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For this study, we investigated comprehensive expression of conjoined genes (CGs) in non-Hodgkin B-cell lymphoma (B-NHL) cell line KPUM-UH1 by using paired-end RNA sequencing. Furthermore, we analyzed the expression of these transcripts in an additional 21 cell lines, 37 primary samples of various malignancies and peripheral blood mononuclear cells of four normal individuals. Seventeen CGs were detected in KPUM-UH1: CTBS-GNG5, SRP9-EPHX1, RMND5A-ANAPC, OTX1-EHBP1, ATF2-CHN1, PRKAA1-TTC33, LARP1-MRPL22, LOC105379697-BAK1, TIAM2-SCAF8, SPAG1-VPS13B, WBP1L-CNNM2, NARS2-GAB2, CTSC-RAB38, VAMP1-CD27-AS1, LRRC37A2-NSF, UBA2-WTIP and ZNF600-ZNF611. To our knowledge, 10 of these genes have not been previously reported. The various characteristics of the CGs included in- and out-of-frame fusions, chimeras involving non-coding RNA and transcript variants. A finding of note was that LARP1-MRPL2 was characterized as in-frame fusion and was recurrently expressed in B-NHL samples. In this study, variety of CGs was expressed both in malignant and normal cells, some of which might be specific to lymphoma.

Extended post-therapy long-term survival of patients with diffuse large B cell lymphoma (DLBCL) may also lead to an increase of late adverse events. We retrospectively investigated the frequency and clinical manifestation of second primary malignancy (SPM) after rituximab-containing immunochemotherapy in patients with DLBCL treated at seven institutes belonging to the Kyoto Clinical Hematology Study Group (KOTOSG) from the perspective of the existence of past or synchronous cancer history. In a median follow-up period of 899 days, 69 SPMs were observed in 58 of 809 patients. The most frequent SPM was gastric cancer, followed by lung cancer and colorectal cancer. The cumulative incidence of SPM increased steadily over time and was not significantly influenced by the presence or absence of past or synchronous cancer history. Our study suggests the need for careful attention to SPM in patients with DLBCL in daily practice.

RSK2 is a serine/threonine kinase downstream signaling mediator in the RAS/ERK signaling pathway and may be a therapeutic target in mantle cell lymphoma (MCL), an almost incurable disease subtype of non‐Hodgkin lymphoma. In this study, serine‐227 (RSK2Ser227) in the N‐terminal kinase domain (NTKD) of RSK2 was found to be ubiquitously active in five MCL‐derived cell lines and in tumor tissues derived from five MCL patients. BI‐D1870, an inhibitor specific to RSK2‐NTKD, caused RSK2Ser227 dephosphorylation, and thereby, induced dose‐dependent growth inhibition via G2/M cell cycle blockade and apoptosis in four of the five cell lines, while one cell line showed only modest sensitivity. In addition, RSK2 gene knockdown caused growth inhibition in the four BI‐D1870‐sensitive cell lines. Comparative gene expression profiling of the MCL‐derived cell lines showed that inhibition of RSK2Ser227 by BI‐D1870 caused downregulation of oncogenes, such as c‐MYC and MYB; anti‐apoptosis genes, such as BCL2 and BCL2L1; genes for B cell development, including IKZF1, IKZF3, and PAX5; and genes constituting the B cell receptor signaling pathway, such as CD19, CD79B, and BLNK. These findings show that targeting of RSK2Ser227 enables concomitant blockade of pathways that are critically important in B cell tumorigenesis. In addition, we found favorable combinatory growth inhibitory effects of BI‐D1870 with inhibitors of BTK (ibrutinib), AKT (ipatasertib), and BCL2 (venetoclax) in cell characteristic‐dependent manners. These results provide a rationale for RSK2Ser227 in the NTKD as a potential therapeutic target in MCL and for future development of a novel bioavailable RSK2 NTKD‐specific inhibitor., RSK2Ser227 in the N‐terminal kinase domain (NTKD) of RSK2 was found to be ubiquitously active in mantle cell lymphoma (MCL). RSK2 concomitantly regulates series of genes involved in oncogenesis, apoptosis, B cell development, and B cell receptor signaling. RSK2Ser227 in the NTKD is a potential therapeutic target in MCL.

An increase in immunosuppressive myeloid-derived suppressor cells (MDSCs) is associated with disease progression and treatment resistance in multiple myeloma (MM). We investigated the mechanisms underlying MDSC induction, and sought to discover a strategy for prevention of MDSC induction in MM. Using a transwell co-culture system, four of nine examined human myeloma-derived cell lines (HMCLs) were potent in inducing monocytic (M)-MDSCs from normal peripheral blood mononuclear cells (PBMCs). As the results, we identified that secretion of C-C motif chemokine ligand 5 (CCL5) and macrophage migration inhibitory factor (MIF) by myeloma cells is a prerequisite for induction of MDSCs in MM. The immunomodulatory drug (IMiD) compounds, such as lenalidomide (LEN) and pomalidomide (POM), were identified as potent inhibitors of MDSC induction through bidirectional molecular effects of cereblon (CRBN)-dependent and -independent downregulation of CCL5 and MIF in myeloma cells; and downregulation of C-C motif chemokine receptor 5, a receptor for CCL5, and induction of interferon regulatory factor 8, a critical transcription factor for monocytic differentiation, in PBMCs. In the present study of the molecular mechanisms underlying MDSC induction, we identified a novel effect of LEN and POM of inhibiting MDSC induction via overlapping regulatory effects in myeloma cells and normal PBMCs.

Carfilzomib (CFZ) constitutes powerful combinatory therapy for relapsed/refractory multiple myeloma (RRMM); however, cardiovascular adverse events (CVAEs) have been shown as major treatment obstacles with the use of CFZ. Along with our multi-institutional prospective observational study by the Kyoto Clinical Hematology Study Group on the efficacy and safety of CFZ-based treatments (UMIN000025108), we here performed an ad hoc analysis of CFZ-related CVAEs in 50 patients with RRMM. We analyzed the association between CFZ-related CVAEs and pre-planned examinations, including patients' background, electrocardiographic findings, echocardiographic findings, and serum/plasma levels of 18 potential candidate biomarkers. The common CVAEs were hypertension (42%), arrhythmia (14%), and prolongation of QT corrected interval (10%), whereas no serious CVAEs occurred. The pretreatment serum level of interleukin-6 was identified as a significant risk factor for CFZ-related hypertension. This study revealed hypertension as the most frequent CFZ-related CVAE and suggested that baseline serum interleukin-6 is a useful predictor for CFZ-induced hypertension.

Background Combinatory strategies with carfilzomib (CFZ), a second‐generation proteasome inhibitor, plus dexamethasone (DEX) with or without lenalidomide (LEN) have shown promising efficacy for patients with relapsed/refractory multiple myeloma (RRMM) in pivotal clinical trials. However, their effects on patients who were resistance to bortezomib (BTZ) and/or LEN have not been fully evaluated in a daily practice setting. Aims To evaluate the real‐world efficacy and safety of CFZ‐based treatments; that is, CFZ with LEN plus DEX (KRD therapy) and CFZ with DEX (KD therapy), in Asian patients, we conducted a multicenter pilot prospective observational study in the Kyoto Clinical Hematology Study Group. Methods and Results All 50 patients with RRMM enrolled in this study were treated with CFZ‐based treatments between 2017 and 2019. KRD and KD were administered to 31 and 19 patients, respectively. The overall response rates (ORRs) were 80.6% with KRD and 73.7% with KD. Two‐year progression‐free survival (PFS) and overall survival (OS) were 58.5% and 79.7% with KRD, and 23.1% and 52.6% with KD. By multivariate analysis, refractoriness to BTZ and to LEN were identified as independent unfavorable factors for both PFS and OS. The common non‐hematologic AEs included hypertension (42.0%), fever (24.0%), fatigue (24.0%), and infection (16.0%). No serious heart failure was observed. This study is registered as UMIN000025108. Conclusion This study suggests the need of the development of novel CFZ‐containing strategy which can overcome the refractoriness to BTZ and/or LEN, while both KRD and KD were shown to be mostly feasible in Asian patients in a daily practice setting.

