ALBERT

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

Description: A randomized study had been performed between December 2001 and December 2005 to assess the optimal post remission therapy for adult AML in the first CR. The updated results are here presented, after a median follow-up of 48 months. JALSG AML201 enrolled 1064 previously untreated AML patients (pts) aged 15–64 yrs. The induction therapy consisted of cytarabine (Ara-C 100 mg/m2 day1–7) and idarubicin (IDR 12 mg/m2 day1–3) (arm A) or cytarabine (100 mg/m2 day1–7) and daunorubicin (DNR 50 mg/m2 day1–5) (arm B). If the patients did not achieve remission after the first induction therapy, then the same therapy was given once more. Pts were categorized into good, intermediate or poor risk groups by risk factors based on the criteria established in previous JALSG AML studies (Miyawaki et al. Cancer 2005). All CR pts were stratified according to the induction, the number of courses of induction, age and karyotype and were randomly assigned to the high dose Ara-C (HDAC) post remission regimen (arm C) or the conventional JALSG post remission regimen (arm D). Arm C: the three courses of HDAC which consisted of Ara-C 2.0 g/m2 q12h day1–5, arm D: the first course consisted of Ara-C 200 mg/m2 day1–5+ mitoxantrone (MIT) 7 mg/m2 day1–3, 2) Ara-C 200 mg/m2 day1–5+ DNR 50 mg/m2 day1–3, 3) Ara-C 200 mg/m2 day1–5+ aclarubicin (ACR) 20 mg/m2 day15, 4) Ara-C 200 mg/m2 day1–5+ etoposide (ETP) 100 mg/m2 day1–5 + vincristine (VCR) 0.8 mg/m2 day 8 + vindesine (VDS) 2 mg/m2 day10. Results: Of the 1064 pts registered, 1057 pts (median age: 47 years) were evaluable. 823 pts (78%) achieved CR after one or two courses of induction therapy. Of the 823 pts in CR, 781 pts were assigned to arm C (n=389) or arm D (n=392). The 5-year OS rate of arm C was 57.8% while that of arm D was 55.9% (p=0.96). The 5-year RFS rate of the CR pts was 42.7% in arm C and 38.9% in arm D (p=0.73). Among the good risk group (n=155), the 5-year OS rate of arm C was 69.9% while that of arm D was 80.5 % (p=0.11), and the 5-year RFS rate of arm C was 54.5% while that of arm D was 55.7% (p=0.53). Among the intermediate risk group (n=439), the 5-year OS rate of arm C was 50.9% while that of arm D was 48.5% (p=0.59), and the 5-year RFS rate of arm C was 41.5% while that of arm D was 36.5% (p=0.50). Among the poor risk group (n=49), the 5-year OS rate of arm C was 12.9% while that of arm D was 17.2% (p=0.58), and the 5-year RFS rate of arm C was 14.3% while that of arm D was 15.5% (p=0.78). In the CBF leukemia group (n=218), the 5-year OS rate of arm C was 75.0% while that of arm D was 65.8% (p=0.17), and the 5-year RFS rate of arm C was 56.5% while that of arm D was 38.7% (p=0.05). Among the young group (=50 yrs) (n=314), the 5-year OS rate of arm C was 51.3% while that of arm D was 40.1% (p=0.16), and the 5-year RFS rate of arm C was 40.0% while that of arm D was 28.1% (p=0.23). After all of consolidation, the lowest WBC count and the duration of neutropenia in arm C were significantly lower and longer than those in arm D. There was a higher rate of documented infection in arm C (20.9%) than in arm D (14.5%) (p〈 0.001). Conclusion: The conventional post remission therapeutic regimen established by JALSG consisting of 4 courses of consolidation was found to be as effective as the three courses of HDAC therapy. HDAC therapy produced a slightly positive effect on RFS in only the CBF leukemia group.

