We have previously identified NME2 (Nm23-H2) as a tumor antigen in a patient with chronic myeloid leukemia (CML). Here we investigated the association between NME2 and Bcr-Abl. NME2 protein was highly overexpressed in the cytoplasm of peripheral blood mononuclear cells from 29/30 patients with CML at diagnosis and 10/10 patients resistant to imatinib. Protein was overexpressed in the absence of increased levels of mRNA and was limited to Bcr-Abl + populations, being absent from Bcr-Abl - patient cells, normal donors and 14/15 acute myeloid leukemia (AML) samples. Furthermore, the Bcr-Abl dependent overexpression of NME2 protein was reversed specifically by tyrosine kinase inhibitor (TKI) treatment of Ba/F3 expressing wild-type and TKI-sensitive, but not TKI-resistant, mutants of Bcr-Abl. The post-transcriptional up-regulation of the tumor antigen NME2 is therefore a common and specific property of CML closely associated with Bcr-Abl activity.

Introduction: The human Nm23 family consists of 8 genes, termed Nm23-H1 through -H8. H1 and H2 are 88% identical and localized to chromosome 17. Both Nm23-H1 and -H2 display nucleotide-diphosphate kinase (NDPk) activity responsible for balancing NTP levels by transferring gamma-phosphates between nucleotides, and individually demonstrate a variety of other activities including DNA binding, transcriptional regulation and apoptotic cleavage of DNA. A variety of studies imply a role for Nm23 proteins in development and in a range of cancers, in which level of expression rather than mutation correlates to prognosis. Specifically, a mutual transcriptional activation between the nm23-H2 and c-myc genes has been implicated in transformation. Our group described the identification of Nm23-H2 as a candidate tumor-associated antigen in a case of CML and demonstrated the presence of Nm23-H2 reactive T cells 5 years after transplantation. Here, we compare the expression of Nm23-H2 and H1 proteins in leukaemic patients and healthy donors. Materials and Methods: PBMC were collected from 10 CML patients at initial diagnosis, from 4 Glivec-resistant CML patients, from 12 AML patients at diagnosis and from 5 healthy donors. PBMC, CD3+, CD14+, CD33+ and CD34+ populations were stained, fixed and sorted by FACS onto microscope slides. Immunocytochemical analysis was performed with Nm23-H1 (Novacastra), Nm23-H2 (Santa Cruz) and β-Actin (Sigma) specific antibodies. Evaluation was carried out using a confocal laser scanning microscope LSM 510 (Zeiss). Results: PBMC from 9 out of 10 untreated CML patients (90%) showed a strong Nm23-H2 staining. Fractionation of the PBMC population revealed that CD14+, CD33+ and CD34+ but not CD3+ cells are Nm23-H2 positive in the majority of these patients. Nm23-H2 was also detected in the PBMC of 3 out of 4 Glivec-resistant CML patients. In most cases, Nm23-H2 co-localized with β-actin. Under the same staining conditions, PBMC and sorted subpopulations from 11 of 12 AML patients (92%) and from all healthy donors were Nm23-H2 negative, while very weak staining was detected in PBMC of one AML patient. Nm23-H1 protein was clearly detectable in a number of cell lines (HL-60, U-937, K562 and T47D), but was very weak or absent in PBMC from CML patients and healthy donors. Conclusion: Taken together, our results show that those myeloid populations functionally affected by the bcr/abl mutation specifically over express Nm23-H2 but not Nm23-H1 protein in 9 out of 10 cases of CML. While the molecular basis of this expression pattern and the possible involvement of c-Myc remains to be resolved, these results support the proposal that immune responses to Nm23-H2 may mediate GvL effects and that peptides derived from Nm23-H2 might therefore be attractive targets for specific immunotherapy in CML patients.