Multiple myeloma (MM) is cytogenetically, genetically and molecularly heterogenous even among subclones in one patient, therefore, it is essential to identify both frequent and patient-specific drivers of molecular abnormality. Following previous molecular investigations, we in this study investigated the expression patterns and function of the Ewing sarcoma breakpoint region 1 (EWSR1) gene in MM. The EWSR1 transcriptional level in CD138-positive myeloma cells was higher in 36.4% of monoclonal gammopathy of undetermined significance, in 67.4% of MM patients compared with normal plasma cells, and significantly higher in ten human myeloma-derived cell lines (HMCLs) examined. EWSR1 gene knockdown caused growth inhibition with an increase of apoptotic cells in NCI-H929 and KMS-12-BM cells. Gene expression profiling using microarray analysis suggested EWSR1 gene knockdown caused transcriptional modulation of several genes associated with processes such as cell proliferation, cell motility, cell metabolism, and gene expression. Of particular, EWSR1 gene knockdown caused upregulation of let-7c and downregulation of its known targets K-RAS and AKT. Finally, our analysis using community database suggested that high EWSR1 expression positively associates with poor prognosis and advanced disease stage in MM. These findings suggest that EWSR1 overexpression is a pro-oncogenic molecular abnormality that may participate in MM progression.

Diffuse large B‐cell lymphoma (DLBCL), which is the most prevalent disease subtype of non‐Hodgkin lymphoma, is highly heterogeneous in terms of cytogenetic and molecular features. This study retrospectively investigated the clinical impact of G‐banding‐defined chromosomal abnormality on treatment outcomes of DLBCL in the era of rituximab‐containing immunochemotherapy. Of 181 patients who were diagnosed with DLBCL and treated with R‐CHOP or an R‐CHOP‐like regimen between January 2006 and April 2014, metaphase spreads were evaluable for G‐banding in 120. In these 120 patients, 40 were found to harbor a single chromosomal aberration type; 63 showed chromosomal abnormality variations (CAVs), which are defined by the presence of different types of chromosomal abnormalities in G‐banding, including 19 with two CAVs and 44 with ≥3 CAVs; and 17 had normal karyotypes. No specific chromosomal break point or numerical abnormality was associated with overall survival (OS) or progression‐free survival (PFS), but the presence of ≥3 CAVs was significantly associated with inferior OS rates (hazard ratio (HR): 2.222, 95% confidence interval (CI): 1.056–4.677, P = 0.031) and tended to be associated with shorter PFS (HR: 1.796, 95% CI: 0.965–3.344, P = 0.061). In addition, ≥3 CAVs more frequently accumulated in high‐risk patients, as defined by several conventional prognostic indices, such as the revised International Prognostic Index. In conclusion, our results suggest that the emergence of more CAVs, especially ≥3, based on chromosomal instability underlies the development of high‐risk disease features and a poor prognosis in DLBCL.

Cytogenetic/molecular heterogeneity is the hallmark of multiple myeloma (MM). However, we recently showed that the serine/threonine kinase PDPK1 and its substrate RPS6KA3 (also termed RSK2) are universally active in MM, and play pivotal roles in myeloma pathophysiology. In this study, we assessed involvement of aberrant miR-375 repression in PDPK1 overexpression in MM. An analysis of plasma cells from 30 pre-malignant monoclonal gammopathies of undetermined significance and 73 MM patients showed a significant decrease in miR-375 expression in patient-derived plasma cells regardless of the clinical stage, compared to normal plasma cells. Introduction of miR-375 reduced PDPK1 expression in human myeloma cell lines (HMCLs), indicating that miR-375 is the dominant regulator of PDPK1 expression. In addition, miR-375 introduction also downregulated IGF1R and JAK2 in HMCLs. CpG islands in the MIR375 promoter were pathologically hypermethylated in all 8 HMCLs examined and in most of 58 patient-derived myeloma cells. Treatment with SGI-110, a hypomethylating agent, and/or trichostatin A, a histone deacetylase inhibitor, increased miR-375 expression, but repressed PDPK1, IGF1R and JAK2 in HMCLs. Collectively, these results show the universal involvement of overlapping epigenetic dysregulation for abnormal miR-375 repression in MM, which is likely to contribute to myelomagenesis and to subsequent myeloma progression by activating oncogenic signalling pathways.

Extranodal marginal zone lymphoma (EMZL) of mucosa-associated lymphoid tissue (MALT) of the uterus is rare, and the etiology, pathophysiology and cytogenetic features remain unknown at present. The present study reports a case of a 71-year-old female with EMZL of the uterine cervix that was 80 mm in diameter and invaded directly into the rectal serosa. Complete remission was successfully induced by 6 courses of immunochemotherapy with rituximab plus cyclophosphamide, doxorubicin, vincristine and prednisolone. Although the metaphase spread of the tumor cells was unavailable for whole cytogenetic analysis, fluorescence in situ hybridization (FISH) detected triple signals for MALT1 and B-cell lymphoma 2, located at chromosome 18q21, and the centromere of chromosome 18, which was suggestive of trisomy 18, and in combination with previous studies, suggested a possible association between trisomy 18 and the large tumor at initial presentation in the present patient. In addition, FISH examination detected immunoglobulin heavy chain gene rearrangement, although the translocation partner was unconfirmed. A total of 18 previously-studied patients with EMZL of the uterus, including that of the present study, were reviewed with respect to their clinical features and treatment and cytogenetic abnormality. In the evaluation of the English scientific literature, this is the first reported patient with EMZL of the uterus with partly determined cytogenetic abnormalities.

Background Since bromodomain-containing protein 4 (BRD4) facilitates the transcription of genes important for neoplastic cells in a cancer-type specific manner, BRD4-regulated molecules may also include therapeutic targets for mantle cell lymphoma (MCL), a treatment-refractory subtype of malignant lymphoma. Materials and methods In order to uncover direct BRD4-regulated targets in MCL, we performed integrated analysis using the pathway database and the results of both gene-expression profiling and chromatin immunoprecipitation with parallel sequencing for BRD4. Results Treatment with BRD4 inhibitor I-BET151 exerted a dose-dependent inhibitory effect on cell proliferation in MCL cell lines. BRD4 was found to directly regulate series of genes involved in the B-cell receptor (BCR) signaling pathway, including B-cell linker (BLNK), paired box 5 (PAX5), and IKAROS family zinc finger 3 (IKZF3), and several oncogenes, such as MYB. Indeed, the combinatory inhibition of BCR pathway and IKZF showed an additive antitumor effect. Conclusion Concomitant targeting multiple BRD4-regulated molecules may constitute a rational therapeutic strategy for MCL.

Imatinib mesylate (IM) is a first generation ATP-competitive selective inhibitor for tyrosine kinases (TK) such as ABL, ARG, PDGFRα and b, and c-KIT.[1][1] While use of IM has markedly improved treatment outcomes of Philadelphia chromosome (Ph)-positive leukemias, including chronic myeloid leukemia

Several novel fusion transcripts were identified by next-generation sequencing in gastric cancer; however, the breakpoint junctions have yet to be characterized. The present study characterized a plethora of APIP-FGFR2 genomic breakpoints in the SNU-16 gastric cancer cell line, which harbored homogeneously staining regions (hsrs) and double minute chromosomes. Oligonucleotide microarrays revealed high-level amplifications at chromosomes 8q24.1 (0.8 Mb region), 10q26 (1.1 Mb) and 11p13 (1.1 Mb). These amplicons contained MYC and PVT1 at chromosome 8q24.1, BRWD2, FGFR2 and ATE1 at chromosome 10q26, and 24 genes, including APIP, CD44, RAG1 and RAG2, at chromosome 11p13. Based on these findings, reverse transcription-polymerase chain reaction (PCR) was performed using various candidate gene primers to detect possible fusion transcripts, and several products using primer sets for the APIP and FGFR2 genes were detected. Eventually, three in-frame and two out-of-frame fusion transcripts were detected. Notably, PCR analysis of the entire genomic DNA detected three distinct genomic junctions. The breakpoints were within intron 5 of APIP, which contained three distinct breakpoints, and introns 5, 7 and 9 of FGFR2. Fluorescence in situ hybridization showed several fusion signals within hsrs using two short probes (~10-kb segments of a bacterial artificial chromosome clone) containing exons 2-5 of APIP or exons 11-13 of FGFR2. Although, for any given fusion, a multiplicity of transcripts is thought to be created by alternative splicing of one rearranged allele, the results of the present study suggested that genomic fusions of APIP and FGFR2 are generated in hsrs with a diversity of breakpoints that are then faithfully transcribed.