Description: The outcome of elderly patients with acute myeloid leukemia (AML) has not been improved for these three decades, although the incidence is getting increased with the aging of the population in Japan. In the JALSG GML200 study, we prospectively registered all elderly patients with AML during this study irrespective of the eligible criteria,. Since elderly patients are less able to tolerate to intensive cyottoxic intensive chemotherapy, we conducted a randomized study to assess an individualized induction treatment. After consolidation chemotherapy, we evaluated the adjuvant effect of ubenimex (UBX), an anti-leukemia immunomodulator. Method: From April 2000 to August 2005, the JALSG GML200 study registered all newly diagnosed AML patients aged 65 years and over. Patients who satisfied the eligibility criteria were randomized to receive either the individualized therapy (arm B) or non-individualized therapy (arm A). Patients who were not eligible were classified in arm C. Induction therapy of Arm A: enocitabine (BH-AC) 200 mg/m2 on days 1–8, 3 h infusion + daunorubicin (DNR) 40 mg/m2 on days 1–3, 30 min infusion. Arm B: BH-AC 200 mg/m2 on days 1–8, with extension to day 12 according to the results of bone marrow examination on days 7 and 10, + DNR 40 mg/m2 on days 1–3, with extension on day 12 depending on the results of bone marrow examination on days 7 and 10. Patients who achieved a complete response (CR) were randomized again to receive immuno-therapy with ubenimex (group UY) or without ubenimex (group UN). Consolidation therapy consisted of three courses: BH-AC 200 mg/m2 on days 1–5 + Mitoxantrone (MIT) 7 mg/m2 on days 1–3, BH-AC 200 mg/m2 on days 1–5 + DNR 30 mg/m2 on days 1–2, ETP 100 mg/m2 on days 1–3, BH-AC 200 mg/m2 on days 1–5 + ACR 14 mg/m2 on days 1–5. Result: Of the 374 patients registered, 244 patients were eligible for randomization to arms A and B, and 235 patients were evaluable. The median patients’ age of arms A, B and C were 70 (range, 65–79), 71 (range, 65–79), and 74 years (range, 65–92), respectively. CR rate of arm A was 63.2% and that of arm B was 65.3% (p = 0.75). CR rates of male and female patients significantly differed in each arm (arm A: male 55.4% vs. female 76.3%, p=0.053, arm B: male 53.1% vs. female 80.0%, p=0.008). Overall survival rates at 3 years of arms A and B were 25.5% and 21.5% (p = 0.56), respectively. With reference to survival with or without ubenimex in patients who achieved CR, the overall survival rate at 3 years was 43.1% for arm UY and 26.9% for arm UN (p=0.073). The number of patients in arm C was 130, and most patients in this arm received similar induction chemotherapy using DNR and BH-AC. The survival rate of this group at 3 years was unexpectedly high at 29.4%. Conclusion: Induction chemotherapy using DNR and BH-AC is one of the superior methods to achieve CR in elderly AML patients. However, individualized treatment has little advantage over non-individualized therapy. The remission rate of the female patients is higher than male patients, and it may have relations with a long life span of the Japanese female. Long-term survival rate is still unfavorable and search for a better post-remission therapy is warranted. Immunomodulator therapy using ubenimex provides a possible benefit for longer survival in elderly AML patients at CR.

Description: Neutrophils, one kind of granulocytes, are the most abundant type of white blood cells in human peripheral blood and form an integral part of the immune system. In addition, the majority of acute myelogeneous leukemia (AML) cells are from the granulocyte lineage. Granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor (GM-CSF) control migration, proliferation and survival of granulocytes. G-CSF and GM-CSF activate the transcription factors STAT5A/B (STAT5), which are essential for the development of T and B cells and the erythroid lineage. However, it is not clear to what extent G-CSF or GM-CSF signaling through STAT5 controls the differentiation, proliferation, survival in granulocyte lineage. STAT5 is not only essential for normal development and its constitutive activation has been linked to AML patients with Flt3 mutations. The objective of this study was to explore the contribution of STAT5 in G-CSF- and GM-CSF-induced granulopoiesis and to elucidate the underlying molecular mechanisms. Towards this goal, the Stat5a/b genes were deleted in mouse hematopoietic stem cells in vivo using Cre-loxP-mediated recombination (mutant mice). Injection of 5-FU resulted in a cytokine storm, which in controls, but not in mutant mice, led to a 10-fold elevation of neutrophils. Strikingly, the distribution of myeloid progenitor populations in bone marrow was not altered in STAT5-null animals in homeostasis. Colony assays were performed to address which cytokine controls granulopoiesis from these progenitors. While common multipotent progenitor cells (CMPs) and granulocyte macrophage progenitor cells (GMPs) from control mice formed large colonies in the presence of GM-CSF, mutant cells responded poorly. No difference between control and mutant colonies was observed in the presence of G-CSF. To investigate GM-CSF-mediated survival, apoptosis-assays were performed with peritoneal neutrophils. Greatly elevated apoptosis was observed with STAT5-null neutrophils. To further dissect the contribution of apoptosis and/or proliferation in the observed defects, long-term time-lapse imaging and single cell tracking was applied. Control and STAT5-null GMPs were cultured with GM-CSF and individual cells and all their progeny were continuously observed for 5 generations. Despite an equal number of initial GMPs responding to GM-CSF, the generation time of STAT5-null GMP-derived progeny was significantly prolonged in each generation and the number of cell death events increased dramatically from generation to generation. Therefore, GM-CSF-mediated STAT5 signaling is necessary to generate high numbers of granulocytic cells from GMPs by providing pro-survival and pro-proliferation signals. To identify GM-CSF-mediated and STAT5-dependent genetic cascades that control proliferation and survival of the granulocyte lineage, we performed gene expression profiling and ChIP-seq of control and STAT5-null CMPs, GMPs and neutrophils. STAT5 target genes specific to CMPs, GMPs and neutrophils were identified and their contribution to normal granulopoiesis is currently being investigated.

