Overexpression of the cellular nuclear exportin 1, more commonly called chromosomal region maintenance 1 (CRM1), has been associated with malignant progression and mortality. Therefore, activation of nuclear export can play a significant etiologic role in some forms of human neoplasia and serve as a novel target for the treatment of these cancers. Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma that remains incurable. The objective of this study was to investigate the functional significance of CRM1 in MCL by evaluating the therapeutic efficacy of CRM1 inhibition in MCL in vitro and in vivo. Our results showed that CRM1 is highly expressed in MCL cells and is involved in regulating growth and survival mechanisms through the critical nuclear factor-kappa B survival pathway, which is independent of p53 status. Inhibition of CRM1 by two novel selective inhibitors of nuclear export (SINE), KPT-185 and KPT-276, in MCL cells resulted in significant growth inhibition and apoptosis induction. KPT-185 also induced CRM1 accumulation in the nucleus, resulting in CRM1 degradation by the proteasome. Oral administration of KPT-276 significantly suppressed tumor growth in an MCL-bearing severe combined immunodeficient mouse model, without severe toxicity. Our data suggest that SINE CRM1 antagonists are a potential novel therapy for patients with MCL, particular in relapsed/refractory disease. (c) 2013 ISEH - Society for Hematology and Stem Cells. Published by Elsevier Inc.

Epithelial cancer cells are likely to undergo epithelial–mesenchymal transition (EMT) prior to entering the peripheral circulation. By undergoing EMT, circulating tumor cells (CTCs) lose epithelial markers and may escape detection by conventional methods. Therefore, we conducted a pilot study to investigate mRNA transcripts of EMT‐inducing transcription factors (TFs) in tumor cells from the peripheral blood (PB) of patients with primary breast cancer (PBC). PB mononuclear cells were isolated from 52 patients with stages I–III PBC and 30 healthy donors (HDs) and were sequentially depleted of EpCAM+ cells and CD45+ leukocytes, henceforth referred to as CD45−. The expression levels of EMT‐inducing TFs (TWIST1, SNAIL1, SLUG, ZEB1 and FOXC2) in the CD45− cells were determined using quantitative real‐time polymerase chain reaction. The highest level of expression by the CD45− cell fraction of HD was used as “cutoff” to determine if samples from patients with PBC overexpressed any EMT‐inducing TFs. In total, 15.4% of patients with PBC overexpressed at least one of the EMT‐inducing TF transcripts. Overexpression of any EMT‐inducing TF transcripts was more likely to be detected in patients with PBC who received neoadjuvant therapies (NAT) than patients who received no NAT (p = 0.003). Concurrently, CTCs were detected in 7 of 38 (18.4%) patients by CellSearch® and in 15 of 42 (35.7%) patients by AdnaTest™. There was no association between the presence of CTCs measured by CellSearch® or AdnaTest™. In summary, our results demonstrate that CTCs with EMT phenotype may occur in the peripheral circulation of patients with PBC and that NAT is unable to eliminate CTCs undergoing EMT.

Mammalian cells fuel their growth and proliferation through the catabolism of two main substrates: glucose and glutamine. Both nutrients are required for the synthesis of glucosamine, the precursor substrate of the hexosamine biosynthetic pathway. The hexosamine signaling pathway terminating in O-linked N-acetyl glucosamine (O-GlcNAc) cycling has been implicated in cellular signaling cascades and regulation of transcription factors involved in cancer biology. Biological functions of the hexosamine biosynthetic signaling pathways need elucidation, to determine whether altered O-GlcNAc metabolism plays a significant role in hematologic tumors such as diffuse large B-cell lymphoma (DLBCL), and utilize this bifunctional pathway as a targeted therapeutic strategy in DLBCL. We have identified a key enzyme of the hexosamine biosynthetic pathways to be highly-expressed in DLBCL cell lines and patient tumor cells. In contrast to normal circulating and tonsillar B cells, DLBCL cells expressed high levels of the terminating enzyme O-GlcNAc transferase (OGT). OGT mRNA expression is highly expressed in DLBCL in comparaison to other cancers. We discovered that several key growth and survival transcription factors, such as NF-kB and NFAT, known to be highly-activated in DLBCL, are linked to the hexoasmine biosynthetic pathway. We demonstrated that both NF-kB (p65) and NFATc1 directly associated with OGT, and down-regulation of OGT by siRNA inhibits these transcription factors activation, suggesting that both NF-kB-p65 and NFATc1 require O-GlcNAc glycosylation by OGT for their activation. These results suggest that the hexosamine pathway is highly active and utilized in DLBCL, and that exploiting this bi-functional pathway(s) as a therapeutic approach is feasible. We have previously developed an imaging agent, 99mTc-ethylenedicysteine-glucosamine (99mTc-EC-G) because EC-G mimics phosphorylated N-acetylglucosamine. ECG treatment in DLBCL cells enhances p65 and NFATc1 nuclear translocation. For therapeutic strategies, we developed metallic unlabeled Platinum (Pt) derivatives-EC-G as potential therapeutic agents. Pre-clinical in vitro studies have shown that our two lead compounds, Pt-9 and Pt-DACH-EC-G effectively inhibit lymphoma cell growth and induce apoptosis. These lead compounds can also induce DNA damage in DLBCL cells, through the up-regulation of phosphorylated histone 2AX (pH2AX), leading to the disruption of p65 and NFATc1 binding to DNA. This data importantly demonstrates that the hexosamine biosynthetic pathway is linked to key growth and survival pathways involved in the pathophysiology of DLBCL. Targeting these pathways with novel platinum EC-G compounds as a theranostic approach should lead to new, more effective treatments and diagnosis for DLBCL, particularly for relapsed/refractory DLBCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 218. doi:1538-7445.AM2012-218

