ALBERT

Description: Using the intra-bone marrow injection (IBMI) method, we have identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with multi-lineage repopulating ability (Blood101:2924,2003). Functional studies revealed that these CD34− SRCs have different hematopoietic stem cell (HSC) characteristics from CD34+ SRCs. In order to further clarify the HSC characteristics of CD34− SRCs, here we investigate the proliferative potential and redistribution kinetics of human CB-derived CD34− SRCs, and compare them with those of CD34+CD38+/− SRCs using IBMI. First, we performed limiting dilution analyses and revealed that the incidence of CD34+CD38− SRCs in CB-derived Lin−CD34+CD38− cells was 1 out of 41 cells by IBMI. In contrast, the incidence of CD34− SRCs in Lin−CD34− cells was 1 out of 24,100, as we previously reported. Based on these data, we transplanted 200 to 5,000 Lin−CD34+CD38− cells (containing 5 to 120 SRCs), 15,000 to 50,000 Lin−CD34+CD38+ cells (containing 10 to 30 SRCs), or 60,000 to 70,000 Lin−CD34− cells (containing 3 SRCs) into primary recipient NOD/Shi-scid mice. After 5 weeks, all mice that received transplants of Lin−CD34+CD38+/− cells showed the human CD45+ cell repopulation in the other bones as well as the injected left tibiae. However, the human CD45+ cells were only detected in the injected left tibiae in mice that received transplants of Lin−CD34− cells 5 weeks after the transplantation. In the mice that received transplants of 200 Lin−CD34+CD38− cells (containing 5 SRCs), the CD45+CD34+ as well as CD45+CD34− cells were detected in both sites. In contrast, only CD45+CD34− cells were detected in the mice that received transplants of 70,000 Lin−CD34− cells (3 SRCs). These results suggested that CD34− SRCs might remain or slowly proliferate as CD34− cells at the site of injection for at least 5 weeks. Next, we serially investigated the human CD45+ cell repopulation in the injected site and the other bones, separately. Very interestingly, CD34+CD38+/− SRCs began to migrate 2 weeks after the transplantation. The human cell repopulation in these mice was observed in other bones by 3 weeks after transplantation. Moreover, these CD34+ SRCs actively proliferated at both sites and produced CD34+ progenies. In contrast, CD34− SRCs began to migrate 5 weeks after the transplantaion. Furthermore, these CD34− SRCs showed significantly higher proliferative potential 8 weeks after transplantation than CD34+ SRCs and produced more CD34+ progenies not only at the site of injection, but also in the other bones. These results indicated that CD34− SRC as well as CD34+CD38+/− SRCs could actively migrate from the injected site to the other bones. However, the time of initiation of migration was different between CD34+/− SRCs. All these findings indicate that CD34− SRCs show different proliferative potential and redistribution kinetics, and suggest that our identified CD34− SRCs are distinct class of primitive HSCs in comparison with CD34+CD38+/− SRCs. We are now in the progress of clarifying whether the CD34− SRCs migrate to other bones with the CD34− immunophenotype or after their conversion (differentiation) to the CD34+ cells.

