Masaharu Akiyama

Inhibition of the insulin-like growth factor receptor-1 tyrosine kinase activity as a therapeutic strategy for multiple myeloma, other hematologic malignancies, and solid tumors.

Insulin-like growth factors and their receptor (IGF-1R) have been implicated in cancer pathophysiology. We demonstrate that IGF-1R is universally expressed in various hematologic (multiple myeloma, lymphoma, leukemia) and solid tumor (breast, prostate, lung, colon, thyroid, renal, adrenal cancer, retinoblastoma, and sarcoma) cells. Specific IGF-1R inhibition with neutralizing antibody, antagonistic peptide, or the selective kinase inhibitor NVP-ADW742 has in vitro activity against diverse tumor cell types (particularly multiple myeloma), even those resistant to conventional therapies, and triggers pleiotropic antiproliferative/proapoptotic molecular sequelae, delineated by global transcriptional and proteomic profiling. NVP-ADW742 monotherapy or its combination with cytotoxic chemotherapy had significant antitumor activity in an orthotopic xenograft MM model, providing in vivo proof of principle for therapeutic use of selective IGF-1R inhibitors in cancer.

Activation of NF-kappaB and upregulation of intracellular anti-apoptotic proteins via the IGF-1/Akt signaling in human multiple myeloma cells: therapeutic implications.

Interleukin-6 (IL-6) and insulin-like growth factor-1 (IGF-1) promote the proliferation of multiple myeloma (MM) cells and protect them against dexamethasone (Dex)-induced apoptosis. We have previously shown that Apo2 ligand/TNF-Related apoptosis inducing ligand (Apo2L/TRAIL) induces apoptosis of MM cells, including cells either sensitive or resistant to Dex and cytotoxic drugs, and overcomes the growth and survival effect of IL-6; conversely, NF-κB transcriptional activity attenuates their Apo2L/TRAIL-sensitivity. In the current study, we demonstrate that IGF-1 stimulates sustained activation of NF-κB and Akt; induces phosphorylation of the FKHRL-1 Forkhead transcription factor; upregulates a series of intracellular anti-apoptotic proteins including FLIP, survivin, cIAP-2, A1/Bfl-1, and XIAP; and decreases Apo2L/TRAIL-sensitivity of MM cells. In contrast, IL-6 does not cause sustained NF-κB activation, induces less pronounced Akt activation and FKHRL-1 phosphorylation than IGF-1, and increases the expression of only survivin. Forced overexpression of constitutively active Akt in MM-1S cells reduced their sensitivity to Apo2L/TRAIL and to doxorubicin (Doxo). In contrast, the Akt inhibitor IL-6-Hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate induced cell death of both Dex- and Doxo-sensitive and -resistant cells; opposed the protective effect of constitutive Akt activity against Apo2L/TRAIL; and abrogated the NF-κB activation, increase of anti-apoptotic proteins and protection against Apo2L/TRAIL induced by IGF-1. These findings therefore define an important role of the Akt pathway in modulating tumor cell responsiveness to Apo2L/TRAIL, delineate molecular mechanisms for the survival effects of IGF-1, and characterize differential pathophysiologic sequelae of IGF-1 vs IL-6 on MM cells. Importantly, they provide the basis for future clinical trials in MM combining conventional or novel agents with strategies designed to neutralize IGF-1.

Molecular mechanisms whereby immunomodulatory drugs activate natural killer cells: Clinical application

Thalidomide and immunomodulatory drugs (IMiDs), which target multiple myeloma (MM) cells and the bone marrow microenvironment, can overcome drug resistance. These agents also have immunomodulatory effects. Specifically, we have reported that thalidomide increased serum interleukin‐2 (IL‐2) levels and natural killer (NK) cell numbers in the peripheral blood of responding MM patients. In this study, we investigated the mechanisms whereby IMiDs augment NK cell cytotoxicity. NK cytotoxicity and antibody‐dependent cell‐mediated cytotoxicity (ADCC) of peripheral blood mononuclear cells cultured with IMiDs were examined in the presence or absence of anti‐IL‐2 antibody, ciclosporin A or depletion of CD56‐positive cells. IMiDs‐induced signalling pathways, triggering IL‐2 transcription in T cells, were also delineated. IMiDs facilitated the nuclear translocation of nuclear factor of activated T cells‐2 and activator protein‐1 via activation of phosphoinositide‐3 kinase signalling, with resultant IL‐2 secretion. IMiDs enhanced both NK cell cytotoxicity and ADCC induced by triggering IL‐2 production from T cells. These studies defined the mechanisms whereby IMiDs trigger NK cell‐mediated tumour‐cell lysis, further supporting their therapeutic use in MM.

Proteasome inhibitor PS-341 abrogates IL-6 triggered signaling cascades via caspase-dependent downregulation of gp130 in multiple myeloma.