Chromosome instability (CIN), the hallmarks of cancer, reflects ongoing chromosomal changes caused by chromosome segregation errors and results in whole chromosomal or segmental aneuploidy. In multiple myeloma (MM), CIN contributes to the acquisition of tumor heterogeneity, and thereby, to disease progression, drug resistance, and eventual treatment failure, however, the underlying mechanism of CIN in MM remains unclear. Faithful chromosomal segregation is tightly regulated by a series of mitotic checkpoint proteins, such as budding uninhibited by benzimidazoles 1 (BUB1). In this study, we found that BUB1 was overexpressed in patient-derived myeloma cells, and BUB1 expression was significantly higher in patients in an advanced stage compared to those in an early stage. This suggested the involvement of aberrant BUB1 overexpression in disease progression. In human myeloma-derived cell lines (HMCLs), BUB1 knockdown reduced the frequency of chromosome segregation errors in mitotic cells. In line with this, partial knockdown of BUB1 showed reduced variations in chromosome number compared to parent cells in HMCLs. Finally, BUB1 overexpression was found to promote the clonogenic potency of HMCLs. Collectively, these results suggested that enhanced BUB1 expression caused an increase in mitotic segregation errors and the resultant emergence of subclones with altered chromosome numbers and, thus, was involved in CIN in MM.

Cyclin D1 (CCND1) overexpression is an early and unifying oncogenic event in mantle cell lymphoma (MCL) and multiple myeloma (MM) with chromosome 11q13 abnormalities. Herein, we report newly discovered transcript variants of the CCND1 gene in MCL and MM cells with chromosome 11q13 abnormalities. These transcript variants, designated CCND1.tv., covered the full-length coding region of CCND1 with longer 5'-untranslated regions (5'-UTRs) of CCND1 and occasionally contained a novel exon. CCND1.tv. was specifically detectable in patient-derived primary MCL or MM cells with chromosomal translocation t(11;14)(q13;q32), but not in t(11;14)-negative cells. The lengths of the 5'-UTR sequences of CCND1.tv. differed among patients and cell lines. Introduction of CCND1.tv. led to increased expression of normal-sized CCND1 protein in HEK293 cells. Furthermore, mTOR inhibition by rapamycin or serum starvation reduced ectopic expression of CCND1.tv.-derived CCND1 protein, but not 5'-UTR less CCND1-derived CCND1 protein in HEK293 cells, suggesting that the protein expression of CCND1.tv. is regulated by the mTOR pathway. Our results suggest that the aberrant expression of CCND1.tv. may contribute to the understanding of the pathogenesis of MCL and MM with 11q13 abnormalities.

An individual with a twin who has developed leukemia or non-Hodgkin lymphoma (NHL) has an increased risk of developing the same disease, particularly with monozygotic twins. The few reported pairs of twins who developed NHL had similar primary sites and pathological subtypes. Here, we present the first reported cases of primary conjunctival NHL in both female monozygotic twins. Twin 1 was diagnosed with an extranodal marginal zone lymphoma (EMZL; Ann Arbor stage IE) in the right conjunctiva at 25 years old and a subsequent tumor in the left conjunctiva at 39 years, and was also histopathologically diagnosed as EMZL. No infiltration of other organs was detected and both lesions were surgically excised. At the age of 40 years, Twin 2 was diagnosed with an EMZL (Ann Arbor stage IE) in the right conjunctiva without infiltration of other organs and was treated with external beam radiation therapy rather than surgery. Complete remission was achieved in both twins; neither developed conjunctival recurrences. This study highlights the importance of examining the other, apparently healthy twin when one twin develops conjunctival lymphoma.

Despite the recent therapeutic progress, the prognoses of diffuse large B-cell lymphomas (DLBCLs) that concomitantly overexpress c-MYC and BCL2, i.e., double hit lymphoma (DHL) and double expressing lymphoma (DEL), remain poor. This study examined triple targeting of c-MYC, BCL2 and the B-cell receptor (BCR) signaling pathway for DHL and DEL. We first used AZD5153, a novel bivalent inhibitor for bromodomain-containing 4 (BRD4), in DHL- and DEL-derived cell lines, because BRD4 regulates disease type-oriented key molecules for oncogenesis. AZD5153 was more effective than conventional monovalent BRD4 inhibitors, JQ1 and I-BET151, in inhibiting cell proliferation of a DHL-derived cell line and two DEL-derived cell lines, with at least 10-fold lower half growth inhibitory concentrations. AZD5153 caused G1/S cell cycle blockade, while the apoptosis-inducing effect was relatively modest. At the molecular level, AZD5153 was potent in downregulating various molecules for oncogenesis, such as c-MYC, AKT2 and MAP3K; those involved in the BCR signaling pathway, such as CD19, BLNK and CD79B; and those associated with B-cell development, such as IKZF1, IKZF3, PAX5, POU2AF1 and EBF1. In contrast, AZD5153 did not decrease anti-apoptotic BCL2 proteins, and did not activate pro-apoptotic BH3-only proteins, except BAD. To augment cell death induction, we added a novel BH3-mimicking BCL2 inhibitor AZD4320 to AZD5153, and found that these two agents had a mostly synergistic antitumor effect by increasing cells undergoing apoptosis in all three cell lines. These results provide a rationale for dual targeting of BRD4 and BCL2 using AZD5153 and AZD4320 as a therapeutic strategy against DHL and DEL.

Background/Aim: Bendamustine hydrochloride (BH) is a key therapeutic agent for mantle cell lymphoma (MCL), while the mechanism underlying BH-resistance has not been verified. Materials and Methods: We compared molecular/biological characteristics of a newly-generated MCL-derived cell line KPUM-YY1 and its BH-resistant subline KPUM-YY1R. Results: The growth-inhibitory IC50 for BH was 20 μM in KPUM-YY1 cells, while cell proliferation was not inhibited by up to 60 μM BH in KPUM-YY1R cells. Compared to KPUM-YY1 cells, gene expression profiling in KPUM-YY1R cells revealed up-regulation of 312 genes, including ABCB1 encoding P-glycoprotein (P-gp), and microsomal glutathione S-transferase 1 (MGST1). Addition of either a P-gp inhibitor or a GST inhibitor, at least partly, restored sensitivity to BH in KPUM-YY1R cells. In addition, KPUM-YY1R cells showed cross-resistance against various anti-MCL chemotherapeutics. Conclusion: BH resistance is mediated by overlapping mechanisms with overexpression of ABCB1 and MGST1, and is potentially accompanied by multidrug resistance in MCL.

The deleted in colorectal carcinoma (DCC) gene at 18q21 encodes a netrin-1 receptor, a tumor suppressor that prevents cell growth. While allele loss or decreased expression of DCC has been associated with the progression of solid tumors and hematologic malignancies, including leukemias and malignant lymphomas, its involvement has not been evaluated in multiple myeloma (MM), a plasma cell malignancy characterized by complex and heterogenous molecular abnormalities. We here show that 10 of 11 human myeloma-derived cell lines (HMCLs) expressed non-translated aberrant DCC transcriptional variants, in which exon 2 fuses with intron 1 instead of exon 1 (mt.DCC). Among them, two co-expressed wild type transcripts (wt.DCC), while eight co-expressed the splicing variant (sv.DCC) lacking exon 1. The remaining HMCL expressed only sv.DCC. In addition, analyses revealed that there were two types of mt.DCC that differed in their fusion of intron 1 with exon 2. In patient-derived samples from 30 MM and 8 monoclonal gammopathy of undetermined significance (MGUS) patients, wt.DCC was expressed in 53% of MM, but not in MGUS, while 23% of MM and 75% of MGUS expressed only sv.DCC. Considering that 25% of MGUS, 57% of MM, and 91% HMCLs expressed mt.DCC, our results suggest that the acquisition of mt.DCC might be a secondary genetic change in plasma cell dyscrasia.

Acute lymphoblastic leukemia (ALL) occasionally develops before or after the onset of Langerhans cell histiocytosis (LCH). The mechanism of LCH developing after ALL remains unclear; thus the clonality of LCH developing during maintenance chemotherapy for T-cell ALL (T-ALL) was investigated. The T-ALL and LCH cells tested had the same T-cell receptor (TCR) gamma rearrangement. Mutation analysis of the NOTCH1 gene revealed 7213C>T (Q2405X) in exon 34 in T-ALL and LCH cells, but 5156T>C (I1719T) in exon 27 only in T-ALL. Polymerase chain reaction-restriction fragment length polymorphism analysis revealed three patterns of NOTCH1 mutations in T-ALL cells. The results suggest that the T-ALL and LCH cells were derived from a common precursor with TCR rearrangement and a single NOTCH1 mutation, rather than LCH cells developing from a minor clone of T-ALL with single NOTCH1 mutation.