Diffuse Large B cell lymphomas (DLBCL) are the most prevalent of the non-Hodgkin lymphomas and are currently initially treated fairly successfully, but frequently relapse as refractory disease, resulting in poor salvage therapy options and short survival. The greatest challenge in improving survival of DLBCL patients is overcoming chemo-resistance, whose basis is poorly understood. Among the potential mediators of DLBCL chemo-resistance is the thioredxoin (Trx) family, primarily because Trx family members play critical roles in the regulation of cellular redox homeostasis, and recent studies have indicated that dysregulated redox homeostasis also plays a key role in chemoresistance. In this study, we showed that most of the DLBCL-derived cell lines and primary DLBCL cells express higher basal levels of Trx-1 than normal B cells and that Trx-1 expression level is associated with decreased patients survival. Our functional studies showed that inhibition of Trx-1 by small interfering RNA or a Trx-1 inhibitor (PX-12) inhibited DLBCL cell growth, clonogenicity, and also sensitized DLBCL cells to doxorubicin-induced cell growth inhibition in vitro. These results indicate that Trx-1 plays a key role in cell growth and survival, as well as chemoresistance, and is a potential target to overcome drug resistance in relapsed/refractory DLBCL.

Abstract Mantle cell lymphoma (MCL) is an aggressive B-cell non-Hodgkin lymphoma (NHL) with poor prognoses; novel agents are needed for its therapy. Bruton's tyrosine kinase (Btk) has been identified as an essential kinase for B-cell survival and it is activated through the B-cell receptor (BCR) pathway. Btk has recently emerged as a promising target in MCL, as demonstrated by recent clinical trials on the Btk inhibitor PCI-32765 (Pharmacyclics, Sunnyvale, CA), suggesting that elucidating critical signaling pathways emanating through Btk will hold an important key to deciphering the pathogenesis of MCL that can lead to the development of more effective targeted therapies. In this study, we showed that Btk is constitutively phosphorylated in most MCL cell lines except Rec1 and DB SP53 and is variable among primary cells from patients. We demonstrated that knockdown of Btk by siRNA diminished Btk expression, reduced constitutive NF-kB activation by luciferase assays, leading to the cell growth inhibition and induction of apoptosis in MCL cell lines. MCL cells treated with the Btk inhibitor PCI-32765 effectively inhibits Btk activity, leading to reduced MCL cell growth with IC50 values range from 2-6 uM in Mino, Jeko1, Z138 and JMP1. Interestingly, the IC50 value in DB SP53, which lacks both phosphorylated Btk and membrane immunoglobulins, is 35 uM, suggesting that BCR signaling is not active in these cell lines. PCI-32765 also induced caspase-dependent apoptosis in a dose- and time-dependent manner in representative MCL cell lines and in patient primary cells. We further demonstrated that PCI-32765 down-regulates NF-kB activity through both, the canonical and alternative NF-kB pathways. Since previous studies have indicated synergy between proteasome inhibitors and tyrosine kinase inhibitors, we evaluated whether there is synergism between PCI-32765 and the next generation proteasome inhibitor Carfilzomib (Onyx Pharmaceuticals, South San Francisco, CA). Our data suggest potential synergy between Carfilzomib and PCI-32765 in represented MCL cell lines in terms of inhibiting cell growth and induction of apoptosis. Both compounds also synergize to inhibit NF-kB activation in MCL cells. In summary, our data suggest that Btk is a key survival kinase in MCL and strategic targeting of growth/survival Btk-mediated NF-kB pathways with novel therapeutic agents such as PCI-32765 should provide a novel therapy regimen for MCL patients. Combining PCI-32765 with the proteasome inhibitor Carfilzomib can synergize its effect in MCL and may be a useful therapeutic strategy, particularly for patients with relapsed/refractory MCL. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2854. doi:1538-7445.AM2012-2854