Description: Introduction: Chronic Lymphocytic Leukemia (CLL) is the most frequent leukemia in adults of western countries. Its clinical course is variable with some patients presenting an indolent disease while others have an aggressive disease requiring prompt treatment. Biomarkers to identify patients with poor prognosis, especially in early clinical stages, are being studied. Increase of angiogenesis, as an apoptosis deregulator, has been associated to poor prognosis in some malignant diseases such as Multiple Myeloma and Non-Hodgkin Lymphoma. Expression of Vascular Endothelial Growth Factor (VEGF) in CLL cells seems to have an unfavorable impact on prognosis, even though only a few studies have addressed this issue. AIMS: to analyze the expression of VEGF and its relation to Binet clinical stage, mutation status of IgVH and event free survival. Patients and Methods: 52 CLL patients diagnosed at UNIFESP and HSPE were studied. VEGF expression was evaluated on blood samples stained with Biotinylated Human VEGF (R&D Systems) monoclonal antibody and acquired with FACScalibur® and analyzed CellQuest® software. The expression of VEGF was considered positive when its mean fluorescence intensity was 〉200. IgVH mutation of 25 cases status was also studied by sequencing of the amplified samples by RT-PCR using VH family specific primers. The sequences were analysed with lg Blast® software and considered mutated (M) when there was a germline homology of 98% or greater and those displaying homology

Description: Interleukin-2 receptor (IL2R) is usually expressed on activated T cells by antigen stimulation and on pre-B cells during B cell differentiation. Soluble IL-2R (sIL-2R) in serum is a truncated form of the 55-kDa chain of IL-2R, which is believed to be produced by cleavage by proteases. The concentration of sIL-2R in serum has been an index of tumor burden in adult T cell leukemia/lymphoma (ATL), in which CD4-positive T cells express IL-2R (CD25) on the cell surface. Subsequently, analysis of serum sIL-2R concentration has also been useful in predicting disease activity and response to treatment in B cell lymphoma. However, it is still unclear whether B cell lymphoma cells express IL-2R (CD25) or whether serum sIL-2R concentration is due to IL-2R on B cell lymphoma cells. Furthermore, it is unclear how sIL-2R is released from IL-2R in ATL. First, we examined whether serum sIL-2R concentration is a prognostic factor in previously untreated patients with DLBCL (n = 105, median age 67.0 (18–91 years)) or FL (n = 30, median age 60.0 (40–82 years)) diagnosed between January 2001 and December 2005, and who received six cycles of R + CHOP or R + THP-COP therapy. Patients who relapsed or had disease progression after R + CHOP or R + THP-COP received R + EDAP or R + ICE for DLBCL, and R + FND for FL. The 5-year OS rates for patients with sIL-2R levels of 〈 1500 U/ml and ⊠ 1500 U/ml were 76% and 62%, respectively (p 〈 0.05) in DLBCL, and 100% and 79.3%, respectively (p = 0.19) in FL. Next, we analyzed IL-2R (CD25) expression on lymphoma cells by flow cytometry. Nine of 25 patients with DLBCL and 4 of 11 patients with FL showed CD25 expression. Some T cells (CD3-positive cells) expressed CD25 in both lymphomas. On the other hand, 7 of 7 patients with MCL expressed CD25. There was no significant relationship between serum sIL-2R concentrations and CD25 expression on lymphoma cells or clinical stage in either DLBCL or FL. Metalloproteinase-9 (MMP-9) is reported to be an important protease for releasing sIL-2R from IL-2R. However, there was no significant relationship between MMP-9 and sIL-2R levels in sera from patients with DLBCL or FL. On the other hand, 7 of 7 patients with ATL showed high concentration of MMP-9 (〉 128 ng/ml) irrespective of sIL-2R levels. We then confirmed MMP-9 expression in 3 ATL cell lines by Western blotting, and addition of MMP-9 inhibitor to culture media of these cell lines significantly decreased sIL-2R levels in supernatants. On immunohistochemical staining (IHC) using anti-MMP-9 antibody, macrophages not lymphoma cells or T cells were positive for MMP-9 in DLBCL and FL. These findings suggest that high serum sIL-2R concentrations in ATL are due to the cleavage of IL-2R by MMP-9 produced by ATL cells. On the other hand, the main source of sIL-2R may be due to release from activated T cells in DLBCL and FL. Therefore, serum sIL-2R levels may indicate activity of neoplastic cells in ATL; however, its levels may reflex the activity of microenviromental non-neoplastic cells in DLBCL and FL.