We investigated clinical and genetic characteristics of high-risk follicular lymphoma (FL), that lacked evidence of large cell transformation at diagnosis, in the rituximab era. First, we retrospectively analysed the clinical features of 100 patients with non-transformed FL that were consecutively treated with rituximab-containing therapies in a discovery cohort. The presence of either peripheral blood and/or bone involvement was associated with short progression-free survival. This was confirmed in a validation cohort of 66 FL patients. Then, whole exome sequencing was performed on randomly selected 5 high- and 9 standard-risk FL tumours. The most common mutational signature was a CG > TG substitution-enriched signature associated with spontaneous deamination of 5-methylcytosine at CpG, but mutations in WA and WRC(Y) motifs (so-called activation-induced cytidine deaminase (AID) motifs) were also enriched throughout the whole exome. We found clustered mutations in target sequences of AID in the IG and BCL2 loci. Importantly, high-risk FLs harboured more somatic mutations (mean 190 vs. 138, P = 0.04), including mutations in WA (33 vs. 22, P = 0.038), WRC (34 vs. 22, P = 0.016) and WRCY motifs (17 vs. 11, P = 0.004). These results suggest that genomic instability that allows for emergence of distinct mutations through AID activity underlies development of the high-risk FL phenotype.

Mantle cell lymphoma (MCL) is a relatively rare subtype of B-cell non-Hodgkin lymphoma (NHL) that has a poor prognosis despite recent advances in immunochemotherapy and molecular targeted therapeutics against NHL. Therefore, the development of a new therapeutic strategy for MCL is urgently needed. In this study, we show for the first time that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), an oncogenic serine–threonine protein kinase, is commonly expressed in its phosphorylated active form in patient-derived tumor cells of various types of B-cell NHL cells, including diffuse large B-cell lymphoma, follicular lymphoma, and MCL. Blockade of PDPK1 activity by small-molecule inhibitors specific for PDPK1 (BX-912 and GSK2334470) or by RNA interference exerted antiproliferative effects in all four MCL-derived cell lines examined and these growth-inhibitory effects were mediated by both induction of apoptosis and G 2 /M cell cycle blockade. In addition, blockade of PDPK1 led to inactivation of its downstream effector kinase RSK2, but not AKT, suggesting the importance of the PDPK1/RSK2 signaling pathway in the proliferation and survival of MCL cells. Finally, when combined with anticancer agents, including genotoxic agents, a proteasome inhibitor, and a BH3 mimetic in vitro, the PDPK1 inhibitor BX-912 showed additive growth-inhibitory effects against MCL-derived cell lines in most settings. In particular, the combination of BX-912 and ABT-263, a BH3 mimetic, resulted in the enhancement of the induction of apoptosis. In conclusion, our results suggest that PDPK1 is a potential novel therapeutic target in MCL and indicate that clinical development of PDPK1-targeted therapy for MCL is desirable.

The monocytic leukemia zinc finger protein KAT6A (formerly MOZ) gene is recurrently rearranged by chromosomal translocations in acute myeloid leukemia (AML). KAT6A is known to be fused to several genes, all of which have histone acetyltransferase (HAT) activity and interact with a number of transcription factors as a transcriptional coactivator. The present study shows that the leucine twenty homeobox (LEUTX) gene on 19q13 is fused to the KAT6A gene on 8p11 in a therapy-related AML with t(8;19)(p11;q13) using the cDNA bubble PCR method. The fusion transcripts contained 83 nucleotides upstream of the first ATG of LEUTX and are presumed to create in-frame fusion proteins. LEUTX is known to have a homeobox domain. Expression of the LEUTX gene was only detected in placenta RNA by RT-PCR, but not in any tissues by Northern blot analysis. The putative LEUTX protein does not contain any HAT domain, and this is the first study to report that KAT6A can fuse to the homeobox gene. The current study, with identification of a new partner gene to KAT6A in a therapy-related AML, does not elucidate the mechanisms of leukemogenesis in KAT6A-related AML but describes a new gene with a different putative function.

Among various cytogenetic abnormalities involved in lymphomagenesis and disease progression of highly aggressive and treatment-resistant B-cell lymphomas (HABCLs), classified as the so-called aggre...

Multiple myeloma is an entity of cytogenetically and genetically heterogenous plasma cell neoplasms. Despite recent improvement in the treatment outcome of multiple myeloma by novel molecular-targeted chemotherapeutics, multiple myeloma remains incurable. The identification of a therapeutic target molecule in which various signaling for cell-survival converge is a core component for the development of new therapeutic strategies against multiple myeloma. RSK2 is an essential mediator of the ERK1/2 signaling pathway for cell survival and proliferation. In this study, we discovered that RSK2Ser227, which is located at the N-terminal kinase domain and is one site responsible for substrate phosphorylation, is activated through phosphorylation regardless of the type of cytogenetic abnormalities or upstream molecular signaling in all 12 multiple myeloma–derived cell lines examined and 6 of 9 patient-derived CD138-positive primary myeloma cells. The chemical inhibition of RSK2Ser227 by BI-D1870 or gene knockdown of RSK2 inhibits myeloma cell proliferation through apoptosis induction, and this anti-myeloma effect was accompanied by downregulation of c-MYC, cyclin D, p21WAF1/CIP1, and MCL1. RSK2Ser227 inhibition resulting from BI-D1870 treatment restored lenalidomide-induced direct cytotoxicity of myeloma cells from interleukin-6–mediated cell protection, showed no cross-resistance to bortezomib, and exerted additive/synergistic antiproliferative effects in conjunction with the mTOR, histone deacetylase, and BH3-mimicking BCL2/BCLXL inhibitors. These results suggest that RSK2Ser227 is a potential therapeutic target not only for newly diagnosed but also for patients with later phase multiple myeloma who are resistant or refractory to currently available anti-myeloma therapies. Mol Cancer Ther; 11(12); 2600–9. ©2012 AACR.

Primary cutaneous gamma-delta T-cell lymphoma (PCGD-TCL), which originates from activated mature gamma-delta T cells with a cytotoxic phenotype is a rare T-cell lymphoproliferative disease. The prognosis of PCGD-TCL has been rather unfavorable due to poor response to conventional chemotherapy, and its molecular features and pathophysiology underlying disease development remain unknown. We report here a case with primarily treatment-resistant PCGD-TCL featuring highly complex cytogenetic and genetic aberrations detected by spectral karyotyping and genome-wide single nucleotide polymorphism (SNP) array. Chromosomal aberrations included several chromosomal translocations involving breakpoints at 9p21, 14q11.2, 14q32.1, or 16q23.1, suggesting the involvement of WWOX , TCL gene cluster, and BCL11B , which are crucial for tumorigenesis in T-cell lymphomas. SNP analysis also identified genome copy number gains and losses in various regions, which can potently deregulate expression of various pro- and anti-oncogenic genes involved in RAS-related protein pathways, PI3K/AKT/MTOR-related pathways, MYC-related signaling, or TP53 - related signaling. Thus, this case report may shed some light on the complex molecular abnormalities involved in the development of PCGD-TCL and on information that can aid the search for druggable target molecules in this disease.

The AML1 gene is frequently rearranged by chromosomal translocations in acute leukemia. We identified that the LAF4 gene on 2q11.2-12 was fused to the AML1 gene on 21q22 in a pediatric patient having T-cell acute lymphoblastic leukemia (T-ALL) with t(2;21)(q11;q22) using the bubble PCR method for cDNA. The genomic break points were within intron 7 of AML1 and of LAF4, resulting in the in-frame fusion of exon 7 of AML1 and exon 8 of LAF4. The LAF4 gene is a member of the AF4/FMR2 family and was previously identified as a fusion partner of MLL in B-precursor ALL with t(2;11)(q11;q23), although AML1-LAF4 was in T-ALL. LAF4 is the first gene fused with both AML1 and MLL in acute leukemia. Almost all AML1 translocations except for TEL-AML1 are associated with myeloid leukemia; however, AML1-LAF4 was associated with T-ALL as well as AML1-FGA7 in t(4;21)(q28;q22). These findings provide new insight into the common mechanism of AML1 and MLL fusion proteins in the pathogenesis of ALL. Furthermore, we successfully applied bubble PCR to clone the novel AML1-LAF4 fusion transcript. Bubble PCR is a powerful tool for detecting unknown fusion transcripts as well as genomic fusion points.