Abstract Acquired chemoresistance (ACR) is currently the most important cause of treatment failure and early mortality in DLBCL, arguably the most important but greatest unmet need in lymphoma therapy today. Diffuse Large B cell Lymphoma (DLBCL), the most common human lymphoma, comprises a genetically and clinically diverse group of aggressive B cell non-Hodgkin lymphomas (NHL-B), among a small group of important human cancers increasing in incidence in the US over the last four decades. NHL-B are the fifth most common cancers in the USA (>62,000 new cases/20,000 deaths) expected in 2011. The molecular biologic and genetic basis of the patho-physiology of these important lymphoid tumors is still mostly unresolved. Relapsed/refractory (r/rDLBCL) DLBCL is one of the most difficult scientific challenges and severe unmet therapeutic needs in clinical oncology today. This is due largely to 1.) very drug resistant tumor cells with very poor responses (<20% PR/CR) to current “salvage” therapies; and 2.) non-existent, invalid, or inadequate disease models, that we have recently been able to overcome, with development of valid chemo-refractory DLBCL a unique cell model library, derived from our patients, that now provides the opportunity to delineate critical previously unknown patho-physiologic biomarkers and in vivo molecular model mechanisms, while concurrently providing excellent models for new agent experimental therapeutics. We designed and synthesized novel inhibitors named Turmerax (TMX), when encapsulated with nano-liposomes to overcome solubility/bioavailability issues, is >1000x more effective than curcumin in inhibiting DLBCL growth and survival in vitro, and show no toxicities in normal peripheral blood lymphocytes. Liposomal-TMX (L-TMX) also targets multiple growth and survival signaling pathways, such as NF-kB and AKT, and key cellular regulatory proteins in drug resistance (MDR) in r/r DLBCL. We have also shown in vivo that L-TMX increases in survival time with decreases in lymphoma tumor burden in preliminary studies in SCID/MCL xeno-transplant (XT-SCID) models, without evidence of significant host toxicities. The development of our new agent nano-liposomal Turmerax, has provided a multi-targeted, non-toxic nano-therapeutic small molecule with the critical growth/survival (G/S) targeting specificities similar to natural product therapeutic agents (curcumin) but without the many foibles involving solubility, bioavailability, and poor in vivo potency problems, by providing a well-tolerated, well-delivered (in vivo) effective small molecule therapeutic agent, showing very promising preliminary activities on r/r DLBCL in xeno-transplant human DBLCL models, that appears to be an excellent candidate for future clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr A129.

Abstract 4971 Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non-Hodgkin lymphoma with poor prognoses. Discovery of less-toxic and better therapeutic agents is an ongoing challenge. Bruton tyrosine kinase (BTK), is identified as an essential kinase for B-cell survival, activated through the B-cell receptor (BCR) pathway. Recent studies have indicated that BTK mediates NF-kB activation. The potential therapeutic agent PCI-32765, a novel BTK inhibitor (Pharmacyclics, Sunnyvale, CA) have shown effective preliminary targeting of these pathways in MCL. Carfilzomib, a 2nd generation proteasome inhibitor (Onyx Pharm. Inc., Emeryville, CA) proven effective in myeloma in clinical trial is also effective in MCL in vitro and in vivo in pre-clinical studies. The objective of our study was to evaluate the therapeutic efficacy of PCI-32765 and carfilzomib in MCL, and elucidate the mechanisms of their actions. We initially screened 12 MCL cell lines and discovered that Btk is constitutively phosphorylated in all MCL cells. Representative MCL cell lines, both classic and blastoid variant, were utilized for in vitro studies. PCI-32765 effectively inhibited phospho-BTK, leading to reduced MCL cell growth with IC:50 values range from 10–50 uM. PCI-32765 also induced MCL cell apoptosis in both dose- and time-dependent manner as well as carfilzomib. Using luciferase pGL3 reporter plasmid (6xNF-kB-CD40L/TKm), we demonstrated that both PCI-32765 and carfilzomib down-regulated the reporter with a dosage dependent manner. Our data suggest that strategic targeting of growth/survival pathways with novel therapeutic agents PCI-32765 and carfilzomib should provide a novel therapy regimen for patients with relapsed/refractory MCL. Disclosures: Wang: Onyx Pharmaceuticals: Research Funding.