Proteasome inhibitor PS-341 is one of the most promising novel agents against multiple myeloma (MM). We have previously shown that PS-341 inhibits IL-6 triggered phosphorylation of extracellular signal-regulated kinases (ERK) 1/2 (also known as p42/44 mitogen-activated protein kinases) in MM cells. In this study, we further examined whether clinically achievable concentrations of PS-341 could inhibit IL-6 triggered signaling cascades in MM. We found that PS-341 inhibited not only ERK, but also signal transducers and activators of transcription (STAT) 3 as well as Akt phosphorylation. Since gp130 (CD130) dimerizes and is phosphorylated after IL-6 binding to gp80 (IL-6 receptor), we hypothesized that gp130 could be involved in PS-341-induced blockade of signaling cascades mediating MM cell growth, survival, and drug resistance in the bone marrow (BM) microenvironment. In this study, we first demonstrate that PS-341 induces downregulation of gp130 in a time- and dose-dependent manner in vitro, prior to MM cell death. Conversely, downregulation of gp130 is completely abrogated by the pan-caspase inhibitor Z-VAD-FMK, suggesting that downregulation of gp130 is mediated via caspase activation. Z-VAD-FMK also abrogates the inhibitory effect of PS-341 on IL-6-triggered signaling cascades. Importantly, we demonstrate that phosphorylation of ERK, STAT3, and Akt in MM.1S cells induced by either exogenous IL-6 or by binding of MM cells to BM stromal cells is abrogated by PS-341. These studies, therefore, define another novel mechanism whereby PS-341 can overcome the growth and survival advantage in MM cells conferred by the BM milieu. Importantly, this effect on cytokine-induced gp130 signaling cascades may account, at least in part, for the remarkable preclinical sensitivity and clinical responses achieved in MM with PS-341 treatment.

Changes of telomere length in children after hematopoietic stem cell transplantation

Telomeres are responsible for keeping the stability not only of chromosomes but also of genes. To investigate the effect of hematopoietic stem cell transplantation (HSCT) on telomeres, we studied telomere length in the peripheral blood mononuclear cells of 31 children who received HSCT. In the auto-HSCT groups telomere length ranged from 8.6 to 12.0 kb and in the allo-HSCT groups from 8.4 to 12.0 kb. Comparison of the telomere length between before and after auto-HSCT showed shorting up to 1.0 kb. Moreover, comparison between donors and recipients in allo-HSCT revealed that telomeres of recipients were up to 1.0 kb shorter than those of the donors. Patients who received allo-HSCT from donors older than 18 years had significantly shorter telomeres than those transplanted from donors under 18 years old (P < 0.05), indicating that donor age is an important factor for recipient’s telomere length. these findings suggest that the effects which might be induced by shortening of telomeres in recipients are within the biologically tolerable range. however, if hematopoietic stem cells from elderly donors are transplanted into younger patients, the telomere length may become too short for acceptable lifetime risks of genetic instability in the recipient.

Shortening of telomeres in recipients of both autologous and allogeneic hematopoietic stem cell transplantation

Telomere length of peripheral blood mononuclear cells (PBMCs) from 23 autologous HSCT patients ranging from 4 to 61 years old, and 46 allogeneic HSCT recipients from 6 to 52 years old were studied to confirm whether excessive shortening of telomeres is associated with HSCT. After autologous HSCT, telomere length of PBMCs ranged from 6.8 to 12.0 kb. The comparison between transplanted PBMCs and PBMCs after autologous HSCT showed shortening by up to 1.9 kb (mean ± s.d.: 0.64 ± 0.50 kb). There was a difference between autologous HSCT patients and normal volunteers in the slopes of regression lines. After allogeneic HSCT, telomere length of PBMCs ranged from 6.8 to 12.0 kb. Telomeres of recipients were up to 2.1 kb (0.60 ± 0.468 kb) shorter than those of donors. The slope of regression lines for allogeneic HSCT patients and normal volunteers were parallel. Although all patients were transplanted with more than 2.0 × 108 cells/kg, telomere length did not correlate with the number of transplanted cells. There was no significant correlation between telomere length and recovery of hematological parameters. However, three patients with an average telomere length of 6.8 kb after HSCT took a longer period to reach the normal hematological state. Taken together, these data suggest that most HSCTs are performed within the biological safety range of telomeres, while the patients who have telomeres shorter than 7.0 kb after HSCT should be observed carefully for long-term hematopoiesis and the occurrence of hematopoietic disorders. Bone Marrow Transplantation (2000) 25, 441–447.

Biologic sequelae of c-Jun NH(2)-terminal kinase (JNK) activation in multiple myeloma cell lines.