Description: The use of imatinib, an ABL tyrosine kinase inhibitor, has led to a dramatic change in the management of BCR-ABL positive leukemia patients. However, the resistance to imatinib mediated by mutations in the BCR-ABL domain has become a major problem in the treatment. Histone deacetylase (HDAC) inhibitors have been shown to mediate the regulation of gene expression, induce cell growth, cell differentiation and apoptosis of tumor cells. Vorinostat (suberoylamide hydroxamic acid:SAHA) is a hydroxamic acid based polar HDAC inhibitor. Vorinostat have shown efficacy in a wide range of cancers such as cutaneous T-cell lymphoma (CTCL). However, efficacy of vorinostat against the BCR-ABL mutants has fully not known. Here we report on the studies performed against murine Ba/F3 cell line which was transfected wild type (Wt) p210 and p185 BCR-ABL or imatinib resistant BCR-ABL mutants such as G250E, Q252H, Y253F, E255K, M294V, T315I, T315A, F317L, F317V, M351T, H396P and T315I(p185). 48 hours treatment of vorinostat exhibits cell growth inhibition and proapoptotic activity murine Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation in a dose dependent manner. IC50 of these cell lines are Wt(720nM), G250E(625nM), Q252H(220nM), Y253F(525nM), E255K(685nM), M294V(785nM), T315I(500nM), T315A(715nM), F317L(560nM), F317V(565nM), M351T(375nM) and H396P(485nM). Aurora kinases play a pivotal role in the regulator of mitotic processes during cell division. MK-0457 is a small molecule inhibitor of the Aurora kinase family and was found to be active against the cells from BCR-ABL positive patients with T315I mutation in clinical trial. Because vorinostat also depleted BCR-ABL, as well as induced apoptosis and sensitized BCR-ABL-expressing leukemia cells, we examined whether vorinostat and MK-0457 enhances the apoptosis in imatinib resistant BCR-ABL-expressing cells. 48 hours treatment of MK-0457 exhibits cell growth inhibition of Ba/F3 cells ectopically expressing Wt and imatinib resistant BCR-ABL mutants including T315I mutation. IC50 of MK-0457 is Wt(215nM), G250E(205nM), Q252H(185nM), Y253F(245nM), E255K(185nM), M294V(238nM), T315I(205nM), T315A(165nM), F317L(200nM), F317V(200nM), M351T(225nM) and H396P(195nM). We examined the intracellular signaling by using these cell lines. We found that caspase 3, and poly (ADPribose) polymerase (PARP) were activated after MK-0457 treatment in a dose dependent manner. Phosphorylation of BCR-ABL and Crk-L which is downstream target of BCR-ABL was reduced after MK-0457 treatment. We found that combination of vorinostat and MK-0457 synergistically cell growth inhibition of Wt and BCR-ABL mutants Ba/F3 cells in 48 hours treatment. Phosphorylation of Crk-L was reduced after vorinostat and MK-0457 treatment. Caspase 3 and PARP activation were also synergistically increased after vorinostat and MK-0457 treatment. We evaluated the activity of MK-0457 and vorinostat in primary BCR-ABL positive acute lymphoblastic leukemia (ALL) cells with the T315I mutation. We found that MK-0457 potently induced cell growth inhibition of primary T315I cells in 48 hours treatment. Moreover, combination of vorinostat and MK-0457 synergistically increased the cell growth inhibition in primary T315I cells. This study demonstrate monotherapy of vorinostat and the combination of vorinostat and MK-0457 are more potent efficacy not only wild type BCR-ABL but also imatinib resistant BCR-ABL mutants cells and represents a promising new strategy for treatment of imatinib resistant BCR-ABL positive leukemias, including those harboring the T315I mutation.