Abstract 1428 Acquired chemo-resistance (ACR) is currently the most important cause of treatment failure and early mortality in DLBCL, arguably the most important unmet need in lymphoma therapy today. Diffuse Large B cell Lymphoma (DLBCL), the most common human lymphoma, comprises a genetically and clinically diverse group of aggressive B cell non-Hodgkin lymphomas (NHL-B), among a small group of important human cancers increasing in incidence in the US over the last four decades. NHL-B are the fifth most common cancers in the USA (>62,000 new cases/20,000 deaths) expected in 2011. The molecular biologic and genetic basis of the patho-physiology of these important lymphoid tumors is still mostly unresolved. This is due primarily to the lack of valid patho-biologic experimental models allowing for identification of the key patho-physiologic molecular/genetic mechanisms involved in chemo-resistance, resulting in mostly unsuccessful empiric new drug salvage trials, rather than efficient drug-targeting key growth/survival/chemo-resistance (GSC) pathways essential for effective salvage therapies. We have been developing such novel translational experimental DLBCL systems (>25 DLBCL cell lines derived from relapsed DLBCL patients) and novel agents as the conceptual basis of this model. We have distinguished a set of cell lines that are more resistant to chemo-therapy and identified that the transcription factor p52 component of the alternative NF-kB pathway is highly expressed in DLBCL cell lines that show the highest chemo-resistance characteristics. Down-regulation of p52 sensitizes resistant cells to chemotherapy. This is of particular interest since previous studies have not as yet established definitive role(s) for the alternative NF-kB pathway, particularly p52, in chemo-resistance development. We have discovered that the second generation proteasome inhibitor, Carfilzomib can target the alternative NF-kB-p52 pathway by down-regulating the TNF-receptor family BAFF-R, resulting in lymphoma cell growth inhibition and apoptosis induction. NFATc1, another important multifunctional regulatory molecule (transcription factor (TF), chromatin remodeler, etc), that we have shown to be intrinsically involved with NF-kBs in most DLBCL, and whose involvement in DLBCL is becoming increasingly important on multiple levels, that was recently confirmed genetically, identifying NFATc1 expression as a candidate oncogene in ABC DLBCLs. We have also discovered that GSK3b, a key upstream natural inhibitor of NFATc1, is constitutively phosphorylated in DLBCL cells and can negatively regulate NFATc1 activation. The PKC beta II inhibitor Enzastaurin, affectively inhibits pGSK3b, leading to NFATc1 inactivation and inhibiting cell growth/survival in a broad range of DLBCL cell lines, both GCB and ABC subtypes, with IC: 50 values in the low uM ranges. Enzastaurin strongly synergizes with Carfilzomib to inhibit DLBCL cell growth and induce apoptosis, particularly in chemo-resistant DLBCL cells. Carfilzomib alone enhances pGSK3b and NFATc1 activation, while Enzastaurin abolishes CFZ-induced pGSK3b and NFATc1, suggesting a mechanism for the synergistic activity of the drugs. Novel drug combinations with agents that target multiple growth, survival, and chemo-resistance pathways, such as Carfilzomib and Enzastaurin, represent promising, emerging therapeutic options for reversing chemo-resistance in relapsed/refractory DLBCL patients. Disclosures: No relevant conflicts of interest to declare.