Description: We have identified a specific dual Bcr-Abl/Lyn inhibitor, NS-187 (elsewhere described as CNS-9), which is 25–55 times more potent than imatinib against wild type Bcr-Abl in vitro. To evaluate the potential of NS-187 as a therapeutic agent, we assessed its in vivo activity. When Balb/c mice were given NS-187 orally at a dose of 30 mg/kg, the pharmacokinetic parameters were as follows: Tmax, 2 h; Cmax, 586 ng/ml; AUC0-∝, 2999 ng•h/ml; T1/2, 1.0 h; and bioavailability value (BA), 33%. The maximal tolerated dose (MTD) of NS-187 in Balb/c or Balb/c-nu/nu mice was 200 mg/kg/day (100 mg/kg, twice daily). To test the effect of NS-187 on in vivo tumor growth, Balb/c-nu/nu mice were injected subcutaneously with Bcr-Abl-positive KU812 cells on Day 0 and given NS-187 or imatinib orally twice a day from Day 7 to Day 17. At 20 mg/kg/day, imatinib inhibited tumor growth slightly, while at 200 mg/kg/day, it inhibited tumor growth almost completely. In contrast, at only 0.2 mg/kg/day NS-187 significantly inhibited tumor growth, while at 20 mg/kg/day it completely inhibited tumor growth without any adverse effects. The body weights of the treated tumor-bearing mice were not significantly different from those of untreated mice, even at a dosage of 200 mg/kg/day NS-187. Thus, NS-187 was at least 10-fold more potent than imatinib in vivo with complete inhibition of tumor growth as the end-point. We also tested the ability of NS-187 to suppress tumor growth in another murine tumor model, namely, Balb/c-nu/nu mice intravenously transplanted with BaF3 cells harboring wild type Bcr-Abl. The mice were treated orally with NS-187 or imatinib for 11 days starting on Day 1. All eight untreated mice and all eight mice treated with 400 mg/kg/day imatinib had died by Day 25 due to leukemic cell expansion, and NS-187 significantly prolonged the survival of the mice in a dose-dependent manner. We next examined the ability of NS-187 to block the in vivo growth of BaF3 cells harboring one of the Abl point-mutants M244V, G250E, Q252H, Y253F, T315I, M351T and H396P in Balb/c-nu/nu mice. These mice were treated with NS-187 or imatinib for 11 days starting on Day 1. NS-187 at 200 mg/kg/day significantly prolonged the survival of mice inoculated with BaF3 cells harboring any of these mutants except T315I compared with untreated or imatinib-treated mice (see Figure for an example). Thus, NS-187 was more potent than imatinib and could override the point-mutation-based imatinib-resistance mechanism in vivo. The efficacy and safety of NS-187 for Ph+ leukemias is expected to be verified by early-phase clinical trials. Figure Figure

Description: Recently, we have successfully identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) method (Blood101:2924,2003). These CD34− SRCs could home into the BM niche only by IBMI, because they expressed lower levels of homing receptors including CXCR4. These CD34− SRCs did not express CD38 as well as c-kit. It is well documented that the tyrosine kinase receptors c-kit and flt3 are expressed and function in early mouse and human hematopoiesis. In murine model, it was reported that Lin−CD34−Sca−1+c-kit+flt3− cells supported long-term multilineage hematopoietic reconstitution. In contrast, Lin−CD34−Sca-1+c-kit+flt3+ cells are progenitors for the common lymphoid progenitor. More recently, these Lin−CD34−Sca-1+c-kit+flt3+ hematopoietic stem cells (HSCs) have been revealed to lack erythro-megakaryocytic potential. In this study, we have investigated the function of flt3 in our identified human CB-derived CD34− SRCs. First, we studied the SRC activity of CB-derived Lin-CD34+Flt3+/− or CD34−Flt3+/− cells using IBMI. Both CD34+FLT3+/− cells repopulated all 13 recipient mice. The level of human CD45+ cells in murine BMs received transplants of CD34+Flt3+ cells (29.3~90.8%, median 62.8%) was higher than those received transplants of CD34+Flt3− cells (9.8~45.1%, median 17.7%). On the other hand, only CD34−Flt3− cells repopulated all 7 recipient mice and the level of human CD45+ cells in murine BMs was 11.7~63.3% (median 37.9%). To further evaluate the long-term repopulating potential of these three populations, including CD34+Flt3+/− and CD34−Flt3− cells, BM cells obtained from each primary recipient mice were accessed for their SRC activities by secondary transplantation by IBMI. While CD34+Flt3+ cells did not show secondary repopulating activity, CD34+Flt3− cells could repopulate 83% (5/6) of secondary recipients. Moreover, CD34−Flt3− cells did repopulate all 5 secondary recipient mice with higher repopulating rate. Next, we cocultured CD34−Flt3−cells with the murine stromal cell line, HESS-5 in the presence of SCF, TPO, IL-3, IL-6, and G-CSF. After one week, significant numbers of CD34+Flt3− and CD34+Flt3+ cells were generated. Then we sorted these two populations, CD34+Flt3+/− cells, and tested their SRC activities by IBMI. Seven out of 10 and 5 out of 10 mice received CD34+Flt3+/− cells were repopulated with human cells, respectively. These results indicated that human CB-derived Lin−CD34−Flt3− cells produced CD34+Flt3− as well as CD34+Flt3+ SRCs in vitro. Our present study has demonstrated that human CB-drived CD34− SRCs do not express Flt3 tyrosine kinase receptor as did murine CD34− KSL cells. Based on these data, we propose that the immunophenotype of very primitive long-term repopulating human HSC is Lin−CD34−CD38−c-kit-Flt3−.

