BAX, BAK, PUMA, and NOXA, conferring cells in part with therapy resistance. The BCL6 inhibition (FX1) alone was able to upregulate TP53 and restore TP53 expression that ruxolitinib had diminished. In addition, ruxolitinib and FX1 concertedly downregulated MYC. As a result, FX1 treatment alone had growth-inhibitory and apoptosis-sensitizing effects, but the combination of ruxolitinib and FX1 more potently inhibited leukemia cell growth, enhanced apoptosis sensitivity, and prolonged xenografted mice survival. These findings provide one mechanism for the insufficiency of JAK inhibition for CRLF2-rearranged ALL treatment and BCL6 inhibition as a potentially helpful adjunctive therapy combined with JAK inhibition.

Zinc-finger protein 384 (ZNF384) fusion (Z-fusion) genes have recently been identified as recurrent fusion genes in B-cell precursor acute lymphoblastic leukaemia (BCP-ALL) and have been detected in 7-17% of Philadelphia chromosome-negative BCP-ALL cases. We selected SALL4 and ID2 as potential Z-fusion-specific transcriptional targets that might lead to the differentiation disorder of Z-fusion-positive ALL. The introduction of EP300-ZNF384 and SYNRG-ZNF384 induced the expression of these genes. Z-fusion proteins exhibited stronger transcriptional activities on the promoter or enhancer region of these genes than Wild-Z. Furthermore, GST pull-down assay revealed that Z-fusion proteins associated more strongly with EP300 than Wild-Z. Coexpression of EP300 specifically enhanced the transcriptional activities of Z-fusion proteins. We propose the increased EP300 binding of Z-fusion proteins as a mechanism for their increased transcriptional activities.

The genetic basis of leukemogenesis in adults with B-cell acute lymphoblastic leukemia (B-ALL) is largely unclear, and its clinical outcome remains unsatisfactory. This study aimed to advance the understanding of biological characteristics, improve disease stratification, and identify molecular targets of adult B-ALL. Adolescents and young adults (AYA) (15 to 39 years old, n = 193) and adults (40 to 64 years old, n = 161) with Philadelphia chromosome-negative (Ph−) B-ALL were included in this study. Integrated transcriptomic and genetic analyses were used to classify the cohort into defined subtypes. Of the 323 cases included in the RNA sequencing analysis, 278 (86.1%) were classified into 18 subtypes. The ZNF384 subtype (22.6%) was the most prevalent, with 2 novel subtypes (CDX2-high and IDH1/2-mut) identified among cases not assigned to the established subtypes. The CDX2-high subtype (3.4%) was characterized by high expression of CDX2 and recurrent gain of chromosome 1q. The IDH1/2-mut subtype (1.9%) was defined by IDH1 R132C or IDH2 R140Q mutations with specific transcriptional and high-methylation profiles. Both subtypes showed poor prognosis and were considered inferior prognostic factors independent of clinical parameters. Comparison with a previously reported pediatric B-ALL cohort (n = 1003) showed that the frequencies of these subtypes were significantly higher in AYA/adults than in children. We delineated the genetic and transcriptomic landscape of adult B-ALL and identified 2 novel subtypes that predict poor disease outcomes. Our findings highlight the age-dependent distribution of subtypes, which partially accounts for the prognostic differences between adult and pediatric B-ALL.

The genetic landscape of Burkitt lymphoma (BL) has been elucidated over decade, however, few reports have described genetic basis for its pathogenesis in familial BL [1-5]. Here, we performed targeted deep next generation sequencing of Japanese dizygotic twins who were diagnosed with BL (Supplemental Figure 1a).

MEF2D-fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5 % of acute lymphoblastic leukemia (ALL) cases. Their introduction to cells has been reported to transform cell lines or increase the colony formation of bone marrow cells, suggesting their survival-supporting ability, which prompted us to examine M-fusion-targeting drugs. To identify compounds that reduce the protein expression level of MEF2D, we developed a high-throughput screening system using 293T cells stably expressing a fusion protein of MEF2D and luciferase, in which the protein expression level of MEF2D was easily measured by a luciferase assay. We screened 3766 compounds with known pharmaceutical activities using this system and selected staurosporine as a potential inducer of the proteolysis of MEF2D. Staurosporine induced the proteolysis of M-fusion proteins in M-fusion (+) ALL cell lines. Proteolysis was inhibited by caspase inhibitors, not proteasome inhibitors, suggesting caspase dependency. Consistent with this result, the growth inhibitory effects of staurosporine were stronger in M-fusion (+) ALL cell lines than in negative cell lines, and caspase inhibitors blocked apoptosis induced by staurosporine. We identified the cleavage site of MEF2D-HNRNPUL1 by caspases and confirmed that its caspase cleavage-resistant mutant was resistant to staurosporine-induced proteolysis. Based on these results, we investigated another Food and Drug Administration-approved caspase activator, venetoclax, and found that it exerted similar effects to staurosporine, namely, the proteolysis of M-fusion proteins and strong growth inhibitory effects in M-fusion (+) ALL cell lines. The present study provides novel insights into drug screening strategies and the clinical indications of venetoclax.