The nuclear factor of activated T cells (NFAT) family of transcription factors functions as integrators of multiple signaling pathways by binding to chromatin in combination with other transcription factors and coactivators to regulate genes central for cell growth and survival in hematopoietic cells. Recent experimental evidence has implicated the calcineurin/NFAT signaling pathway in the pathogenesis of various malignancies, including diffuse large B-cell lymphoma (DLBCL). However, the molecular mechanism(s) underlying NFATc1 regulation of genes controlling lymphoma cell growth and survival is still unclear. In this study, we demonstrate that the transcription factor NFATc1 regulates gene expression in DLBCL cells through a chromatin remodeling mechanism that involves recruitment of the SWItch/Sucrose NonFermentable chromatin remodeling complex ATPase enzyme SMARCA4 (also known as Brahma-related gene 1) to NFATc1 targeted gene promoters. The NFATc1/Brahma-related gene 1 complex induces promoter DNase I hypersensitive sites and recruits other transcription factors to the active chromatin site to regulate gene transcription. Targeting NFATc1 with specific small hairpin RNA inhibits DNase I hypersensitive site formation and down-regulates target gene expression. Our data support a novel epigenetic control mechanism for the transcriptional regulation of growth and survival genes by NFATc1 in the pathophysiology of DLBCL and suggests that targeting NFATc1 could potentially have therapeutic value.

Abstract Abstract 2004 Diffuse large B cell lymphomas (DLBCL) are the most common non-Hodgkin (NHL-B) lymphoma. Current treatment is fairly successfully (∼70-80% remission with R-CHOP frontline chemotherapy), but relapse is common (∼50% after 2–3yrs) with poor salvage therapy options and short survival in relapsed/refractory (r/r) DLBCL. The greatest challenge in improving survival of DLBCL patients is overcoming chemo-resistance, which we currently have very little understanding of the basis. Elucidation of molecular pathways and tumor-encoded genes whose expression contribute to the intrinsic resistance and rapid cell growth of lymphoma cells could yield immediate clinical benefits and reveal new therapeutic targets for effective control and treatment of r/r lymphomas. Thioredoxin (Trx) family members play critical roles in the regulation of cellular redox homeostasis. Cancer cells exist in a stressed environment and rely on the Trxs for protection against stress-disregulated redox signaling. The most extensively studied member of the family is Trx-1 whose levels are increased in many human cancers, most likely in direct response to stress. Trx-1 contributes to many of the hallmarks of cancer including increased proliferation, resistance to cell death and increased angiogenesis. Trx-1 is a validated cancer drug target associated with aggressive tumor growth, resistance to standard therapy and decreased patient survival. In this study we showed that the majority of the DLBCL-derived cell lines express high basal levels of thioredoxin-1 (Trx-1) when compared to normal B cells by both Western blotting and real-time PCR. Tissue microarray (TMA) and immunohistochemistry analysis of 100 cases of DLBCL showed that 77% of the cases were positive for Trx-1 and 40% of the cases over-expressed Trx-1. Based on these observations, we hypothesize that aberrant expression of Trx-1 contributes to the development of drug resistance and cell growth and survival phenotype in DLBCL. Inhibition of Trx-1 by small interfering RNA (siRNA) inhibited DLBCL cell line growth in vitro. Down-regulation of Trx-1 also sensitized lymphoma cells to doxorubicin-induced cell growth inhibition. A doxorubicin-resistant human DLBCL cell line (McA), that was highly sensitive to doxorubicin, has been selected by continuously exposing cells to gradually increasing doses of doxorubicin. The resistant phenotype has been retained for over 6 months despite growth in drug-free medium. Western blot analysis detected increased expression of the Trx-1 in the resistant subline. These results indicate that Trx-1 plays a key role in growth and survival, as well as chemoresistance in the pathophysiology of DLBCL. Based on these data, we plan to use small molecule inhibitors in vitro as a proof of principle for possible future human phase I studies. Disclosures: Ford: Cell Point: Research Funding.

Abstract Mantle cell lymphoma (MCL) is an aggressive histotype of B-cell non–Hodgkin lymphoma that has increased in incidence over the past few decades and is incurable, usually poorly responsive to standard chemotherapy combinations, and associated with poor prognoses. Discovering new therapeutic agents with low toxicity that produce better outcomes in patients with MCL is an ongoing challenge. Recent studies showed that degrasyn, a novel small-molecule inhibitor of the Janus kinase/signal transducer and activation of transcription (JAK/STAT) pathway, exerts antitumor activity in lymphoid tumors by inhibiting key growth and survival signaling (JAK/STAT) pathways. In the present study, we found that treatment of both typical and blastoid-variant MCL cells with degrasyn in combination with bortezomib resulted in synergistic growth inhibition and apoptosis induction in vitro. The apoptosis in these cells was correlated with the downregulation of constitutive NF-κB and phosphorylated STAT3 activation, leading to the inhibition of c-Myc, cyclin D1, and bcl-2 protein expression and the upregulation of bax protein expression. In vivo, degrasyn and bortezomib interacted to synergistically prevent tumor development and prolong survival durations in a xenotransplant severe combined immunodeficient mouse model of MCL. These findings suggest that agents such as degrasyn that can pharmacologically target constitutively expressed NF-κB and STAT3 in MCL cells may be useful therapeutic agents for MCL when administered together with bortezomib. Mol Cancer Ther; 9(7); 2026–36. ©2010 AACR.