Multiple myeloma is a cytogenetically/molecularly heterogeneous hematologic malignancy that remains mostly incurable, and the identification of a universal and relevant therapeutic target molecule is essential for the further development of therapeutic strategy. Herein, we identified that 3-phosphoinositide–dependent protein kinase 1 (PDPK1), a serine threonine kinase, is expressed and active in all eleven multiple myeloma–derived cell lines examined regardless of the type of cytogenetic abnormality, the mutation state of RAS and FGFR3 genes, or the activation state of ERK and AKT. Our results revealed that PDPK1 is a pivotal regulator of molecules that are essential for myelomagenesis, such as RSK2, AKT, c-MYC, IRF4, or cyclin Ds, and that PDPK1 inhibition caused the growth inhibition and the induction of apoptosis with the activation of BIM and BAD, and augmented the in vitro cytotoxic effects of antimyeloma agents in myeloma cells. In the clinical setting, PDPK1 was active in myeloma cells of approximately 90% of symptomatic patients at diagnosis, and the smaller population of patients with multiple myeloma exhibiting myeloma cells without active PDPK1 showed a significantly less frequent proportion of the disease stage III by the International Staging System and a significantly more favorable prognosis, including the longer overall survival period and the longer progression-free survival period by bortezomib treatment, than patients with active PDPK1, suggesting that PDPK1 activation accelerates the disease progression and the resistance to treatment in multiple myeloma. Our study demonstrates that PDPK1 is a potent and a universally targetable signaling mediator in multiple myeloma regardless of the types of cytogenetic/molecular profiles. Cancer Res; 74(24); 7418–29. ©2014 AACR.

Abstract 4418 Chromosomal translocations play a crucial role in tumorigenesis, often resulting in the formation of chimeric genes or in gene deregulation through position effects. Hematologic malignancies are associated with a large number of such rearrangements. In this study, we have identified 28S ribosomal DNA (RN28S1) gene as a novel fusion partner of the B-cell leukemia/lymphoma 11B (BCL11B), immunoglobulin kappa variable 3–20(IGKV3–20), and component of oligomeric golgi complex 1 (COG1) locus in T-cell acute lymphoblastic leukemia (T-ALL), Burkitt lymphoma (BL) and multiple myeloma (MM), respectively. RN28S1-BCL11B fusion gene was identified in T-ALL with t(6;14)(q25;q32) by cDNA bubble PCR using BCL11B primers, whereas the breakpoint of 14q32 was near, but out of, the BCL11B. BLAST search revealed that the RN28S1 was fused to BCL11B within exon3 (coding region). During the expression analysis of RN28S1-BCL11B, We identified the same type of RN28S1-BCL11B fusion transcripts in a BL and 2 MM cell lines, and unexpectedly identified other RN28S1-fusion transcripts in hematologic malignancy cell lines. Fusion of RN28S1 and IGKV3–20 exon2 (coding region) was detected in 2 BL and a MM cell lines, and that of RN28S1 and COG1 within exon 13 (coding region) in a MM cell line. Furthermore, RN28S1 breakpoint was concentrated in between 1.4∼1.8kb. RN28S1 is located in the p12 region of chromosomes 13, 14, 15, 21 and 22. The ribosomal RNA (rDNA) gene repeats are essential housekeeping genes found in all organisms. A gene amplification system maintains large clusters of tandemly repeated copies in the chromosome, and as the product, ribosomal RNA accounts for ∼70% of total RNA in the cell. rDNA exists in high copy number in most eukaryotic cells. Repeatitive genes tend to lose copies through homologous recombination among the repeats. The rDNA has a gene amplification system, thought to be present in most eukaryotes, that function to keep high copy numbers. In the tumor cells, rDNA is transcribed higher than normal cells for cell proliferation. Thus, that may be associated with chromosome instability. RN28S1 degradation was reported to be associated with apoptosis of hematologic malignancies, and previously, another group was detected RN28S1-BCL6 fusion genes in gastric lymphoma. This case was no expression of BCL6 protein. In conclusion, these results suggest that RN28S1 fusions are concerned with the play something role of tumorigenesis. Because of RN28S1 fused to BCL11B, IGKV3–20 and COG1 whose breakpoints were involved in the coding region, we speculate that these fusion genes are not produce normal structure. Disclosures: No relevant conflicts of interest to declare.

JAK2/STAT signaling promotes survival and expansion of myelodysplastic syndrome (MDS) clones, but little is known about the potential of JAK2/STAT as a therapeutic target in MDS. We investigated the effect of NS-018, a novel antagonist for JAK2, on the colony-forming ability of bone marrow mononuclear cells (BMMNCs) from high-risk MDS patients. NS-018 decreased colony-forming unit-granulocyte/macrophage (CFU-GM) colony numbers from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent than against normal BMMNCs. In addition, NS-018 suppressed the phosphorylation of STAT3 in colony-forming cells from MDS patients. Collectively, NS-018 could be a new therapeutic option for high-risk MDS.

PP2A activator FTY720 has been shown to possess the anti-leukemic activity for chronic myelogenous leukemia (CML), however, the cell killing mechanism underlying its anti-leukemic activity has remained to be verified. We investigated the precise mechanisms underlying the apoptosis induction by FTY720, especially focusing on the roles of BH3-only proteins, and the therapeutic potency of FTY720 for CML. Enforced expression of either BCL2 or the dominant-negative protein of FADD (FADD.DN) partly protected CML cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways. FTY720 activates pro-apoptotic BH3-only proteins: BIM, which is essential for apoptosis by BCR-ABL1 tyrosine kinase inhibitors (TKIs), and BID, which accelerates the extrinsic apoptosis pathway. Gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the extent of cell protection was not as much as that by overexpression of either BCL2 or FADD.DN. Moreover, knockdown of both BIM and BID did not provide additional protection compared with knockdown of only BIM or BID, indicating that BIM and BID complement each other in apoptosis by FTY720, especially when either is functionally impaired. FTY720 can overcome TKI resistance caused by ABL kinase domain mutations, dysfunction of BIM resulting from gene deletion polymorphism, and galectin-3 overexpression. In addition, ABT-263, a BH3-mimetic, significantly augmented cell death induction by FTY720 both in TKI-sensitive and -resistant leukemic cells. These results provide the rationale that FTY720, with its unique effects on BIM and BID, could lead to new therapeutic strategies for CML.

Galectin-3 is induced in chronic myelogenous leukemia (CML) cells by co-culture with bone marrow stromal cells, making paracrine growth promotion of CML cells in conditioned medium (CM) from galectin-3 overexpressing CML cells more potent. We used gel filtration chromatography to demonstrate that the bovine SERPINA1-fetal bovine serum albumin (BSA) complex was specifically suppressed in CM from galectin-3 overexpressing cells. The SERPINA1-BSA complex as well as human plasma SERPINA1 inhibited the growth of CML cells, while exogenous galectin-3 partly offset this effect. These findings suggest that galectin-3 overexpression promotes paracrine growth of CML cells by interfering with the action of the growth inhibitory SERPINA1-albumin complex.

[Introduction] Multiple myeloma (MM) remains mostly incurable despite major advances in treatment strategies. The complex interplay among various cell-intrinsic and -extrinsic molecular mechanisms confers inter-patients diversity and intraclonal heterogeneity in MM. However, as the universal and relevant therapeutic target molecule against MM, we have recently identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1) and its major downstream substrate RSK2 were universally active in MM (Shimura Y, Mol Caner Ther 2012; Chinen Y, Cancer Res 2014), while we also identified that the repression of miR-375 was found to be the universal underlying mechanism for the overexpression/activation of PDPK1 in MM (Tatekawa S, ASH meeting 2015). In this study, we further extended our study to assess the clinical relevance of miR-375 repression and the molecular mechanisms for the miR-375 repression in MM for the future clinical translation of miR-375/PDPK1/RSK2 signaling axis in the diagnosis and treatment development for MM. [Methods] The miR-375 expression level was analyzed by the quantitative RT-PCR in 11 HMCLs and 113 patient-derived myeloma cells isolated by CD138-positive cell sorting (normal plasma cells; N=10, MGUS; N=30, newly diagnosed MM (NDMM); N=34, relapsed/refractory (RRMM); N=39). The level of miR-375 expression was calculated with 2-ΔΔCt methods. Human U6 snRNA was examined as the reference and cDNA of RPMI8226 was used as a calibrator sample. The methylation status of miR-375 upstream regions including promoter site were analyzed by methylation-specific PCR (MSP) and bisulfite sequence. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) was performed to examine the histone modification status in miR-375 upstream regions. The circulating plasma RNA samples were converted to cDNA libraries followed by sequencing using the multiplex small RNA library primer set. This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Boards. Patient-derived samples were obtained with informed consent. [Result] When compared to normal plasma cells, the miR-375 expression was significantly decreased in CD138 positive plasma cells from MGUS (p [Conclusion and Discussion] This study revealed that the expression level of miR-375 decreased specifically in clonal plasma cells from MGUS to RRMM patients, and the repression of miR-375 was caused by the overlapping epigenetic dysregulations. The abnormal repression of miR-375 which is the major causative for the constitutive hyperactivation of the PDPK1/RSK2 signaling axis in MM is likely to be involved in the myelomagenesis in MGUS as well as the disease progression in MM, and the epigenetic deregulations may be a novel therapeutic target for MM. Disclosures Iida: Celgene: Honoraria, Research Funding; Janssen Pharmaceuticals: Honoraria, Research Funding. Kuroda:Celgene: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding; Astra Zeneca: Research Funding; Janssen: Honoraria.