DUX4 fusions are novel oncogenes in a subset of B-cell acute lymphoblastic leukemia (B-ALL) clinical cases, generally in older children and adolescent patients.[1][1] DUX4 fusion transcripts are generated from insertions of wild-type (WT) DUX4 , mainly into the IGH locus.[1][1]–[4][2] Notably, the

[Background] ETV6-FRK is a rare kinase-related fusion gene which was identified only in acute myeloid leukemia (Hosoya N, et al. Genes Chromosomes Cancer. 2005). Herein, we firstly identified ETV6-FRK fusion gene in a patient with high-risk pediatric B cell precursor ALL (B-ALL). Because FRK is Src family tyrosine kinase, we performed functional analysis of ETV6-FRK to establish molecular targeting therapy. [Patient] A 11-year-old boy with B-ALL was refractory to conventional chemotherapy and received allogeneic bone marrow transplantation (allo-BMT) following two courses of blinatumomab. This patient maintains complete remission for three months after allo-BMT. Cytogenetic analysis demonstrated t(6;12)(q21;p13) as a part of complex karyotype. Targeted capture mRNA sequencing identified ETV6-FRK fusion transcript in this patient. [Materials and methods] ETV6-FRK fusion was validated by RT-PCR of the diagnostic leukemic sample from this patient. Full length of ETV6-FRK cDNA was cloned into retroviral vector with Tet-On system. Then, Ba/F3 cells, which are IL-3 dependent murine pro B-ALL cells, were transduced with retroviral vector to establish Ba/F3 cells expressing ETV6-FRK (Ba/F3-ETV6-FRK) under doxycycline (DOX) dependent manner. Ba/F3-ETV6-FRK was analyzed whether IL-3 independent growth was achieved. To determine whether aberrant activation of FRK-STAT pathway as the downstream effects of ETV6-FRK, activation of FRK-STAT pathway was evaluated by western blot. Finally, in cytotoxic assay, proliferation of Ba/F3-ETV6-FRK was assessed under the media with various concentrations of dasatinib, a tyrosine kinase inhibitor. [Results and discussions] Sequencing of RT-PCR product revealed that ETV6 exon 4 was fused in-frame to FRK exon 3, creating an ETV6-FRK fusion gene. The ETV6-FRK fusion gene produced a chimeric protein consisting of the entire pointed (PNT) oligomerization domain of ETV6 and the kinase domain of FRK (Fig 1). The expression of ETV6-FRK in Ba/F3 cells under DOX dependent manner was confirmed by western blot. Ba/F3-ETV6-FRK proliferated without IL-3 in contrast to Ba/F3 cells not expressing ETV6-FRK (p [Conclusion] We identified the first patient of pediatric high-risk B-ALL harboring ETV6-FRK fusion by targeted capture mRNA sequencing, who was refractory to the conventional chemotherapy. We also provide the first evidence that dasatinib could abrogate proliferation activity of ETV6-FRK in vitro, suggesting that dasatinib might be effective for the patient with B-ALL carrying a ETV6-FRK fusion. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Plasma cell differentiation is initiated by antigen stimulation of the B cell receptor (BCR) and is regulated by BLIMP1. Prior to the stimulation of BCR, BLIMP1 is suppressed by PAX5, which is a key transcriptional repressor that maintains B cell identity. The upregulation of BLIMP1 and subsequent suppression of PAX5 by BLIMP1 are observed after the BCR stimulation. These events are considered to trigger plasma cell differentiation; however, the mechanisms responsible currently remain unclear. We herein demonstrated that the BCR signaling component, SYK, caused PAX5 tyrosine phosphorylation in vitro and in cells. Transcriptional repression on the BLIMP1 promoter by PAX5 was attenuated by this phosphorylation. The BCR stimulation induced the phosphorylation of SYK, tyrosine phosphorylation of PAX5, and up-regulation of BLIMP1 mRNA expression in B cells. The tyrosine phosphorylation of PAX5 co-operatively functioned with PAX5 serine phosphorylation by ERK1/2, which was our previous findings, to cancel the PAX5-dependent repression of BLIMP1. This co-operation may be a trigger for plasma cell differentiation. These results imply that PAX5 phosphorylation by a BCR signal is the initial event in plasma cell differentiation.

Most B cell precursor acute lymphoblastic leukemia (BCP ALL) can be classified into known major genetic subtypes, while a substantial proportion of BCP ALL remains poorly characterized in relation to its underlying genomic abnormalities. We therefore initiated a large-scale international study to reanalyze and delineate the transcriptome landscape of 1,223 BCP ALL cases using RNA sequencing. Fourteen BCP ALL gene expression subgroups (G1 to G14) were identified. Apart from extending eight previously described subgroups (G1 to G8 associated with MEF2D fusions, TCF3–PBX1 fusions, ETV6–RUNX1–positive/ETV6–RUNX1–like, DUX4 fusions, ZNF384 fusions, BCR–ABL1/Ph–like, high hyperdiploidy, and KMT2A fusions), we defined six additional gene expression subgroups: G9 was associated with both PAX5 and CRLF2 fusions; G10 and G11 with mutations in PAX5 (p.P80R) and IKZF1 (p.N159Y), respectively; G12 with IGH–CEBPE fusion and mutations in ZEB2 (p.H1038R); and G13 and G14 with TCF3/4–HLF and NUTM1 fusions, respectively. In pediatric BCP ALL, subgroups G2 to G5 and G7 (51 to 65/67 chromosomes) were associated with low-risk, G7 (with ≤50 chromosomes) and G9 were intermediate-risk, whereas G1, G6, and G8 were defined as high-risk subgroups. In adult BCP ALL, G1, G2, G6, and G8 were associated with high risk, while G4, G5, and G7 had relatively favorable outcomes. This large-scale transcriptome sequence analysis of BCP ALL revealed distinct molecular subgroups that reflect discrete pathways of BCP ALL, informing disease classification and prognostic stratification. The combined results strongly advocate that RNA sequencing be introduced into the clinical diagnostic workup of BCP ALL.