Description: Central nervous system (CNS) relapse accompanying prolonged administration of imatinib mesylate, an Abl-specific tyrosine kinase inhibitor, has recently become apparent as an impediment to the therapy of Philadelphia-chromosome-positive (Ph+) leukemia. CNS relapse may be explained by limited penetration of imatinib into the cerebrospinal fluid due to presence of P-glycoprotein (P-gp) at blood-brain barrier. To overcome imatinib-resistance mechanisms such as bcr-abl gene amplification, point mutations within ABL kinase domain, and activation of Lyn, we recently developed a specific dual BCR-ABL/Lyn inhibitor, INNO-406 (formerly NS-187), which is 25–55 times more potent than imatinib in vitro and at least 10 times more potent in vivo (Blood106: 3948–3954, 2005). The aim of this study was to investigate the efficacy of INNO-406 in treating CNS Ph+ leukemia. The intracellular accumulation of [14C]INNO-406 in P-gp overexpressing LLC-GA5-COL150 cells was much less than that in parental LLC-PK1 cells. The addition of 10 mM cyclosporin A (CsA) increased the intracellular accumulation of [14C]INNO-406 in both LLC-PK1 cells and LLC-GA5-COL150 cells. The peak concentration of INNO-406 in the brain when 30 mg/kg INNO-406 was administered p.o. was 50 ng/ g (87 nM), representing only 10% of plasma drug level. These findings suggested that INNO-406 is also a substrate of P-gp, as is imatinib. However, the residual concentration of INNO-406 in the CNS was enough to inhibit the growth of Ph+ leukemic cells according to the in vitro data. To increase the concentration of INNO-406 in CNS, we next examined the combined effects of CsA. In the brain, the concentration of INNO-406 was doubled following prior administration of 50 mg/kg CsA. Since pharmacokinetic studies suggested the possible effects of INNO-406 against CNS Ph+ leukemia, we investigated in vivo anti-CNS Ph+ leukemia effects of INNO-406 alone and combination of INNO-406 and CsA using immunodeficient mice (nude or NOD/SCID) which received Ph+ leukemic cells into the cerebral ventricle. INNO-406 alone inhibited growth of leukemic cells harboring either wild type or mutated BCR-ABL such as E255K and M351T in CNS. Furthermore, CsA significantly enhanced anti-CNS Ph+ leukemia effects of INNO-406 in vivo not only against cells harboring wild type BCR-ABL but also against cells harboring BCR-ABL/M351T (Figure). In conclusion, INNO-406 was found to inhibit Ph+ leukemic cell growth in CNS in spite of efflux of the compound by P-gp, and CsA augmented the anti-CNS Ph+ leukemia effects of INNO-406. Phase I clinical study on INNO-406 was initiated in the U.S.A. in July 2006. The efficacy and safety of INNO-406 in the treatment of leukemias is expected to be verified by early-phase clinical trials. Figure Figure

Description: Survivin, a member of the inhibitors of the apoptosis family, is overexpressed frequently in a variety of cancers and hematological malignancies, but not in normal tissues. Murine in vivo and human in vitro studies have suggested that immunotherapy of cancer patients using survivin peptide might be feasible. In the present study, we examined whether HLA-A24 restricted cytotoxic T lymphocytes (CTL) which recognize survivin peptide can be generated from peripheral blood of lymphoma patients. HLA-A24 positive four lymphoma patients and two healthy volunteers were enrolled. Three immunodominant 9-mer candidate peptides (2B, 3A, 3B) were selected on the basis of anchoring motif of peptide binding