Description: Introduction. Myelodysplastic Syndromes (MDS) are hemopoietic clonal disorders characterized by a heterogeneous clinical course, which is currently predicted on the basis of clinical, morphologic and cytogenetic data. Only a few reports have studied the impact of flow cytometry immunophenotypic data on the prognosis of the disease. Objectives: To evaluate the immunophenotypic characteristics of CD34+ myeloid precursor cells in MDS and their association with the prognosis of the disease. Material & Methods: Overall, 39 patients with MDS - 8 with refractory anemia (RA), 9 RA with ringed sideroblasts (RARS), 12 refractory cytopenias with multilineage dysplasia (RCMD), 2 RA with excess of blasts (RAEB)-1, 5 RAEB-2, 2 with 5q- Syndrome and one unclassifiable MDS - were studied. Bone marrow cells were stained with an extensive panel of MoAbs. Results: the most frequent phenotypic abnormalities on CD34+ myeloid cells were: CD7+ (36%), CD25+ (33%) and CD56+ (15%) followed by overexpression of CD13 and/or CD33 (33%). Abnormal expression of CD7, CD25, CD13 and CD33 were evenly distributed within the WHO and IPSS subgroups of the disease, although CD56 expression was more frequently detected in low risk IPSS cases. From the prognostic point of view, patients that showed altered expression of CD25, CD56 and CD33 had a shorter median overall survival (OS): 30 vs 109 months (p = 0.0003), 19 vs 102 months (p=0.009), and 21 vs 102 months (p=0.009), for CD25, CD56 and CD33, respectively. Reactivity for CD7 and overexpression of CD13 did not influence OS. A phenotypic score was built where a score of 1 was given to abnormal CD25, CD56 or CD33 expression. Significantly different median OS rates (p1 (15 ± 5 months). Discussion: Our results show that flow cytometry immunophenotyping of CD34+ myeloid precursors provides clinically useful information for the prognostic stratification of MDS patients. Further studies including larger number of patients with a longer follow-up are necessary to confirm these results.

Description: Pure red cell aplasia (PRCA) has been reported in association with lymphoma as one of the autoimmune diseases seen during the course of lymphoid malignancies. However, the relation of PRCA with the underlying lymphomas remains unclear. The aim of this study was to clarify the histologic subtypes of lymphomas, the chronological sequence of anemia and lymphoma, and the response to treatment, and eventually to establish the treatment guideline for lymphoma-associated PRCA. We conducted a nationwide survey in Japan. From a cohort of 185 patients with PRCA, 8 patients with lymphoma were evaluated. Complete response (CR), partial response (PR) and no response (NR) were defined as the achievement of normal hemoglobin levels without transfusion, the presence of anemia but without transfusion dependence, and the continued presence of transfusion-dependence, respectively. Patient age at the onset of PRCA ranged from 48 to 82 years (median age, 68 years) with an equal male to female ratio. Histologic subtypes varied and the lymphoma was of the B-cell type in four cases and of the T-cell type in four. Four patients had coexisting anemia and lymphoma. One patient developed PRCA before the onset of lymphoma. Three other patients developed PRCA following lymphoma, two of whom developed anemia during remission. Anemia responded to chemotherapy and/or immunosuppressive therapy in seven of eight patients. In four responding patients, PRCA remained in durable remission without maintenance immunosuppressive therapy (27, 76, 97 and 127 months), which is a different feature from that of idiopathic PRCA (Sawada K, et al. Haematologica2007;92:1021). Four patients were alive, and two of these four remained in remission for both lymphoma and PRCA. Effective chemotherapy was associated with remission of anemia in patients with concurrent lymphoma and PRCA. In patients with PRCA presenting before or after the onset of lymphoma, immunosuppressive therapy was effective for improving anemia. In conclusion, chemotherapy should be introduced for patients with coexisting lymphoma and PRCA. Additional immunosuppressive therapy may be necessary for PRCA that has failed to respond to chemotherapy. Durable maintenance-free remission of anemia may be obtained in lymphoma-associated PRCA. Table 1. Patient characteristics and induction therapy for PRCA Age/Sex Histologic subtypes Days from lymphoma to PRCA Disease status of lymphoma Induction therapy for PRCA * Response of PRCA Response of lymphoma *(/), in sequential administration; (-), in combination. 