Mammalian target of rapamycin (mTOR) is a serine-threonine kinase that acts downstream of the phosphatidylinositol 3-kinase signaling pathway and regulates a wide range of cellular functions including transcription, translation, proliferation, apoptosis, and autophagy. Whereas genetic alterations that result in mTOR activation are frequently present in human cancers, whether the mTOR gene itself becomes an oncogene through somatic mutation has remained unclear. We have now identified a somatic non-synonymous mutation of mTOR that results in a leucine-to-valine substitution at amino acid position 2209 in a specimen of large cell neuroendocrine carcinoma. The mTOR(L2209V) mutant manifested marked transforming potential in a focus formation assay with mouse 3T3 fibroblasts, and it induced the phosphorylation of p70 S6 kinase, S6 ribosomal protein, and eukaryotic translation initiation factor 4E-binding protein 1 in these cells. Examination of additional tumor specimens as well as public and in-house databases of cancer genome mutations identified another 28 independent non-synonymous mutations of mTOR in various cancer types, with 12 of these mutations also showing transforming ability. Most of these oncogenic mutations cluster at the interface between the kinase domain and the FAT (FRAP, ATM, TRRAP) domain in the 3-D structure of mTOR. Transforming mTOR mutants were also found to promote 3T3 cell survival, and their oncogenic activity was sensitive to rapamycin. Our data thus show that mTOR acquires transforming activity through genetic changes in cancer, and they suggest that such tumors may be candidates for molecularly targeted therapy with mTOR inhibitors., Cancer Science, 106(12), pp.1687-1692; 2015

MEF2D fusion genes are newly discovered recurrent gene abnormalities that are detected in approximately 5% of acute lymphoblastic leukemia cases. We previously demonstrated that the vector-driven expression of MEF2D fusion proteins was markedly stronger than that of wild-type MEF2D; however, the underlying mechanisms and significance of this expression have yet to be clarified. We herein showed that the strong expression of MEF2D fusion proteins was caused by the loss of the target site of miRNA due to gene translocation. We identified the target region of miRNA located in the coding region and selected miR-122 as a candidate of the responsible miRNA. Mutations at a putative binding site of miR-122 increased MEF2D expression, while the transfection of its miRNA mimic reduced the expression of wild-type MEF2D, but not MEF2D fusion proteins. We also found that MEF2D fusion proteins inhibited the transcriptional activity of PAX5, a B-cell differentiation regulator in a manner that depended on fusion-specific strong expression and an association with histone deacetylase 4, which may lead to the differentiation disorders of B cells. Our results provide novel insights into the mechanisms underlying leukemia development by MEF2D fusion genes and the involvement of the deregulation of miRNA-mediated repression in cancer development.

Fusion genes found in cases of acute lymphoblastic leukemia (ALL) are reported to be associated with age, such as MLL rearrangements in neonates and BCR-ABL1 in adults. However, the pathogenesis of ALL in adolescents and young adults (AYA) remains largely unknown. To investigate the potential role of fusion genes, we performed RNA-sequencing on 73 BCR-ABL1-negative ALL patients who were all AYA. Interestingly, DUX4-IGH was the most frequent fusion gene detected in B-ALL (18.5%) and was preferentially detected in the AYA generation. ZNF384 and MEF2D genes were also recurrently identified as functionally relevant fusion genes in 16.7% and 9.3% of AYA with B-ALL, respectively. Patients with DUX4 and ZNF384 fusion genes displayed better prognosis, while those with the MEF2D fusion gene displayed a worse outcome. To improve treatment outcome, the fusion genes detected in this study will be useful for risk stratification and target therapy.