Follicular lymphoma (FL) is the most prevalent among indolent subtypes of non-Hodgkin lymphomas. While the histologic transformation associates with disease aggressiveness in variant FL, a minor population of non-transformed FL patients also shows a shorter period for disease control despite the use of rituximab (Rit) immunochemotherapy. In this study, we investigated the clinical and genetic features of high-risk non-transformed FL. With the median follow-up period of 61 months for 103 consecutively treated patients with histologically non-transformed FL including 85 patients treated with the first-line Rit-containing therapies and 18 with watch and wait, the 5-year overall survival (OS) and progression-free survival (PFS) of the entire cohort were 93.3% and 54.4%, respectively. Chemotherapy was initiated according to the GELF criteria. Although neither FLIPI nor FLIPI2 sufficiently predicted the risks for either OS or PFS, the presence of two extranodal involvements, including peripheral blood (PB) (N=7, p=0.006) and/or bone (N=6, p=0.007), at diagnosis were found to be significantly associated with poor PFS, but not OS, in our cohort. Indeed, 13 high-risk patients with PB and/or bone involvements at diagnosis showed a significantly shorter median PFS of 27.2 months compared with that of the 90 standard-risk patients, who have not yet reached the median (pTG substitution to be the most common mutational signature, while there was no difference with regard to the composition of the signature between the high- and standard-risk FLs. "Kataegis", a pattern of clustered hypermutations, was observed mostly in the target motif of activation-induced cytidine deaminase (AID), recurrently on chromosome 2p12 (involving the IGK locus), 14q32 (IGH), 18q21 (BCL2), 21p11, and 22q11 (IGLL). The mean number of C>T mutations in the AID-motif was significantly increased in the high-risk FLs over that in the standard-risk FLs (17 vs. 11, p=0.02), whereas there was no difference in the mean number of mutations in the target motif of APOBEC, which is considered to be the major driving agent responsible for kataegis in various solid cancers, between the two risk groups (8 vs. 7, p=0.49). For functional assessment of mutated genes, CREBBP and TNFRSF14 mutations were identified as the driver mutations of FL regardless of the disease risk using background mutation rate analysis. Hypergeometric test on all of the non-synonymous mutated genes from 14 FLs resulted in 71 significantly enriched gene ontologies (GOs), including the terms "cell development and differentiation", "regulation of cell death", "immunity mediated by lymphocytes, including B cells", "hematopoiesis", and "differentiation of leukocytes, including lymphocytes". In addition, pathway analysis demonstrated that mutated genes were enriched in the Notch and B cell receptor signaling pathways. However, we were not able to identify GOs or signaling pathways specific to the high-risk FL patients that could explain the different outcomes of the disease. Non-synonymous mutations in the high-risk FLs were found in genes, such as MEF2B and FUBP1, and were suggested to be associated with cancer aggressiveness, but not with the AID pathway. Overall, our results suggested that genomic instability, which allows for the emergence of distinct mutations not only by kataegis due to AID activity but also by additional mechanisms that increase mutations, underlies development of the high-risk phenotype in FLs. Disclosures Kuroda: Bristol Myers Squibb: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Astra Zeneca: Research Funding; Janssen: Honoraria.

In B-cell malignancies, genes implicated in B-cell differentiation, germinal center formation, apoptosis, and cell cycle regulation are juxtaposed to immunoglobulin loci through chromosomal translocations. In this study, we identified the BTB and CNC homology 2 (BACH2) gene as a novel translocation partner of the immunoglobulin heavy chain (IGH) locus in a patient with IGH-MYC-positive, highly aggressive B-cell lymphoma/leukemia carrying der(14)t(8;14) and del(6)(q15). Fluorescence in situ hybridization analysis using an IGH/MYC probe detected an IGH-MYC fusion signal on der(14) and IGH signal on del(6). Genome copy number analysis showed a deletion in the 6q15-25 region and a centromeric breakpoint within the BACH2 gene. cDNA bubble polymerase chain reaction using BACH2 primers revealed that the first exon of Cδ was fused to the 5'-untranslated region of BACH2 exon 2. The Cδ-BACH2 fusion transcript consisted of exon 1 of Cδ and exons 2 to 9 of BACH2, encompassing the entire BACH2 coding region, and the BACH2 was highly expressed in this patient. These results indicate that Cδ-BACH2 fusion may cause constitutive activation of BACH2. Although additional screening of 47 samples of B-cell non-Hodgkin's lymphoma (B-NHL) patients and 29 cell lines derived from B-cell malignancies by double-color fluorescence in situ hybridization analysis detected a split signal with deletion of centromeric region of BACH2 only in a patient with follicular lymphoma, BACH2 was highly expressed in lymphoma cells of the patient and B-NHL cell lines with IGH-MYC translocation. These findings suggest that BACH2 plays a critical role in B-cell lymphomagenesis, especially related to IGH-MYC translocation in some way.

Multiple myeloma (MM) is a highly molecularly heterogeneous hematologic malignancy and remains mostly incurable despite recent improvement of treatment outcome by novel agents. Therefore, the identification of novel and universal targetable therapeutic molecules is a core component for the therapeutic development. Herein, we identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a member of serine threonine kinase, is a rationalistic candidate of a novel therapeutic target against MM. PDPK1 is universally phosphorylated in all eleven MM-derived cell lines examined regardless of type of cytogenetic/genetic abnormalities or the activation/mutation state of FGFR3, RAS, ERK and AKT. PDPK1 promotes the cell proliferation of myeloma cells by activating RSK2Ser227 at N-terminal kinase domain which is a pivotal regulator of molecules that are essential for myelomagenesis, such as c-MYC, IRF4, or Cyclin Ds (Shimura Y, Mol Cancer Ther 2011), and AKTThr308. In contrast, PDPK1 inhibition by a selective inhibitor, BX-912, caused G2/M arrest, which was accompanied by the inactivation of PLK1, and resulted in cell death via induction of apoptosis which was accompanied by the activation of pro-apoptotic BH3-only proteins, BIM and BAD, in myeloma-derived cell lines. These molecular and cytological effects of PDPK1 inhibition in myeloma cells were also validated by gene knockdown by means of RNA interference using two different siRNAs specific for PDPK1. In addition, the cytotoxic effect of BX-912 was not hampered in two cell lines acquiring resistance to bortezomib (BTZ) (Ri M, Leukemia 2010), and PDPK1 inhibition by BX-912 showed additive to synergistic in vitro cytotoxic effects on myeloma cells with melphalan, etoposide, bortezomib or BAY11-7085, an inhibitor for NF-κB. BX-912 also induced cell death in eleven patient-derived primary myeloma cells those were positively isolated by CD138-labelling from bone marrow aspirates, while normal peripheral lymphocytes were significantly less sensitive to PDPK1 inhibitor compared with MM cells. In the clinical setting, PDPK1 was active in myeloma cells of 57 of 65 (87.7%) symptomatic MM patients at diagnosis. While patients backgrounds, such as age, gender, and the type of M-protein, were not significantly different between PDPK1-negative (PDPK1(-)) and PDPK1-positive (PDPK1(+)) patients, patients with the disease stage III according to International Staging System were significantly more frequent in the PDPK1(+) cohort compared with PDPK1(-) cohort. The PDPK1(-) patients tended to exhibit longer overall survival (OS) than the patients with PDPK1(+) (median OS: 2925 days vs. 2155 days, p=0.069) with a median follow-up period of 1310 days (range: 228–3317 days). Of particular, the 8-year OS of PDPK1(-) patients was statistically significantly more favorable than those of PDPK1(+) patients (83% vs. 17%, p=0.041). In addition, when we analyzed the impact of PDPK1 activity on the treatment outcome of patients treated by BTZ and dexamethasone therapy (BD), our results revealed that progression free survival (PFS) of patients with PDPK1(-) was a significantly longer than those of patients with PDPK1(+) (median PFS: PDPK1(-) vs. PDPK1(+), not reached vs. 167 days, p=0.049). The PFS of PDPK1(-) patients treated by high-dose therapy/autologous stem cell transplantation (HDT/ASCT) tended to be longer than those of PDPK1(+) (median PFS: PDPK1(-) vs. PDPK1(+), 972 days vs. 567 days, p=0.125). In conclusion, our study provides the rationale for PDPK1 as a possible universal molecular target for MM with various molecular/cytogenetic features. Especially, PDPK1 is a potential therapeutic target for not only newly diagnosed patients but also patients who are resistant or refractory to currently available anti-myeloma therapies. This study was conducted in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. Patient-derived samples were obtained with informed consent. Disclosures No relevant conflicts of interest to declare.