This study investigated the activity of lenalidomide in patients with relapsed/refractory chronic lymphocytic leukemia (CLL). Lenalidomide was given at 10 mg daily with dose escalation up to 25 mg daily. Three patients (7%) achieved a complete response (CR), one a nodular partial remission, and 10 patients a partial remission (PR), for an overall response (OR) rate of 32%. Treatment with lenalidomide was associated with an OR rate of 31% in patients with 11q or 17p deletion, of 24% in patients with unmutated V-H, and of 25% in patients with fludarabine-refractory disease. The most common toxicity was myelosuppression, and the median daily dose of lenalidomide tolerated was 10 mg. Plasma levels of angiogenic factors, inflammatory cytokines, and cytokine receptors were measured at baseline, day 7, and day 28. There was a dramatic increase in median interleukin (IL)-6, IL-10, IL-2, and tumor necrosis factor receptor-1 levels on day 7, whereas no changes were observed in median vascular endothelial growth factor levels (20 patients studied). According to our experience, lenalidomide given as a continuous treatment has antitumor activity in heavily pretreated patients with CLL. This trial is registered at http://www.clinicaltrials.gov as no. NCT00267059.

Peptides derived from BCR-ABL have been used to trigger immune responses in CML pts. The native peptides may have low immunogenicity because they bind with low affinity to the major histocompatibility complex (MHC) molecules. Synthetic peptides containing mutations in the sequences of these peptides can bind with higher affinity than parental peptides and may generate a cross-reactive T cell response to the native sequence (known as a heteroclitic response). We used a mixture of heteroclitic and native peptides derived from both b3a2 and b2a2 sequences in a pilot study to vaccinate pts with CML in complete cytogenetic remission on imatinib (IM) therapy with stable bcr-abl transcript levels. Montanide ISA51 and GM-CSF were used as immunological adjuvants. Pts were required to have received IM for ≥12 mo with no change in dose for at least 6 mo and were not allowed any dose increases after registration. Pts were vaccinated with the peptides corresponding to their BCR-ABL breakpoint as follows: every 2 wks x4, then once 3 wks later, followed by 10 monthly vaccinations for a total of 15 vaccinations in 12 mo. After registration, pts had 3 additional measures of bcr-abl transcripts to better define baseline values. 8 pts have been vaccinated. At enrollment, pts had a median age of 45 yrs (range 29–63) and had been receiving IM for a median of 63 mo (range 35–68 mo) on a median dose of 600mg (range 300–800mg). Pts have been followed for a median of 15 wks (range 2–20) and have received a median of 7 vaccinations (range 1 to 8). To determine the effect of vaccination on immune leukocytes, we measured peripheral blood T-cell subsets, B, NK, and dendritic cells by FACS at baseline and at 3-month intervals thereafter. The percentage of myeloid DC (DC1) and plasmacytoid DC (DC2) cells were identified by the mutually exclusive expression of CD11c and CD123; Treg cells by the surface co-expression of CD3, CD4 and CD25 together with cytoplasmic expression of CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) and FoxP3 (forkhead/winged helix transcription factor). Vaccinations have been well tolerated. The only adverse event attributable to the vaccine is local site reactions grade 1 or 2 in 5 pts. Among the 5 pts who have had an evaluation at 3 mo from the 1st vaccination, 1 has achieved a major molecular response (bcr-abl/abl <0.05%). The Wilcoxon signed ranks test was used to assess changes in the immunophenotype of peripheral leukocytes of 5 pts evaluated for leukocyte FACS analyses at baseline and after 3 mo on study. Compared with baseline, there were significant decreases in the percentages of B (p = 0.042) and NKT (p = 0.043) cells, and significant increases in the percentages of DC2 (p = 0.043) and Treg (p = 0.043) cells. All pts continue receiving vaccination as planned. We conclude that vaccination with b2a2 and b3a2 synthetic peptides is well tolerated. Further follow-up is required to determine the immunogenecity and efficacy of the vaccine in this setting.