Although c-Jun NH2-terminal kinase (JNK) is activated by treatment with therapeutic agents, the biologic sequelae of inhibiting constitutive activation of JNK has not yet been clarified. In this study, we examine the biologic effect of JNK inhibition in multiple myeloma (MM) cell lines. JNK-specific inhibitor SP600125 induces growth inhibition via induction of G1 or G2/M arrest in U266 and MM.1S multiple myeloma cell lines, respectively. Neither exogenous IL-6 nor insulin-like growth factor-1 (IGF-1) overcome SP600125-induced growth inhibition, and IL-6 enhances SP600125-induced G2/M phase in MM.1S cells. Induction of growth arrest is mediated by upregulation of p27Kip1, without alteration of p53 and JNK protein expression. Importantly, SP600125 inhibits growth of MM cells adherent to bone marrow stromal cells (BMSCs). SP600125 induces NF-κB activation in a dose-dependent fashion, associated with phosphorylation of IκB kinase α (IKKα) and degradation of IκBα. In contrast, SP600125 does not affect phosphorylation of STAT3, Akt, and/or ERK. IKK-specific inhibitor PS-1145 inhibits SP600125-induced NF-κB activation and blocks the protective effect of SP600125 against apoptosis. Our data therefore demonstrate for the first time that inhibiting JNK activity induces growth arrest and activates NF-κB in MM cells.

Telomerase overexpression in K562 leukemia cells protects against apoptosis by serum deprivation and double-stranded DNA break inducing agents, but not against DNA synthesis inhibitors

Telomeres are specialized DNA/protein structures that act as protective caps to prevent end fusions. The maintenance of telomeres is essential for chromosomal stability. Telomerase is regulated by human telomerase reverse transcriptase (hTERT). c-Myc oncoprotein is also implicated in the positive regulation of hTERT expression. We show here that two clones of hTERT-transfected K562 erythroleukemia cells have elongated telomeres (22.5 and 24.0 kb), whereas telomere length of both c-Myc-transfected K562 cells and parental K562 cells is 6.5 kb. Telomerase activity and hTERT mRNA expression increased in hTERT-transfected K562 cells, while the expression levels of telomerase activity and hTERT in c-Myc-transfected K562 cells were similar to that in parental K562 cells, despite an overexpression of c-Myc. Importantly, we found that hTERT-transfected K562 cells are protected against apoptosis induced by serum deprivation and double-stranded DNA break inducing agents (ionizing irradiation, and etoposide (VP-16)), but not against DNA synthesis inhibitors (1-beta-D-arabinofuranosylcytosine and hydroxyurea). These findings suggest that overexpression of telomerase by transfecting hTERT confers telomere-elongation and resistance to double-stranded DNA break inducing agents.

Recombinant humanized anti-CD40 monoclonal antibody triggers autologous antibody-dependent cell-mediated cytotoxicity against multiple myeloma cells

Summary. Multiple myeloma (MM) is currently incurable, and novel therapies are needed. In this study, we examined a novel recombinant humanized monoclonal antibody against CD40 (rhuCD40 mAb) and demonstrate for the first time that rhuCD40 mAb induces antibody‐dependent cell‐mediated cytotoxicity (ADCC) against CD40‐positive MM cells. Importantly, we show that rhuCD40 mAb induces autologous ADCC against primary patient MM cells, without triggering ADCC against normal B cells. This study, therefore, both demonstrates that rhuCD40 mAb triggers autologous ADCC against patient MM cells and provides the framework for the clinical evaluation of rhuCD40 mAb immunotherapy to improve patient outcome in MM.

Genetic instability in intestinal metaplasia is a frequent event leading to well-differentiated early adenocarcinoma of the stomach

To understand the development of well-differentiated adenocarcinoma in the stomach, we examined genetic instability in 31 patients with stage Ia gastric cancer. Triplets of tissue specimens (normal/metaplasia/tumour) from 33 lesions were examined for microsatellite instability (MSI) and loss of heterozygosity (LOH), using nine microsatellite loci. Frameshift mutations in the transforming growth factor beta receptor type II (TGF-betaRII) (A)(10), Bcl-2-associated X protein (BAX) (G)(8), hMSH3 (A)(8) and hMSH6 (C)(8) genes were also studied. In this study, a high incidence of MSI (MSI-H) was defined as samples containing 30% or more MSI positive loci, and a low incidence of MSI (MSI-L) as samples which had less than 30% MSI. MSI-L was observed in 19 cancerous lesions (58%), and MSI-H in three (9%). Eleven intestinal metaplasia lesions (33%) showed MSI-L, but no metaplasia lesions exhibited MSI-H. Frameshift mutation was observed in only one cancerous lesion (3%) at the (A)(10) tract of TGF-betaRII. In contrast, LOH was observed in 24 cancerous lesions (73%), and in 15 (45%) of intestinal metaplasia lesions. Intriguingly, these alterations tend to be coincident between metaplasia and cancerous lesions in the same sets of specimens, and there was no case that showed alterations in metaplasia, but not in cancerous lesions. These findings suggest that metaplasia and well-differentiated adenocarcinoma in the stomach may have the same molecular backgrounds, and that these two lesions may be chronologically connected.