Background Cell signaling mediated by the JAK2-STAT (Janus kinase 2-signal transducer and activator of transcription) pathway plays a critical role in hematopoiesis, and its aberrant activation is associated with the progression of hematological malignancies. For instance, somatic mutations in the JAK2 gene that lead to constitutive activation of STATs are involved in the pathogenesis of myeloproliferative neoplasms (MPN) and refractory anemia with ringed sideroblasts with thrombocytosis, one of the myelodysplastic/myeloproliferative neoplasms, unclassifiable (MDS/MPN, U). In addition, insufficient inactivation of the JAK-STAT pathway due to repression of SOCS-1 (suppressor of cytokine signaling 1) through hypermethylation of the SOCS-1 gene is involved in the disease progression of high-risk myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) (Brakensiek et al.; Br. J. Haematol. 2005;130:209). These observations prompted us to investigate the effect of the JAK2-selective inhibitor NS-018, which is in an early-phase clinical trial for MPN, on the colony formation by bone marrow hematopoietic progenitor cells from high-risk de novo MDS patients and the phosphorylation status of STAT3 in these cells. Methods Bone marrow mononuclear cells (BMMNCs) from six MDS patients and three healthy volunteers were collected with informed consent in accordance with the Declaration of Helsinki and with the approval of the Institutional Review Board. The MDS subtypes of the six patients included two RCMD, three RAEB-1 and one RAEB-2 according to the WHO classification. All MDS patients had complex cytogenetic abnormalities and their prognostic risks were defined to be high (N=1) or very high (N=5) in the IPSS-R. Commercially available normal human CD34+ BM cells were also examined for comparison. Cells were incubated in methylcellulose medium containing cytokines with or without NS-018. Burst forming unit-erythroid (BFU-E) and colony forming unit-granulocyte/macrophage (CFU-GM) were scored on day 14 of culture. Colony-forming cells were then collected and subjected to western blotting analysis. Results We first examined the colony-formation capacity of MDS-derived BMMNCs. CFU-GM colony formation was observed in four of six MDS samples, although the absolute CFU-GM numbers from MDS BMMNCs were less than those from normal BMMNCs and normal CD34+BM cells. MDS-derived BMMNCs produced larger numbers of CFU-GM colonies (78–99% of total colonies) than BFU-E, similar to the findings usually observed in AML, whereas normal BMMNCs and normal CD34+BM cells formed virtually the same numbers of CFU-GM colonies and BFU-E colonies. These results suggest that BMMNCs from four of the MDS patients enrolled in this study had characteristics similar to AML BMMNCs, although they showed less proliferation than AML BMMNCs. We next examined the effect of NS-018 on CFU-GM colony formation. NS-018 treatment decreased the numbers of CFU-GM colonies from MDS-derived BMMNCs in a dose-dependent manner, and this effect was significantly more potent against MDS-derived than against normal cells (57.2% inhibition in MDS cells vs. 12.8% inhibition in normal cells at 0.5 mM NS-018; see Figure). These results indicate that NS-018 is preferentially efficacious in inhibiting CFU-GM formation from BMMNCs from high risk MDS. In addition, the level of phospho-STAT3 in MDS-derived colony-forming cells was twice as high as in colony-forming cells from normal BMMNCs, and 1.0 mM NS-018 completely suppressed the phosphorylation of STAT3 in CFU-GM colony-forming cells from MDS. Conclusion Our results show for the first time that a JAK2 inhibitor, NS-018, potently suppresses the formation of MDS colonies. NS-018 could be a new therapeutic option for high-risk MDS patients. Disclosures: Kodama: Nippon Shinyaku: Employment. Naito:Nippon Shinyaku: Employment. Nakaya:Nippon Shinyaku: Employment. Taniwaki:Novartis: Honoraria.

Abstract 2945 Background: Despite recent advances of therapeutic strategies, multiple myeloma (MM) still remains mostly incurable due to its drug resistance by both various cell intrinsic molecular abnormalities and the supports of myeloma microenvironment factors. The search for new therapeutic target molecule upon which various MM-promoting signalings converge is essential for the development of more effective therapy for MM. RSK2 is a member of the 90 kDa ribosomal S6 kinases (RSKs) family of serine (Ser) /threonine (Thr) kinases and lies downstream of RAS/ERK-1/2 signaling cascade. In MM, it has been shown that RSK2 mediates signaling initiated by FGFR3 activation for cell survival, but the functional role and the value as the therapeutic target of RSK2 have not been clearly defined. In this study, we precisely studied the association with RSK2 activation and chromosomal status, especially focusing on RSK2Ser227, which is responsible for downstream signaling phosphorylation, and the value of RSK2 as the therapeutic target in MM. Methods: Twelve human myeloma-derived cell lines and primary MM cells were utilized in this study. The use of human samples was approved by the ethical review board of our institute. Cell killing assay was performed using flow cytometry. Growth inhibitory assay was performed with modified MTT assay. Protein levels were examined by Western blotting. BI-D1870, a RSK2 inhibitor, was purchased from Symansis Limited (Auckland, New Zealand). RNA interference for RSK2 was performed by transfection of small interfering RNA (siRNA) into NCI-H929 cells by means of CLB-Transfection Kit (Lonza, Swiss) with protocol 9. Drug combination assays were evaluated with CalcuSyn software (Biosoft, Cambridge, UK). Results: RSK2Ser227 was shown to be phosphorylated in all 12 MM cell lines and 6 of 9 primary MM cells. t(4;14) involving IgH/FGFR3 was detected only in 7 cell lines and high FGFR3 expression was identified only in 6 cell lines. RSK2Ser227 was phosphorylated even in the absence of ERK1/2 or RSK2Tyr529 phosphorylation in several MM cell lines, indicating that their activation was not mandatory to RSK2Ser227 phosphorylation. Our study also did not show any positive relationship between RSK2Ser227 phosphorylation and other so-called “high-risk” cytogenetic abnormalities, such as 13q-, t(11;14), t(8;14), t(14;16), 1q21+, or 17p-. Treatment with BI-D1870 (RSK2 inhibitor) resulted in complete dephosphorylation of RSK2Ser227, but not RSK2Tyr529. The phosphorylation status of ERK1/2, or related signaling kinases, such as AKT, p38 MAPK, or JNK was also not affected. BI-D1870 showed dose-dependent growth inhibition effect in 6 MM-derived cell lines by inducing apoptosis. At molecular level, Mcl-1, p21WAF1/CIP1, cyclin D2 and c-Myc was significantly down-regulated and Bim was up-regulated by BI-D1870 intervention in NCI-H929 cell. BI-D1870 didn't modulate expression level of IRF-4. These change were also observed in other MM cell lines (KMS-34, AMO-1). To exclude the possibility that above BI-D1870 induced changes in protein expression and cell death are due to its off-target effects, we performed gene knockdown experiments of RSK2. Transient gene knockdown of RSK2 by means of RNAi caused downregulation of cyclin D2, p21WAF1/CIP1, c-Myc, while it induced cell death in NCI-H929 cells. In contrast, Bim expression was not altered by RSK2 knockdown. Lenalidomide (LEN) is the most powerful currently available IMiD for MM treatment. While cell death induction by LEN (100μM) was partly prevented by IL-6 (50ng/ml) in NCI-H929 cells, co-administration of BI-D1870 (0.2 μM, at the degree of low concentration not to affect cell viability when used solely) overcame the resistance to LEN induced by IL-6. KMS-11/BTZ were highly resistant to bortezomib (BTZ)-induced cell death, by contrast, it showed the similar sensitivities to BI-D1870-induced cell death when compared with their parental cells. And more, BI-D1870 showed synergistic or additive effects with upcoming new anti-MM agents, such as RAD001, MS-275 and ABT-263 in MM cell lines. Conclusions: RSK2, especially RSK2Ser227, may be a universal therapeutic target for MMs with diverse molecular signatures. Disclosures: Iida: Janssen Pharmaceutical K.K.: Honoraria.