3266 Poster Board III-1 Introduction: NmE2 (Nm23-H2, NDP kinase B) is one of a family of proteins that catalyze the transfer of gamma-phosphate between nucleoside-triphosphates and diphosphates. The two major family members, NmE1 and NmE2 are strongly implicated in the control of differentiation, proliferation, migration and apoptosis via interactions which are often independent of their kinase activity, NmE2 being a transcriptional activator of the c-myc gene. We recently identified NmE2 as a tumour associated, HLA-A32+ restricted, antigen in a patient with CML and found the protein (but not the mRNA) to be generally over expressed in CML but not in other haematological malignancies. We also detected a specific T-cell response in peripheral blood cells of a patient 5 years after transplantation. This identifies NmE2 as a potential target for both molecular and immunotherapy of CML. However, the development of immunotherapeutic approaches will depend on the ability of NmE2 to function as a tumour antigen in common HLA backgrounds. The aims of this study were firstly to investigate the antigenicity of NmE2 in the HLA-A2 background (which accounts for more than 50% of the Caucasian population), and secondly to characterise the regulatory relationship between Bcr/Abl and NmE2 using a cell line model of CML. Materials and Methods: 5 nonameric NmE2 peptides with predicted anchor amino acids for HLA-A2 were loaded at concentrations of 10μM separately onto HLA-A2 expressing antigen presenting cells. Elispot Assays were carried out with CD8+ MLLCs (for the identification of antigenic peptides) or CD8+ cells isolated directly from a CML patient at different time points after HCT. Ba/F3 cells stably expressing wild type and mutant forms of Bcr/Abl were treated with imatinib and nilotinib (0 – 10 μM) for 48h. Bcr/Abl activity was assessed by FACS using antibodies specific for the phosphorylated forms of CrkL and Stat5. NmE2 and c-Myc protein were detected by immunocytochemistry and Western blotting with specific antibodies [Santa Cruz, clones L-16 and 9E10 respectively]. Levels of nme2 and c-myc mRNA were determined by quantitative real time PCR. Results: Full length NmE2 protein and 2 of 5 HLA-A2 anchor-containing peptides tested (NmE2132–140 and NmE2112–120) were specifically recognized by the HLA-A2+ CD8+ MLLC, demonstrating the antigenicity of NmE2 in the HLA-A2 background in vitro. Furthermore, while CD8+ T-cells from a transplanted HLA-A2+ CML patient showed little or no specific reactivity in the first 10 months after HCT, a distinct reactivity (up to 0.6 % NmE2 reactive CD8+ T cells) became apparent at later stages, consistent with the development of an immune response against NmE2-expressing cells in vivo. The patient remained negative for bcr/abl transcripts throughout this period. BA/F3 Bcr/Abl cells expressed increased levels of NmE2 protein (but not mRNA) compared to the parent BA/F3 line. Interestingly, treatment with imatinib or nilotinib reduced NmE2 protein expression in BA/F3 Bcr/Abl, but not in cells expressing Bcr/Abl mutants resistant to the respective inhibitors. Treatment of BA/F3 Bcr/Abl cells with the PI3K inhibitor Ly294002 resulted in reduced Bcr/Abl activity and a corresponding reduction in both c-Myc and NmE2 protein levels, without affecting mRNA levels. Conclusion: The over expression of NmE2 is closely linked to Bcr/Abl kinase activity, the predominant level of regulation being post-transcriptional and dependent on PI-3K activity. The NmE2 protein is restricted by HLA-A2 as well as by HLA-A32. The development of an NmE2-specific T-cell response in a CML patient after stem cell transplantation suggests that NmE2 functions as a tumour antigen in HLA-A2+ patients in vivo and may be relevant to the long term immune control of CML. NmE2 is therefore a promising candidate for the development of new immunotherapeutic strategies for the treatment of CML. Disclosures: Lange: BMS: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Niederwieser:BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Research Funding.

4461 Introduction: We have previously identified Nm23-H2 as a tumor antigen in a case of chronic myeloid leukemia. In the following project we investigated the molecular background for the increased expression of Nm23-H2 in CML cells from a large number of patients. Material and Methods: Clinical samples from 30 patients with CML in chronic phase at diagnosis [Bcr-Abl/Abl transcript levels from 69.5 to 93% IS (international scale)], in addition to patients with molecular response to imatinib [n=10; median 2 (range 0.1–11) % IS Bcr-Abl/Abl transcript level] and to imatinib resistant patients [n=10; median 66 (range 37–100) % IS Bcr-Abl/Abl transcript level] were investigated. Subpopulations were isolated from the blood (CD3+, CD14+) or bone marrow (CD34+) by FACS sorting. AML samples were obtained from 12 patients with de novo AML and 3 patients with secondary AML at diagnosis. Ba/F3 cells expressing either wild-type Bcr-Abl (Bcr-Ablwt Ba/F3), the T315I mutant of Bcr-Abl (Bcr-AblT315I Ba/F3) or the E255K mutant of Bcr-Abl (Bcr-AblE255K Ba/F3) were used in the cell line model. Messenger RNA levels of Nm23-H2 were measured using the QuantiTect Primer Assay (Qiagen). Results were normalized to mRNA for the ribosomal protein RPLP0. Western blotting for Nm23-H2 was performed by standard techniques using chemiluminescence and immunocytochemistry was performed using Nm23-H2 and ß-Actin antibodies. Mean fluorescence intensity was determined using the LSM Image Browser software. Results: Nm23-H2 protein is over-expressed in the cytoplasm of mononuclear cells from 30/30 CML patients at diagnosis and 10/10 patients with resistance to imatinib. In contrast, patients responding to Imatinib had markedly reduced levels of Nm23-H2 protein. Over-expression is limited to Bcr-Abl+ progenitor and myeloid populations and is absent from (Bcr-Abl-) T lymphoid cells, normal donors and 14/15 acute myeloid leukemia samples. The increase in Nm23-H2 protein is due to post-transcriptional regulation, with no detectable increase in mRNA levels. The increase in Nm23-H2 can be reversed by imatinib or nilotinib treatment of Ba/F3 cells expressing wild-type Bcr-Abl, but not of cells expressing the respective resistant Bcr-Abl mutants. Conclusions: The post-transcriptional up-regulation of Nm23-H2 is therefore a common and specific property of CML closely associated with Bcr-Abl activity, making Nm23-H2 a potential target for the development of novel immunotherapeutic or pharmaceutical therapies for CML. Disclosures: Al-Ali: Novartis: Consultancy, Honoraria. Niederwieser:Bristol-Myers Squibb: Speakers Bureau; Novartis: Speakers Bureau.