Triple-negative breast cancer (TNBC) cells do not express estrogen receptors, progesterone receptors, or human epidermal growth factor receptor 2. Currently, apart from poly ADP-ribose polymerase inhibitors, there are few effective therapeutic options for this type of cancer. Here, we present comprehensive characterization of the genetic alterations in TNBC performed by high coverage whole genome sequencing together with transcriptome and whole exome sequencing. Silencing of the BRCA1 gene impaired the homologous recombination pathway in a subset of TNBCs, which exhibited similar phenotypes to tumors with BRCA1 mutations; they harbored many structural variations (SVs) with relative enrichment for tandem duplication. Clonal analysis suggested that TP53 mutations and methylation of CpG dinucleotides in the BRCA1 promoter were early events of carcinogenesis. SVs were associated with driver oncogenic events such as amplification of MYC, NOTCH2, or NOTCH3 and affected tumor suppressor genes including RB1, PTEN, and KMT2C. Furthermore, we identified putative TGFA enhancer regions. Recurrent SVs that affected the TGFA enhancer region led to enhanced expression of the TGFA oncogene that encodes one of the high affinity ligands for epidermal growth factor receptor. We also identified a variety of oncogenes that could transform 3T3 mouse fibroblasts, suggesting that individual TNBC tumors may undergo a unique driver event that can be targetable. Thus, we revealed several features of TNBC with clinically important implications., Author summary Cancer can result from genetic alterations, some of which can be good drug targets. To reveal genetic alterations that provide important information for the development of ideal therapeutic strategies for triple-negative breast cancer (TNBC), TNBC tumor samples were subjected to comprehensive genomic analyses. We identified novel recurrent structural variations associated with enhanced expression of the TGFA gene that encodes one of the high affinity ligands for epidermal growth factor receptor (EGFR). Although TGFA expression is known to be elevated in a subset of TNBC tumors, this is the first report of the mechanistic basis of this phenomenon. It is of particular importance considering that anti-EGFR agents are possible therapeutic options for TNBC patients. Our study also revealed several features associated with “BRCAness”, which is critical for identification of patients who may be responsive to platinum agents and/or poly ADP-ribose polymerase inhibitors. Thus, the data presented in this report may advance our understanding of the pathogenesis of TNBC.

MEF2D fusion (M-fusion) genes are newly discovered recurrent gene abnormalities that are detected in approximately 5% of acute lymphoblastic leukemia (ALL) cases. We previously found that the loss of the micro RNA target site in wild-type MEF2D gene by translocation led to strong expression of M-fusion protein in ALL cells by evasion from micro RNA and that M-fusion protein inhibited the transcriptional activity of PAX5, a B-cell differentiation regulator, in a dose-dependent manner. These findings prompted us to explore drugs that induced proteolysis of M-fusion protein as possible therapeutic agents for M-fusion-positive ALL. We developed a high-throughput screening system to find compounds that reduced protein expression level of MEF2D. The expression vector of the fusion protein of N-terminal half of MEF2D (MEF2D N) and luciferase (MEF2D N-Luc) was stably transfected to 293T cells (MEF2D N-Luc/293T). Stable transfectant of the expression vector of luciferase was also established (Luc/293T). We could easily measure protein expression level in these cells by luciferase assay. We screened 3766 compounds with known pharmaceutical activities with this system and selected staurosporine, a multi-kinase inhibitor, as a possible proteolysis-inducer of MEF2D. Staurosporine strongly reduced the luciferase value in MEF2D N-Luc/293T but not in Luc/293T (Figure 1A). Staurosporine induced proteolysis of MEF2D-HNRNPUL1 (M-H) and MEF2D-DAZAP1 (M-D) in M-fusion-positive ALL cell lines within 6 h. Proteolysis of M-fusion proteins were inhibited not by MG-132, a proteasome inhibitor, but by Z-VAD FMK, a caspase inhibitor, indicating that these proteolyses were caspase-dependent (Figure 1B). Consistent with this, Z-VAD-FMK blocked apoptosis by staurosporine in M-H positive ALL cell lines . We confirmed the cleavage of M-H by caspase 3 and caspase 7 in vitro and identified the cleavage site (Figure 1C). Furthermore, staurosporine demonstrated stronger cytotoxic effect on M-fusion-positive ALL cell lines than M-fusion-negative ones (Figure 1D). These results indicated that staurosporine induced apoptosis of M-fusion-positive ALL cells through caspase-dependent proteolysis of M-fusion protein at least in part. Luciferase-based proteolysis screening provided a novel strategy for the development of anti-cancer drugs. Figure legends Figure 1. A. Staurosporine strongly reduced the luciferase value in MEF2D N-Luc/293T but not in Luc/293T. MEF2D N-Luc/293T were treated with 3766 compounds (2uM each) for 24 h. Then luciferase assays were performed to estimate the amount of MEF2D N-Luc protein. Top 15 compounds which reduced the relative luciferase value were selected for the second screening where compounds were added to MEF2D N-Luc/293T and Luc/293T, then we estimated their effect on the expression of MEF2D N-Luc and luciferase. Results of the second screening were plotted on a scattergram, on which the relative luciferase value in Luc/293T and MEF2D N-Luc/293T were set on the Y-axis and X-axis, respectively. Relative luciferase values are relative values to those of control cells treated with vehicle (DMSO). Staurosporine and K252a, an analog of staurosporine, were selected as hit compounds which reduced MEF2D N-Luc but not luciferase. B. Staurosporine reduced the expression of M-H and M-D, which inhibited by Z-VAD FMK. M-H-positive ALL cell lines, Kasumi-7 and Kasumi-9 and a M-D-positive ALL cell line, TS-2 were treated with 1uM staurosporine, 20uM Z-VAD-FMK, or both for 6 h. Cells were lysed and subjected to immunoblotting with indicated antibodies. C. Caspase-3 and caspase-7 cleaved M-H in vitro. M-H and its mutants with aspartate substitutions at possible caspase cleavage sites were synthesized with [35S]-methionine-labeled in vitro translation and were incubated with purified caspase-3 or -7. Cleaved fragments were resolved on SDS-PAGE and visualized by autoradiography. Mutant 4 was resistant to the cleavage by both caspases. D. Staurosporine demonstrated stronger cytotoxic effect on M-fusion-positive ALL cell lines than M-fusion-negative ones. ALL cell lines were treated with staurosporine at the indicated doses for 24 h. Cell viabilities were measured using MTT assay. Figure 1 Disclosures Kiyoi: Takeda Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Honoraria; Astellas Pharma Inc.: Honoraria, Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Bristol-Myers Squibb: Research Funding; Daiichi Sankyo Co., Ltd: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co.,Ltd.: Research Funding; Perseus Proteomics Inc.: Research Funding.