Abstract 3744 [Introduction] Tyrosine kinase inhibitors (TKIs) for BCR-ABL have dramatically improved the treatment outcome of chronic myelogenous leukemia (CML) during this decade, however, the resistance to TKIs due to various molecular mechanisms, such as BCR-ABL mutations, dysfunction of cell killing mechanisms like BIM deregulation, or the supports by leukemia microenvironment factors, has remained to be overcome. We have identified that BIM, a member of BH3-only proteins, is the essential pro-apoptotic mediator under the blockade of BCR-ABL signaling (Kuroda J, PNAS, 2006). One of the attractive therapeutic strategies for complete eradication of CML cells by simultaneously targeting both TKI-sensitive and TKI-resistant CML cells may be the use of agents that can activate BIM as well as other pro-apoptotic BH3-only protein which is not utilized in apoptosis by TKIs. In this study, we assessed the therapeutic potential of the protein phosphatase 2A (PP2A) activator Fingolimod (FTY720), an immunosuppressive agent currently used for multiple sclerosis, in CML. [Materials and methods] Human CML-derived cell lines, K562, MYL, KBM5, KCL22, and their various subclones, were utilized in this study. Murine Ba/F3 lymphoid cells were transfected with either wild type or mutated bcr-abl gene. FTY720 was purchased from Cayman Chemical (Ann Arbor, MI). Cell death analyses were performed using flow cytometry. Protein levels were determined by conventional Western blotting. [Result and discussion] Treatment with FTY720 impairs phosphorylation of BCR-ABL and its downstream targets, AKT, ERK and STAT5, and inhibited the growth of CML cell lines through the induction of apoptosis. The apoptosis induced by FTY720 was accompanied by the activation of both caspase-8 and caspase-9, while enforced expression of either BCL2 or dominant-negative protein of FADD (FADD.DN) by gene introduction partly protected K562 cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways in FTY720-induced apoptosis. FTY720 treatment resulted in the activation of Bim as well as the processing of Bid into tBid, a BH3-only protein which plays crucial role in accelerating death-receptor-mediated extrinsic apoptosis pathway and is not utilized in apoptosis by TKIs. Importantly, gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the degrees of cell protection by those were not as much as the degree of cell protection by overexpression of either BCL2 or FADD.DN. Moreover, double knockdown of BIM and BID did not further protect K562 cells from apoptosis by FTY720, when compared with single knockdown of BIM. These results indicate that BIM and BID compensate each other for promoting apoptosis by FTY720 in CML cells, especially when either of them is functionally disturbed. Through these mechanisms, FTY720 overcomes TKI resistance by various mechanisms, such as ABL kinase domain (KD) mutations in BCR-ABL-transfected Ba/F3 cells (T315I, G250E, E255K or H396P) and in KBM5 cells with T315I. FTY720 also overcomes TKI resistance due to dysfunction of BIM by gene deletion polymorphism in KCL22 cells. Also, although galectin-3 overexpression has been shown to mediate bone marrow environment-mediated drug resistance in CML cells (Yamamoto-Sugitani M, PNAS, 2011), the addition of FTY720 to imatinib overcomes resistance to imatinib in galectin-3 overexpressing K562 cells and MYL cells. In conclusion, FTY720 induces apoptotic cell death in CML via dual activation of both BIM and BID, and is potent in overcoming various types of cell intrinsic and tumor microenvironment-mediated resistance to TKI. Disclosures: No relevant conflicts of interest to declare.

Abstract 4450 In B-cell malignancies, the genes implicated in B-cell differentiation, germinal center formation, apoptosis, and cell cycle regulation are juxtaposed to immunoglobulin loci through chromosomal translocations. In the present study, we have identified BTB and CNC homology 2 (BACH2) as a novel translocation partner gene of the immunoglobulin heavy chain (IGH) locus, resulting in chimeric Cδ-BACH2 gene in a patient with MYC-IGH-positive highly aggressive B-cell lymphoma/leukemia carrying der(14)t(8;14) and del(6)(q15). A 71-year-old male was diagnosed as having highly aggressive B-cell lymphoma/leukemia. SKY analysis revealed the representative karyotype of tumor cells as 45,X,-Y,der(3)t(3;X)(p21.2;q24),del(6)(q15),der(14)t(8;14)(q24;q32),del(16)(q22),der(20) t(3;20)(q21;p13). FISH and long-distance PCR analyses identified a fusion of MYC with Cγ. FISH analysis also detected a small IGH signal on del(6), and a VH on del(6)(q15). Genome copy number analysis using SNP-array detected an approximately 60Mb deletion in 6q15–25 region, and its centromeric breakpoint within BACH2 gene. The cDNA bubble PCR using BACH2 primers detected multiple PCR products, and sequence analysis has revealed that one of the products contained a sequence of the first exon of IGHCδ fused to 5’ untranslated region of BACH2 exon 2. Genomic fusion point of Cδ-BACH2, was within intron 1 of Cδ and intron 1 of BACH2. Cδ-BACH2 fusion transcript consisted of exon 1 of Cδ and exons 2 to 9 of BACH2, indicating that whole coding region of BACH2 was fused to Cδ. This suggested that Cδ-BACH2 fusion cause constitutive activation of BACH2. We next analyzed the expression levels of BACH2, MYC, PRDM1, and IRF4 genes in the patient using real-time PCR and compared them with those of several hematologic malignancy cell lines, including 14 non-Hodgkin's lymphoma (NHL), 10 multiple myeloma (MM), and 3 B-lineage acute lymphoblastic leukemia, and 3 EB-virus transformed B-cell lines from normal healthy volunteers. IGH-MYC-positive MM cell lines showed increased levels of MYC expression compared with the other cell lines. The MYC expression level in our patient was lower than those in IGH-MYC-positive MM cell lines; however, it was similar to 5 IGH-MYC-positive NHL cell lines [3 Burkitt lymphoma (BL) and 2 diffuse large B-cell lymphoma (DLBCL)]. On the other hand, BACH2 expression levels were higher in NHL cell lines, especially in BL cell lines, but not correlated with the IGH-MYC translocation status. Our IGH-BACH2-positive case also showed a higher level of BACH2 expression. The expression levels of IRF4 and PRDM1 were higher in MM cell lines than in other cell lines. PRDM1 is activated by MYC through IRF4, and the MYC is negatively regulated by activated PRDM1. In MM cell lines with IGH-MYC translocation, MYC was highly expressed regardless of high expression of PRDM1, indicating that MYC activated by IGH translocation could not be inhibited by PRDM1. Unexpectedly, the expression levels of PRDM1 and IRF4 were very low in NHL including our patient, suggesting that the regulation of MYC in NHL is different from that in MM. BACH2 is a B cell-specific transcription repressor, and is specifically required for class switch recombination, somatic hypermutation, and germinal center formation. One of the target genes of BACH2 is PRDM1 at 6q21-q22.1 that is required for plasma cell differentiation. In this patient, deletion of 6q15–25 was found, indicating loss of one PRDM1 allele. It was reported that PRDM1 is inactivated by chromosomal alterations in 24% of activated B cell–like DLBCL, suggesting that PRDM1 acts as a tumor suppressor gene, and its inactivation may contribute to lymphomagenesis by blocking post–GC differentiation. The combination of BACH2 and MYC in double IGH translocations is unique and consistent with previous reports demonstrating that each partner gene found in double or multiple IGH translocations is exclusively specific to certain types of B-cell lymphoma. These results suggest that the promoter of IGHCδ and/or Eμ enhancer of IGH activate the expression of BACH2, and that BACH2 may act as oncogene in some cases with B-cell lymphoma. Although the IGH-BACH2 translocation is rare in NHL, our data suggest that the BACH2 plays a critical role in B-cell lymphomagenesis through not only IGH translocation but also activation by some other mechanisms. Disclosures: No relevant conflicts of interest to declare.

The pathogenetic roles of 8q24 amplified segments in leukemic cells with double minute chromosomes remain to be verified. Through comprehensive molecular analyses of 8q24 amplicons in leukemic cells from an acute myelogenous leukemia (AML) patient and AML-derived cell line HL60 cells, we identified two novel fusion genes between NSMCE2 and long noncoding RNAs (lncRNAs), namely, PVT1-NSMCE2 and BF104016-NSMCE2. Our study suggests that 8q24 amplicons are associated with the emergence of aberrant chimeric genes between NSMCE2 and oncogenic lncRNAs, and also implicate that the chimeric genes involving lncRNAs potentially possess as-yet-unknown oncogenic functional roles. Electronic supplementary material The online version of this article (doi:10.1186/s13045-014-0068-2) contains supplementary material, which is available to authorized users.