to HLA-A24 molecule. CD8 T cells from the patients and healthy volunteers were stimulated several times with autologous monocyte-derived dendritic cells pulsed with survivin or control HIV peptides and tested for peptide-specific cytotoxicity by an LDH-release assay. CTL generated with survivin 2B peptide lysed autologous monocytes pulsed with a relevant peptide. However, other survivin peptides did not elicit CTL response. Non-pulsed or HIV peptide-pulsed monocytes were not lysed. On the other hand, CTL generated with HIV peptide only lysed HIV peptide-pulsed monocytes. CTL did not lyse allogeneic monocytes regardless of the peptide pulse. Cytotoxic activity was inhibited by the pretreatment of target cells by anti-HLA class I, not by anti-HLA-DR monoclonal antibody, indicating that the lysis was HLA class I (A24) restricted. These cells did not lyse Daudi and K562, excluding the involvement of LAK or NK activity. Importantly, these survivin peptide-specific CTL showed cytotoxicity to the patient’s lymphoma cells and HLA-A24 positive lymphoma cells. Based on these preclinical data, we have just started a pilot clinical study to examine the safety and the efficacy of peptide vaccination to relapsed, chemotherapy-resistant malignant lymphoma patients who are HLA-A24 and survivin positive. A 46-year old male patient with diffuse large B-cell lymphoma has just completed two courses of four vaccinations at two-week intervals with survivin 2B peptide (1 μg subcutaneously) in an incomplete Freund’s adjuvant (Montanide ISA-51, SEPPIC Co. France). We observed a marked decrease in the size of extra-nodular surface and cervical lymphnodes following vaccinations without serious adverse events. Immunological evaluations using HLA-tetramer and T cell receptor clonality assays revealed an increase in survivin-specific CTL frequency after vaccinations. The in-vitro feasibility study and pilot clinical trial indicate that a vaccination with a survivin peptide is safe and might be a promising novel strategy for the treatment of lymphoma patients.

Description: We recently isolated a novel angiogenesis inhibitor, vasohibin-1, and its homologue, vasohibin-2. In this study we characterize the role of these 2 molecules in the regulation of angiogenesis. In a mouse model of subcutaneous angiogenesis, the expression of endogenous vasohibin-1 was low in proliferating ECs at the sprouting front but high in nonproliferating endothelial cells (ECs) in the termination zone. In contrast, endogenous vasohibin-2 was preferentially expressed in mononuclear cells mobilized from bone marrow that infiltrated the sprouting front. When applied exogenously, vasohibin-1 inhibited angiogenesis at the sprouting front where endogenous vasohibin-1 was scarce but did not influence vascularity in the termination zone where endogenous vasohibin-1 was enriched. Exogenous vasohibin-2 prevented the termination of angiogenesis in the termination zone and increased vascularity in this region. Angiogenesis was persistent in the termination zone in the vasohibin-1 knockout mice, whereas angiogenesis was deficient at the sprouting front in the vasohibin-2 knockout mice. Supplementation of deficient proteins normalized the abnormal patterns of angiogenesis in the vasohibin knockout mice. These results indicate that vasohibin-1 is expressed in ECs in the termination zone to halt angiogenesis, whereas vasohibin-2 is expressed in infiltrating mononuclear cells in the sprouting front to promote angiogenesis.

