Yoshiaki Chinen

Identification of IGHCδ-BACH2 fusion transcripts resulting from cryptic chromosomal rearrangements of 14q32 with 6q15 in aggressive B-cell lymphoma/leukemia.

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‐Hodgkins 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.

Identification of the novel AML1 fusion partner gene, LAF4, a fusion partner of MLL, in childhood T-cell acute lymphoblastic leukemia with t(2;21)(q11;q22) by bubble PCR method for cDNA

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.

8q24 amplified segments involve novel fusion genes between NSMCE2 and long noncoding RNAs in acute myelogenous leukemia

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.

FTY720 induces apoptosis of chronic myelogenous leukemia cells via dual activation of BIM and BID and overcomes various types of resistance to tyrosine kinase inhibitors

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.

RSK2 Ser227 at N-Terminal Kinase Domain Is a Potential Therapeutic Target for Multiple Myeloma

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.

Phosphoinositide Protein Kinase PDPK1 Is a Crucial Cell Signaling Mediator in Multiple Myeloma

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.

The leucine twenty homeobox (LEUTX) gene, which lacks a histone acetyltransferase domain, is fused to KAT6A in therapy-related acute myeloid leukemia with t(8;19)(p11;q13)

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.

Suppression of SERPINA1-albumin complex formation by galectin-3 overexpression leads to paracrine growth promotion of chronic myelogenous leukemia cells

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.

Clinical manifestation of angioimmunoblastic T-cell lymphoma with exuberant plasmacytosis.

Angioimmunoblastic T-cell lymphoma (AITL) is a rare subtype of non-Hodgkin lymphoma characterized by aggressive symptoms and various abnormal laboratory test results. One of the rare immunologic abnormalities in AITL is exuberant polyclonal plasmacytosis, but its clinical significance has not been evaluated. This report concerns three AITL cases with exuberant polyclonal plasmacytosis and investigates its clinical impact by comparison with 12 patients without plasmacytosis. Our study found that the performance status (PS) of the former was significantly worse and their serum immunoglobulin levels were significantly higher. All other parameters, including B symptoms, various prognostic scores, blood cell counts other than plasmacyte, and serum levels of lactate dehydrogenase, C-reactive protein and soluble interleukin-2 receptor, showed no significant differences. More importantly, although the diagnosis of AITL with plasmacytosis was not straightforward in our series, outcomes of treatment with conventional chemotherapy or immunosuppressive therapy with cyclosporine A were favorable. To conclude, AITL should be considered a candidate underlying disease of exuberant polyclonal plasmacytosis. Provided a correct diagnosis is made early and is followed by adequate treatment, the prognosis for AITL with plasmacytosis may not be worse than that for those without plasmacytosis despite the severe exhaustion at first presentation.

Unique clonal relationship between T‐cell acute lymphoblastic leukemia and subsequent Langerhans cell histiocytosis with TCR rearrangement and NOTCH1 mutation

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.