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.

We aimed to evaluate the efficacy and safety of allogeneic hematopoietic stem cell transplantation with targeted oral busulfan (BU) and cyclophosphamide (CY) in a phase II study. Busulfan (1.0 mg/kg) was given initially in six doses. Based on the estimated concentration at steady state after the first dose of BU, subsequent (7th-16th) doses were adjusted to obtain a targeted overall concentration at steady state of 700-900 ng/mL. The primary endpoint was 1-year overall survival (OS). Fifty patients were registered and 46 (median age, 53 years; range, 18-62 years) received planned transplant, including 24 with AML, 16 with myelodysplastic syndrome, and six with CML. Fourteen patients were categorized as standard risk. Nineteen patients received transplant from human leukocyte antigen-identical siblings, 27 from unrelated donors. The BU dose required reduction in 32 patients and escalation in six patients. One-year OS was 65% (95% confidence interval, 50-77%). Cumulative incidence of hepatic sinusoidal obstruction syndrome was 11%. One-year transplant-related mortality was 18%. Both OS and transplant-related mortality were favorable in this study, including patients of older age and with high risk diseases. Individual dose adjustment based on BU pharmacokinetics was feasible and effective in the current phase II study. This trial is registered in the University Hospital Medical Information Network Clinical Trial Registry System (UMIN-CTR, ID:C000000156).

Background Because of the less graft-facilitating effect by bone marrow (BM), we need to assess a dosage of conditioning more accurately particularly in combination with reduced-intensity conditioning. Thus we examined that modified continual reassessment method (mCRM) is applicable for deciding appropriate conditioning of allogeneic BM transplantation. Patients and methods The conditioning regimen consisted of i.v. fludarabine (125 mg/m2) plus an examination dose of i.v. melphalan. The primary endpoint was a donor-type T-cell chimerism at day 28 with successful engraftment defined as >90% donor cells. Five patients per dose level were planned to be accrued and chimerism data were used to determine the next dose. Results Seventeen patients were enrolled at doses between 130 and 160 mg/m2. The dose was changed from 160 to 130 mg/m2 (second level) after five full-donor chimerisms. With one patient of 0% chimera in the second level, the dose was increased to 135 mg/m2 (third level). Following five full-donor chimerisms in the third level, the study was complete as projected. Conclusions mCRM was shown to be a relevant method for dose-finding of conditioning regimen. The melphalan dose of 135 mg/m2 was determined as the recommended phase II dose to induce initial full-donor chimerism.

The effects of macrophage activation on the outcome of allogeneic hematopoietic SCT (allo-HSCT) have yet to be fully examined. A total of 70 adult patients who received a first allo-HSCT for hematological diseases were studied. We counted the number of hemophagocytic cells in BM clot sections on day +14±7, and analyzed its impact on subsequent outcome. In all, 23 patients were diagnosed as having increased numbers of hemophagocytic cells (HP group), whereas 47 were not (non-HP group). The HP group was not associated with an increased incidence of acute or chronic GVHD, but was associated with worse hematopoietic recovery than the non-HP group. The 2-year OS for the HP group and the non-HP group was 30 and 65% (P