76/F DLBCL −114 - Steroid CR - 75/M DLBCL −13 On disease R-CHOP/CsA NR/CR PR 62/M ATLL 0 On disease CHOP-CsA CR PD 82/F MZL 0 On disease Steroid NR NR 58/F AILT 35 On disease CHOP CR CR 48/M Follicular 720 On disease Steroid/CHOP NR/CR CR 64/M T-LBL 205 CR Steroid PR - 71/F AILT 801 CR Steroid/CsA PR/PR -

Description: We have previously reported that zoledoronic acid (ZOL) augmented the in vivo effect of imatinib in a murine chronic myeloid leukemia (CML) model (Blood 2003). ZOL alone induces apoptosis in leukemic cells in vitro by inhibiting prenylation of the Ras-related proteins. In addition to this direct anti-leukemic effect, we hypothesized that ZOL also has some influence in leukemic cells in vivo indirectly by destroying osteoclasts (OCs), which is the primary therapeutic activity of ZOL in osteoporosis patients. Supporting this notion is that by mediating bone resorption, OCs release a variety of cytokines such as IGF- 1, TGF-β, etc. that have accumulated in the bone matrix. It has been reported that OCs play an important role in bone metastasis of solid tumor, especially in cancer stem cells. However, little is known about the role of OCs in leukemia. Therefore, we investigated it in vitro and in vivo. For this purpose, we established an in vitro osteoblasts (OBs) and OCs co-culture system. The stable co-culture system that we developed includes collagen gel and murine primary OBs and OCs. In addition, murine femoral bone sections were sometimes added to this culture system so that the OCs could release the cytokines from the bone matrix. Thus, the collagen gel and OBs were placed in 12-well plates with and without bone sections and/or OCs. The transwell chambers over the wells then received 1×104 Ba/F3 cells that had been transfected with wild type bcr-abl (Ba/F3/bcr-abl cells). OBs markedly enhanced the growth of Ba/F3/bcr-abl cells in this indirect contact coculture system whereas the presence of both OBs and OCs slightly suppressed cell growth. Intriguingly, when bone sections were added (OBs+OCs+bone), Ba/F3/bcr-abl cell proliferation was significantly suppressed compared to the effect of OBs alone or OBs+OCs (Figure). Cell cycle analysis revealed that the G0/G1 population was increased in Ba/F3/bcr-abl cells co-cultured with OBs+OCs+bones. We also observed that the p27 protein levels of Ba/F3/bcr-abl cells increased upon co-culture with OCs or OCs+bones, similar to their response to treatment with purified TGF-β. We performed ELISAs to determine the concentrations of cytokines in the supernatants of co-cultured OBs and OCs. There were higher levels of TGF-β1 in the OBs+OCs+bones supernatant than in the OBs+OCs supernatant. Furthermore, OBs produced high levels of IGF-1. These findings suggest that OBs and OCs affect the proliferation and the cell cycle arrest of leukemic cells by releasing soluble factors, respectively. To more comprehensively elucidate the roles OCs play in leukemia cells in vivo, we used reveromycin A (RM-A) which inhibits bone resorption by specifically inducing apoptosis in OCs (Woo et al, PNAS 2006). RM-A did not have any in vitro effects on the proliferation of Ba/F3/bcr-abl cells. Thus, we could know the unalloyed role of OCs in leukemia with RM-A compared with ZOL which inhibited directly both OCs and leukemic cells. Our preliminary data show that RM-A suppresses the engraftment of inoculated Ba/F3/bcr-abl cells to nude mice. We also present data from ongoing studies showing the effect of RM-A on leukemic cells in murine models. These findings suggested that OCs may be an important constituent of leukemia stem cell niche and destruction of OCs by either ZOL or RM-A is a novel strategy for leukemia treatment. Figure Figure