Description: Neutrophils play a vital role in the immune defense, which is evident by the severity of neutropenia causing life-threatening infections. Granulocyte macrophage-colony stimulating factor (GM-CSF) controls homeostatic and emergency development of granulocytes. However, little is known about the contribution of the downstream mediating transcription factors signal transducer and activator of transcription 5A and 5B (STAT5A/B). To elucidate the function of this pathway, we generated mice with complete deletion of both Stat5a/b genes in hematopoietic cells. In homeostasis, peripheral neutrophils were markedly decreased in these animals. Moreover, during emergency situations, such as myelosuppression, Stat5a/b-mutant mice failed to produce enhanced levels of neutrophils and were unable to respond to GM-CSF. Both the GM-CSF–permitted survival of mature neutrophils and the generation of granulocytes from granulocyte-macrophage progenitors (GMPs) were markedly reduced in Stat5a/b mutants. GMPs showed impaired colony-formation ability with reduced number and size of colonies on GM-CSF stimulation. Moreover, continuous cell fate analyses by time-lapse microscopy and single cell tracking revealed that Stat5a/b-null GMPs showed both delayed cell-cycle progression and increased cell death. Finally, transcriptome analysis indicated that STAT5A/B directs GM-CSF signaling through the regulation of proliferation and survival genes.

Description: RICBT using cyclosporine (CSP) and short-term mycophenolate mofetil (MMF) was performed on adult patients with high-risk hematological malignancies, from which data on complication measures and transplantation indications was obtained. From November 2003 to January 2005, RICBT was performed a total of 16 times on 14 patients. All patients were adults diagnosed with high-risk hematological malignancies, and 12 patients were not in remission. The average age of the patients was 57 years (range: 31–72 years). The average body weight of the patients was 59.1 kg (range: 48–72). The HLA match of two patients was 5/6 and 12 patients was 4/6. The average nucleated cell count was 2.55 x 107 cells/kg (range: 2.12–3.84 x 107 cells/kg). The conditioning regimen consisted of fludarabine and/or busulfan or cyclophosphamide and TBI (total body irradiation), and the intensity of therapy was adjusted based on age, disease, and systemic status. CSP and MMF were administered for GVHD (graft-versus-host disease) prophylaxis. The targeted duration of CSP and MMF administration was set at 100 and 28 days, respectively. Primary graft failure occurred in two patients. RICBT was repeated, and while the second treatment was successful in one patient, the other patient died of brain hemorrhage. Granulocyte recovery was observed in 13 patients on an average of 21 days, and platelet recovery was noted in 11 patients on an average of 43 days. Acute GVHD was assessed in 13 patients and confirmed in 12 patients, but a condition of grade II or above was seen in only three patients. Chronic GVHD was assessed in 12 patients and confirmed in six patients (limited type in five patients and extensive type in one patient). For the 16 treatments, the 100-day TRM (transplantation related mortality) was 19% (3 patients). Of the 14 patients, five remained alive and the statistically calculated one-year survival rate was 37%. Cause of death was recurrence in three patients, acute GVHD in one patient, viral infection in three patients, cerebral hemorrhage in one patient, and sudden death in one patient. Non-recurrence deaths included four patients with a past history of allogeneic transplantation, two patients with serious organ damage, and two patients with diabetic complications (two patients died of multiple causes). Of eight patients, excluding the six patients with past histories of allogeneic transplantation, serious organ damage, or diabetic complications, the 100-day TRM was 0% and the statistically calculated one-year survival rate was 54.7%. Two patients with VZV (varicella-zoster virus) infections died on days 221 and 228. While the risk of graft failure is generally high for RICBT, the success rate for the present RICBT technique was extremely high. This technique also made it possible to induce proper GVHD without steroidal agents. The low recurrence rate appeared to indicate the GVL (graft versus leukemia) effects of RICBT. The high GVL effects and low 100-day TRM observed appeared to be attributable to short-term MMF administration. We believe viral infections must be prevented for a lengthy period after the cessation of immunosuppressant therapy. Our results suggest that a past history of allogeneic transplantation, severe organ damage, or diabetic complications may constitute risk factors.