Although survival rate for children with Acute Lymphoblastic Leukemia (ALL) now exceeds about 90%, the outcome of adult patients with ALL is extremely poor. These differences might be attributed to the lack of insights into pathogenesis and clinical behavior of adult-ALL. Gross chromosomal alterations including chromosome translocations and aneuploidy are considered as early events in ALL and constitute disease subtypes. To identify chromosome translocations underlying adult with Ph-negative B-ALL, we performed RNA-seq analysis on RNA from individuals with B-ALL who had been treated on the Japan Adult Leukemia Study Group (JALSG) ALL202-O protocol (n = 149). We successfully identified chromosome translocations in 100 patients (67.1%). ZNF384 fusions were most frequently detected in 30 patients (20.1%) and they had wide range of fusion partners. DUX4- and MEF2D- fusions were also recurrently found in 7 (4.7%) and 9 (6.0%) patients, respectively. Chromosome translocations activating kinase and cytokine receptor were found in 25 patients (16.8%) with Ph-like gene expression profile. These alterations were almost completely mutually exclusive indicating these are likely to be primary genetic events. For simplicity, here we define (1) fusions involving ZNF384, DUX4, MEF2D, CEBP and PAX5 as well as TCF3-PBX1 and ETV6-RUNX1 as Transcription Factor fusions (TF fusions; 49% of patients), (2) fusions involving CRLF2, JAK2, PDGFRB, EPOR and ABL as Kinase/cytokine-receptor Activating fusions (KA fusions; 15%) and (3) non-recurrent fusions or the absence of fusions/aneuploidy as B-others (30%). First, we analyzed impact of the patient age on types of fusion genes, based of combined data of ALL202-O cohort, childhood B-ALL cohort (Lilljebjörn H, et al. 2016: n = 189) and ALL202-U cohort (Yasuda T, et al. 2016: n = 54). We found that incidence of ZNF384-, CEBP- fusions and B-others increases as patients age, whereas ETV6-RUNX1 and PAX5 fusions were more prevalent in younger patients, exhibiting negative association with age. DUX4 fusions and TF fusions were most prevalent in Adolescent and Young Adult (AYA) generation. JAK2-, PDGFRB-, EPOR- and KA- fusions were positively correlated with age. Next, we analyzed association between patient survival and types of fusions. In Japanese adult B-ALL cohort (ALL202-O and ALL202-U cohort), we observed ZNF384-, DUX4- fusions and TCF3-PBX1 were associated with better disease-free survival than B-others. Furthermore, when combined, MEF2D- (n = 14), CEBP- (n = 4), PAX5- fusions (n = 2) and ETV6-RUNX1 (n = 2) exhibited significantly better disease-free survival than B-others, indicating TF fusions were associated with an improved outcome. In contrast, KA fusions were associated with poorer disease-free survival than B-others. KMT2A fusions were comparable with B-others regarding to patient disease-free survival. These results allowed us to develop a prognostic schema to identify three distinct risk profile groups, based on types of fusion genes and cytogenetics (Table1); favorable-risk (5-year rate of disease-free survival 67.4%), intermediate-risk (5-year rate of disease-free survival 42.5%) and adverse-risk (5-year rate of disease-free survival 9.6%). This prognostic schema predicted the outcome independently of age, sex and methotrexate dose in multivariate analysis (p < 0.001). In conclusion, we promoted a better understanding of the genetic basis of adult B-ALL by focusing on fusion genes. Each chromosome translocations were closely associated with age. ZNF384-, KA fusions and B-others were characteristic for older-adult patients (40-65 years old) with B-ALL. We clearly demonstrated specific primary chromosome abnormalities are strong prognostic marker. Functional properties of primary genetic events (TF fusions vs. KA fusions) might be a key determinant of biological characteristics and clinical outcome. Disclosures Kiyoi: Novartis Pharma K.K.: Research Funding; Celgene Corporation: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; FUJIFILM Corporation: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Bristol-Myers Squibb: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Takeda Pharmaceutical Co., Ltd.: Research Funding; Sanofi K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Phizer Japan Inc.: Research Funding; Eisai Co., Ltd.: Research Funding. Naoe:Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding.

ZNF384 fusion (Z-fusion) genes are recently identified recurrent fusion genes of B-acute lymphoblastic leukemia (ALL) and cause differentiation block of B-cells; however, its molecular mechanisms have yet to be clarified. Common structural character of Z-fusion proteins is that fusion partners are fused to the N-terminal end of full-length ZNF384 (Figure 1A), suggesting that protein-fusions confer specific transcriptional targets on ZNF384. We searched Z-fusion-specific transcriptional targets that could cause differentiation block of B-cells by analyzing the data of gene expression profile of 54 primary B-ALL samples containing 9 Z-fusion positive ALL. We selected ID2 and SALL4 as potential targets. Both genes were expressed markedly higher in Z-fusion-positive ALL. ID2 acts as an inhibitor of E2A, B cell differentiation regulator, and SALL4 plays essential roles in maintaining pluripotency of embryonic stem cells. In the luciferase assays, EP300-ZNF384 (E-Z) and SYNRG-ZNF384 (S-Z) showed stronger transcriptional activities on the promoters of these genes than wild-type ZNF384 (Wild-Z). The introduction of E-Z or S-Z into 293T cells and THP-1 cells induced mRNA expression of these genes more strongly than that of Wild-Z (Figure 1B). We identified Z-fusion binding sites in the promoters of these genes. DNA binding abilities of Z-fusions to these sites were not stronger than that of Wild-Z in electro mobility shift assay. GST-pull down assay showed that E-Z associated with EP300 more strongly than Wild-Z (Figure 1C). Consistent with this, co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z (Figure 1D). These results indicated that ID2 and SALL4 were Z-fusion-specific transcriptional targets and that the high affinity to EP300 was responsible for the strong transcriptional activity of Z-fusions. Our results shed a new insight into the molecular mechanisms of leukemia development by Z-fusions. Figure legends Figure 1. A. Schematic presentation of structures of Wild-Z and Z-fusion proteins. B. Introduction of Z-fusion genes enhanced the mRNA expression of ID2 and SALL4. Wild-Z and Z-fusion genes were introduced to THP-1 cells by nucleofection. Twenty-four hours after gene introduction, the mRNA expression of ID2 and SALL4 were quantified by RQ-PCR and potted on bar charts as relative values to the mRNA expression in the control cells. The expression of ID2 and SALL4 were shown in the left and right bar charts, respectively. C. E-Z associated with HAT more strongly than Wild-Z. Glutathione beads attached with Glutathione S transferase (GST) or GST-fused HAT were incubated with Wild-Z or E-Z synthesized in vitro with [35S]-Methionine labeling. Wild-Z or E-Z associated with GST-HAT were visualized with autoradiography (left panel), quantified, and plotted on the bar charts (right panel). Of note, the quantified association were adjusted for the ratio of the number of methionine containing in Wild-Z and E-Z, 12 to 45, and plotted on the bar charts. D. Co-expression of EP300 enhanced the transcriptional activity of E-Z better than that of Wild-Z. Luciferase assay were performed with or without co-expression of EP300 and the relative value to the control were plotted on the bar charts. Disclosures Naoe: Astellas Pharma Inc.: Research Funding; Fujifilm Corporation: Patents & Royalties, Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Pfizer Japan Inc.: Research Funding; Toyama Chemical Co., Ltd.: Research Funding. Kiyoi:FUJIFILM Corporation: Research Funding; Eisai Co., Ltd.: Research Funding; Astellas Pharma Inc.: Research Funding; Kyowa Hakko Kirin Co., Ltd.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Chugai Pharmaceutical Co., Ltd.: Research Funding; Otsuka Pharmaceutical Co., Ltd.: Research Funding; Zenyaku Kogyo Co., Ltd.: Research Funding; Novartis Pharma K.K.: Research Funding; Nippon Shinyaku Co., Ltd.: Research Funding; Celgene Corporation: Research Funding; Bristol-Myers Squibb: Honoraria; Takeda Pharmaceutical Co., Ltd.: Research Funding; Phizer Japan Inc.: Research Funding; Sanofi K.K.: Research Funding.

