Mio Yamamoto-Sugitani

Galectin-3 (Gal-3) induced by leukemia microenvironment promotes drug resistance and bone marrow lodgment in chronic myelogenous leukemia

Bone marrow (BM) microenvironment (BMME) constitutes the sanctuary for leukemic cells. In this study, we investigated the molecular mechanisms for BMME-mediated drug resistance and BM lodgment in chronic myelogenous leukemia (CML). Gene-expression profile as well as signal pathway and protein analyses revealed that galectin-3 (Gal-3), a member of the β-gal–binding galectin family of proteins, was specifically induced by coculture with HS-5 cells, a BM stroma cell-derived cell line, in all five CML cell lines examined. It was also found that primary CML cells expressed high levels of Gal-3 in BM. Enforced expression of Gal-3 activated Akt and Erk, induced accumulation of Mcl-1, and promoted in vitro cell proliferation, multidrug resistance to tyrosine kinase inhibitors for Bcr-Abl and genotoxic agents as a result of impaired apoptosis induction, and chemotactic cell migration to HS-5–derived soluble factors in CML cell lines independently of Bcr-Abl tyrosine kinase. The conditioned medium from Gal-3–overexpressing CML cells promoted in vitro cell proliferation of CML cells and HS-5 cells more than did the conditioned medium from parental cells. Moreover, the in vivo study in a mice transplantation model showed that Gal-3 overexpression promoted the long-term BM lodgment of CML cells. These results demonstrate that leukemia microenvironment-specific Gal-3 expression supports molecular signaling pathways for disease maintenance in BM and resistance to therapy in CML. They also suggest that Gal-3 may be a candidate therapeutic target to help overcome BMME-mediated therapeutic resistance.

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.

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.

Multifaceted Mechanisms for Cell Survival and Drug Targeting in Chronic Myelogenous Leukemia

Treatment outcomes for chronic myelogenous leukemia (CML) have shown major improvements as a result of the development of the tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib for the disease-specific molecular target BCR-ABL1 tyrosine kinase (TK), but a cure of CML by BCR-ABL1 TKIs has been rarely achieved. CML cells are protected from cytotoxic insults, including those by TKIs, through various collaborative BCR-ABL1- mediated and -independent mechanisms, as well as cell-intrinsic and -extrinsic molecular mechanisms. These protective mechanisms include overlapping cell signaling pathways for normal hematopoietic proliferation, modulation of molecules associated with the BCL2 family protein-regulated programmed cell death pathway, autophagic cell protection capability, bone marrow environment-mediated cell protective signaling, abnormally upregulated genetic instability and other BCR-ABL1- independent kinase activities. To develop a more effective treatment strategy for a cure by means of total leukemic cell killing, a thorough understanding of how CML cells survive and resist cytotoxic insults is essential. In this article, we review current knowledge about multifaceted BCR-ABL1-related and -unrelated mechanisms for survival and death of CML cells and present suggestions for the development of new therapeutic strategies for complete elimination of residual CML cells during TKI treatment.

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.

Deletion or methylation of CDKN2A/2B and PVT1 rearrangement occur frequently in highly aggressive B-cell lymphomas harboring 8q24 abnormality

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 aggressive B-cell lymphomas (BCLs) such as diffuse large B-cell lymphoma (DLBCL) and Burkitt-like lymphoma or Burkitt lymphoma (BL), 8q24 chromosomal abnormality is one of the major genetic/cytogenetic abnormalities, with a frequency of 5–15% [1,2]. Moreover, MYC rearrangements at 8q24 have been occasionally found in combination with a t(14;18)(q32;q21) involving BCL2 in 4% of highly aggressive “double-hit” DLBCLs (DHLs), with an extremely poor prognosis [3–5]. Indeed, MYC and BCL2 synergistically induce tumor formation, as observed in experimental models [6,7]. Therefore, better genetic/molecular diagnostic criteria are needed for the more accurate prediction of outcomes and the development of novel treatments for HABCLs. Because the complex karyotypes often found in HABCLs may cause the gene alterations associated with their biological functions, aberrations of other molecules besides MYC and BCL2 may also further affect the pathogenesis of HABCLs [4,8,9]. In this study, we subjected eight patients with aggressive BCLs with 8q24 abnormality treated at the Kyoto Prefectural University of Medicine (KPUM) between April 2006 and September 2012 (Table I) to a comprehensive molecular analysis using G-banding, spectral karyotyping (SKY), fluorescence in situ hybridization (FISH) for IGH, MYC, PVT-1 [Figure 1(A)], BCL-2, BCL-6, BACH2 and CDKN2A/2B, oligonucleotide array and methylation-specific polymerase chain reaction (PCR) (MSP) (Supplementary Materials and Methods available online at http://www.informahealthcare. com/lal/doi/10.3109/10428194.2013.790543) [8–10]. Patients 1, 2, 3, 7 and 8 were included in our previous publication on survival analysis for DLBCL [5]. We established two cell lines, KPUM-MS3 and KPUM-UH1, from patients 2 and 3, respectively [11]. This study was approved by the ethics committee of KPUM. Three patients had Burkitt-like lymphoma and five had DLBCL. As shown in Table I, all patients presented with advanced disease stage and with unfavorable International Prognostic Index scores. All patients were characterized by systemically disseminated involvements including bone marrow involvement, fluid retention, central nervous system involvement and/or multiple extranodal disease sites. As for response to immunochemotherapy incorporating rituximab and/or irradiation therapy, five patients showed disease progression, while three achieved a complete response. Patient 7 had three disease recurrences prior to a complete response to various salvage chemotherapies. The median survival of eight patients was 6.7 months. In these series, 8q24 abnormalities were detected by G-banding and SKY analysis in five patients, and by FISH in the remaining three. All patients thus showed 8q24 abnormalities, including MYC/PVT1 amplification in patient 4 [Figure 1(B)]. Based on the results obtained by FISH using both PVT1-A and PVT1-S probes, breakpoints were assigned to the PVT1 gene in two patients (2 and 5) and to the region covered by the bacterial artificial chromosome (BAC) CTD-3066D1 containing MYC in four (3, 6, 7 and 8) [Figures 1(A) and 1(C) and Supplementary Table I to be found online at http://www.informahealthcare.com/lal/doi/10.3109/1042 8194.2013.790543]. The MYC-Cg fusion gene was detected in patient 1 by long-distance PCR. Oligonucleotide array analysis demonstrated that the 8q24 rearrangement demarcated copy number changes within PVT1 in intron 1 in patient 2 (Supplementary Figure 1 to be found online at http://www. informahealthcare.com/lal/doi/10.3109/10428194.2013. L eu k L ym ph om a D ow nl oa de d fr om in fo rm ah ea lth ca re .c om b y K yo to F ur its u Id ai L ib ra ry o n 09 /2 8/ 14

Transcriptional dysregulation of the deleted in colorectal carcinoma gene in multiple myeloma and monoclonal gammopathy of undetermined significance.

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.