Description: The blockade of Bcr-Abl signaling suppresses cellular growth and induces cell death in Bcr-Abl+ cells. While they are known to promote caspase-mediated apoptosis, it remains unclear whether caspase-independent cell death-inducing mechanisms are also triggered. Here we assessed the regulatory mechanisms for cellular survival and death of Bcr-Abl+ leukemias more precisely, using a novel Bcr-Abl tyrosine kinase inhibitor, INNO-406 (formerly NS-187) which is more selective and 25-55-fold more active than imatinib (Kimura S, Blood 2005), in four CML-derived Bcr-Abl+ cell lines (K562, KT-1, BV173 and MYL), Ba/F3 harboring wild type bcr-abl (Ba/F3/wt bcr-abl), and in vivo CML mouse model. INNO-406 induces apoptosis in all lines examined, as were demonstrated by typical apoptotic morphology, loss of mitochondrial outer membrane potential (reduction of DiOC6 uptake), increase of cells in subG1 fraction by propidium iodide (PI) staining, DNA fragmentation and caspase-3 activation. However, when we inhibit caspase activity by zVAD-fmk (zVAD), a pan-caspase inhibitor, two modes of cell death execution were observed. In K562, KT-1 and BV173 cells treated with INNO-406, zVAD almost completely prevented apoptosis (i.e. showing atypical feature for apoptosis, no DNA fragmentation and no accumulation of subG1 fraction), with cell death resulting from morphologically non-apoptotic, so-called caspase-independent necrosis-like cell death (CIND). While, in MYL and Ba/F3/wt bcr-abl cells, despite the sufficient inhibition of caspases’ activity, the inhibition of the cell death by zVAD was only partial and these cell lines still underwent apoptosis (i.e. showing DNA fragmentation and the accumulation of subG1 population), suggesting the presence of alter cell death pathway which is caspase-independent apoptosis (CIA) in MYL and Ba/F3/wt bcr-abl. The propensity towards CIND or CIA in cells was strongly associated with cellular dependency on apoptosome-mediated caspase activity, that is CIND with a high apoptosome activity potential while CIA with low. Freshly isolated leukemic cell samples from Bcr-Abl+ leukemia patients also had either low or high apoptosome activity potential. Moreover, cells undergoing CIND exhibited hallmarks of autophagy (i.e. the autophagosome formation, punctate formations of LC3 and the accumulation of LC3-II isoform), suggested the participation of autophagy in response to Bcr-Abl blockade. Inhibition of autophagy with chloroquine enhanced INNO-406-induced cell death, which indicates that the autophagic response of the tumor cells is protective. While, in vivo CML model, INNO-406 treatment increased apoptotic cells regardless of the caspase-3 activation, further implicating the involvement of caspase-independent cell death regulatory pathway in vivo in primary Bcr-Abl+ leukemic cells. These findings suggest new insights into the biology and therapy of Bcr-Abl+ leukemias.

Description: Introduction: Chronic Lymphocytic Leukemia (CLL) is the most frequent adult leukemia in western countries. It is known to have a heterogeneous clinical course, some patients presenting an indolent disease while others have an aggressive disease requiring prompt treatment. Therefore, an individualized approach, especially in early clinical stage patients is necessary. Recent studies suggest that the biological markers LPL and ADAM-29 could be useful to predict prognosis in CLL patients: LPL being associated with an unfavorable prognosis while ADAM-29 to favorable one. Aims: to evaluate the expression of LPL (L), ADAM-29 (A) and the L/A ratio in CLL patients in regard to Binet clinical stage and progression free survival (PFS). Patients and Methods: thirty CLL patients followed at UNIFESP/EPM and HSPE were studied. RNA extraction was done by the TRIZOL method followed by c-DNA synthesis. c-DNA was amplified by PCR using LPL and ADAM-29 specific primers. T-Student’s exact test was used to compare the genes frequencies and Kaplan Meyer analysis to evaluate PFS. The results were considered statistically significant when p