Corticosteroids are often used following allogeneic hematopoietic stem cell transplantation (HSCT) to control complications such as graft-versus-host disease (GVHD). However, there is some concern that corticosteroids may suppress the graft-versus-leukemia effect and increase leukemia relapse. To evaluate the effect of corticosteroids on relapse, we analyzed 112 adult patients who received their first allogeneic HSCT for acute myeloid leukemia at our institution between 1997 and 2007. Fifty-seven patients (50.9%) received corticosteroid therapy. Patients who had corticosteroid therapy included higher proportion of patients who developed GVHD. In multivariate analysis, with corticosteroid administration entered as a time-dependent covariate, corticosteroid administration was not a risk factor for relapse (p = 1.00, hazard ratio [HR] 1.00, 95% confidence interval [CI] 0.53-1.88), but it was associated with higher non-relapse mortality (NRM) (p < 0.001, HR 55.5, 95% CI 7.42-416) and lower overall survival (p < 0.001, HR 2.68, 95% CI 1.56-4.61). The higher NRM associated with corticosteroid administration was mainly due to the increased deaths caused by the complications themselves, which required corticosteroid therapy. The findings of this study indicate the importance of controlling complications after allogeneic HSCT. The strategy of refraining from indispensable corticosteroid therapy because of the excessive concerns about relapse should be avoided.

KAATSU training induces muscle hypertrophy and strengthens muscle in athletes and healthy subjects through short-term and low-intensity exercise. However, it remains uninvestigated whether lowintensity KAATSU resistance training (LIKRT) induces muscle strength and hypertrophy in patients with cardiovascular diseases. We examined the effects of LIKRT on skeletal muscle size/strength and endurance capacity in patients with ischemic heart disease (IHD). Seven male patients with stable IHD performed three kinds of resistance exercises (leg press, leg curl and leg extension) with their femoral muscle blood flow restricted by KAATSU belt two times/week for three months. We measured one RM (1-RM) in each resistance exercises, and evaluated muscle cross-sectional areas (CSA) by MRI before training and after the training. We used cardiopulmonary examinations to measure endurance capacity (Peak VO2 (VO2peak), VO2 at anaerobic threshold (VO2AT)). We performed blood sampling to measure resting plasma level of insulin growth factor-1 (IGF-1) and serum high-sensitive C-reactive protein (hsCRP). LIKRT significantly increased leg press (15%), leg curl (18%) and leg extension (17%) 1-RM strength. Increases of muscle CSA in quadriceps femoris at the proximal lower leg (30%), the mid-thigh (50%), and the proximal lower leg (70%) were 5.1%, 4.6% and 10.4%, respectively. Similarly, hamstring and adductor CSA were also increased by LIKRT. LIKRT significantly increased VO2peak and VO2AT by 10.7% and 10.9%, respectively. IGF-1 and hsCRP were not altered before or after the training. These results suggest that LIKRT increases muscle strength/mass and endurance capacity in patients with IHD. LIKRT appears to be a promising and effective resistance method in cardiac rehabilitation.

Early deaths after allogeneic stem cell transplantation (allo-SCT) are of major concern. On the assumption that both decreased and increased basal metabolism might relate to early deaths, we analyzed the risk factors for overall survival to days 30 (OS30) and 60 (OS60). The Harris-Benedict equation was used to calculate basal metabolism. Comparing a patient's basal metabolism (PBM) calculated from pretransplant body weight with the standard basal metabolism (SBM) calculated from standard body weight (body mass index (BMI) = 22), we defined the basal metabolic ratio (BMR) as a parameter (BMR = PBM/SBM). We retrospectively analyzed 360 adult patients transplanted between 1997 and 2006 at a single center in Japan. A multivariate analysis of OS30 showed risk factors to be: BMRor = 0.95 (low BMR; LBR) (P = 0.01), BMR1.05 (high BMR; HBR) (P = 0.005) and non-complete remission (non-CR) (P 5 0.001), whereas a multivariate analysis of OS60 showed those risk factors to be: LBR (P = 0.02), HBR (P = 0.04), non-CR (P = 0.002), and performance statusor = 1 (P = 0.01). OS30 and OS60 were found to be favorable in 0.95BMRor = 1.05 (average BMR; ABR) (96.8 and 90.3% for ABR, 87.1 and 76.2% for LBR, and 87.8 and 81.1% for HBR). In conclusion, BMR could prove to be a predictor of early death after allo-SCT.

We have been developing a small, lightweight motor-driven pulsatile left ventricular assist device (LVAD) with a ball screw. The motor-driven LVAD consists of a brushless DC motor and a ball screw. The attractive magnetic force between Nd-Fe-B magnets (with a diameter of 5 mm and a thickness of 1.5 mm) mounted in holes in a silicone rubber sheet (thickness 2 mm) and an iron plate adhered onto the a diaphragm of the blood pump can provide optimum active blood filling during the pump filling phase. The LVAD has a stroke volume of 55 ml and an overall volume of 285 ml; it weighs 360 g. The controller mainly consists of a fuzzy logic position and velocity controller to apply doctors' and engineers' knowledge to control the LVAD. Each unit of the controller consists of a functionally independent program module for easy improvement of the controller's performance. The LVAD was evaluated in in vitro experiments using a mock circulation. A maximum pump outflow of 5.1 l/min was obtained at a drive rate of 95 bpm against an afterload of 95 mmHg, and active filling using the attractive magnetic force provided a pump output of 3.6 l/min at a drive rate of 75 bpm under a preload of 0 mmHg. The operating efficiency of the LVAD was measured at between 8% and 10.5%. While the LVAD can provide adequate pump outflow for cardiac assistance, further upgrading of the software and improvement of the blood pump are required to improve pump performance and efficiency.

Transforming growth factor-β (TGF-β) plays central roles in endothelial–mesenchymal transition (EndMT) involved in development and pathogenesis. Although EndMT and epithelial–mesenchymal transition are similar processes, roles of microRNAs in EndMT are largely unknown. Here, we report that constitutively active microRNA-31 (miR-31) is a positive regulator of TGF-β-induced EndMT. Although the expression is not induced by TGF-β, miR-31 is required for induction of mesenchymal genes including α-SMA, actin reorganization and MRTF-A activation during EndMT. We identified VAV3, a regulator of actin remodeling and MRTF-A activity, as a miR-31 target. Global transcriptome analysis further showed that miR-31 positively regulates EndMT-associated unique secretory phenotype (EndMT-SP) characterized by induction of multiple inflammatory chemokines and cytokines including CCL17, CX3CL1, CXCL16, IL-6 and Angptl2. As a mechanism for this phenomenon, TGF-β and miR-31 suppress Stk40, a negative regulator of NF-κB pathway. Interestingly, TGF-β induces alternative polyadenylation (APA)-coupled miR-31-dependent Stk40 suppression without concomitant miR-31 induction, and APA-mediated exclusion of internal poly(A) sequence in Stk40 3′UTR enhances target efficiency of Stk40. Finally, miR-31 functions as a molecular hub to integrate TGF-β and TNF-α signaling to enhance EndMT. These data confirm that constitutively active microRNAs, as well as inducible microRNAs, serve as phenotypic modifiers interconnected with transcriptome dynamics during EndMT.

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.

The oncogenic mechanisms underlying acute lymphoblastic leukemia (ALL) in adolescents and young adults (AYA; 15-39 years old) remain largely elusive. Here we have searched for new oncogenes in AYA-ALL by performing RNA-seq analysis of Philadelphia chromosome (Ph)-negative AYA-ALL specimens (n = 73) with the use of a next-generation sequencer. Interestingly, insertion of D4Z4 repeats containing the DUX4 gene into the IGH locus was frequently identified in B cell AYA-ALL, leading to a high level of expression of DUX4 protein with an aberrant C terminus. A transplantation assay in mice demonstrated that expression of DUX4-IGH in pro-B cells was capable of generating B cell leukemia in vivo. DUX4 fusions were preferentially detected in the AYA generation. Our data thus show that DUX4 can become an oncogenic driver as a result of somatic chromosomal rearrangements and that AYA-ALL may be a clinical entity distinct from ALL at other ages.

Asymmetric selection of single-stranded guide RNAs from double-stranded RNA precursors is crucial in RNA silencing-mediated gene regulation. However, the precise mechanisms of small-RNA asymmetry are unclear, especially because asymmetric selection can still occur when the putative asymmetry sensors Drosophila R2D2 and mammalian Dicer are depleted. Here we report a direct contribution of mammalian Argonaute 2 (Ago2) to microRNA (miRNA) asymmetry. Ago2 selects strands with 5'-uridine or 5'-adenosine and thermodynamically unstable 5' ends in parallel through its two sensor regions, which contact the 5' nucleobases and 5'-phosphates of prospective guide strands. Hence, miRNA asymmetry shows superposed patterns reflecting 5'-end nucleotide identity ('digital' pattern) and thermodynamic stability ('analog' pattern). Furthermore, we demonstrate that cancer-associated miRNA variations reprogram asymmetric selection. Finally, our study presents a model of this universal principle, to aid in comprehensive understanding of miRNA function and development of new RNA-silencing therapies in precision medicine.

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.