ALBERT

Description: Compelling evidences indicate a key role for regulatory T cells (Tregs) on the host response to cancer and recent studies indicated that the generation of effective WT1-specific cytotoxic T cells can be largely affected by the presence of Tregs. This is the first study to describe human Tregs generated specifically against the WT1 antigen which is overexpressed in several human leukemias and provide the mechanism by which these anti-WT1 Tregs inhibit the immune response in leukemia patients. We have generated T cell lines and clones that specifically recognized a WT1-84 peptide in an HLA DRB1*0402/TCR-Vb8-restricted manner. Importantly, they recognized HLADRB1* 04-matched fresh leukemic cells expressing the WT1 antigen. These clones exerted a Th2 cytokine profile, had a CD4+CD25+Foxp3+GITR+CD127− Tregs phenotype, and significantly inhibited the proliferative activity of allogeneic T cells independently of cell-contact. Priming of allo-reactive T cells in the presence of Tregs strongly inhibited the expansion of NK; NK-T and CD8+ T cells, had an inhibitory effect on NK/NK-T cytotoxic activity but not on CD8+ T cells. Furthermore, priming of T cells with the WT1- 126 HLA-A0201-restricted peptide in the presence of Tregs strongly inhibited the induction of anti-WT1-126 CD8+ CTL responses as evidenced by both very low cytotoxic activity and IFN-g production. Moreover, these Tregs clones specifically produced Granzyme-B and selectively induced apoptosis in WT1-84 pulsed-autologous APCs but not in apoptoticresistant DR4-matched leukemic cells. Importantly, we have also detected anti-WT1-84 IL-5+/Granzyme-B+/Foxp3+ CD4+ Tregs in 5 out of 8 HLA-DR4+ AML patients. These findings suggest a critical role for anti-WT1 Tregs in the inhibition of T cell responses against leukemia. This study may have important implications for the clinical manipulation of Tregs such as immuno-targeting of TCR-Vb-8 with mAbs to eliminate anti-WT1 Tregs in leukemia patients in order to enhance GVL before vaccination with the WT1 antigen. On the other hand, leukemia patients with GVHD should be clinically-tried for vaccination with the current WT1-84 peptide or adoptively-treated with ex-vivo anti-WT1 Treg cells to specifically enhance their frequency, which is known to be very low in these patients.

Description: Background Myelodysplastic syndrome (MDS) is a pre-leukemia disease affecting the erythroid, myeloid and megakaryocytic bone marrow production. MDS patients are classified according to the WHO classification of myeloid neoplasms. During the past 15 years management of MDS patients has been stratified according to the International Prognostic Scoring System (IPSS) risk score. Recently a revised version of IPSS has been introduced (IPSS-R). One quarter of LR-MDS in this new IPSS-R were reclassified as having a higher risk and a substantial subset of high risk-MDS (HR-MDS) were reclassified as lower risk. In LR-MDS a differentiation block is observed in the erythroid lineage. The diagnosis and follow up of cytopenias in particular anemia must be the main objective (1). The soluble transferrin receptor (sTfR) directly reflects the erythropoietic activity in individuals without iron defiency and may appreciate ineffective dysplastic erythropoiesis in LR-MDS. In LR-MDS there is also an inverse relationship between EPO level and the degree of anemia but a wide range of EPO levels is found in patients with similar Hb concentrations. Thus the highest EPO levels are found in patients with erythroid hypoplasia in bone marrow. Aims The combination of several biomarkers: Hb, ferritin, EPO and sTfR may be useful in LR-MDS for diagnosis and follow up. Methods A total of 192 patients with LR-MDS were investigated. Median age of the 192 patients was 71 years (21-92) with 56% males, median survival: 54 months, median follow up: 102 months. The stratification according to the WHO criteria and IPSS risk score was realized. Bone marrows were studied and cytogenetic assessment was realized in the same time. Serum concentrations of ferritin, EPO and sTfR has been analyzed by immuno-assays. Hb level was determined on Beckman Coulter apparatus. The follow up of Hb, ferritin, EPO and sTfR was realized every 2 months in patients with supportive care only until the first specific treatment. A multivariate logistic regression analysis to ascertain the correlations between disease progression and studied biological parameters was realized. Results The logistic regression analysis of our results is significant to define a biological evolutive profile of LR-MDS patients with these biomarkers. The combination of these routine parameters may represent a functional erythropoietic follow up in LR-MDS patients (table 1). This biological tool is an easy method to observe the red cell lineage of LR-MDS patients. This combination informs about the progressive ineffective and dysplatic erythropoiesis in LR-MDS patients. The measurement of ferritin which is a correlated parameter in LR-MDS shows the level of iron overload. A normal or high level without inflammation condition excludes an iron deficiency. The EPO level can give a predictive information about the future efficacy of ESA (endogenous EPO 〈 500 U/l). Conclusion With our results and a correlative logistic regression analysis, we can propose a biological scoring system to appreciate the evolutive anemia of LR-MDS progression in patients. In LR-MDS the management of patients may be based on personalized medicine according a risk assessment with IPSS-R, cytogenetics, mutations and HLA typing (2). But an additional biological and functional predictive scoring system informs about the important independent role of dysplasias particularly anemia in LR-MDS patients before to choose a suitable therapy: transfusions, iron chelation, ESA, TGF-ï¢ pathway inhibitors, G-CSF, immun suppressive treatment, lenalidomide, azacytidine, allogeneic HSCT Table 1. Hb ± EPO ±  sTfRDysplastic erythropoiesis without anemia Hb ±  EPO  sTfRStabilized dysplastic erythropoiesis Hb  EPO  sTfRUnstabilized dysplactic erythropoiesis Hb  EPO  sTfRIneffective dysplastic erythropoiesis EPO 〈 500 U/l : ESA may be efficient〉 500 U/l : ESA will be inefficientFerritin level : iron overload References Giagounidis A Management of low-risk myelodysplastic syndromes Hematology Education, 2015, 9 (1), 219-225 Platzbecker U et al Personalized medicine in myelodysplastic syndromes: wishful thinking or already clinical reality? Hematologica, 2015, 100 (5), 568-571 Disclosures No relevant conflicts of interest to declare.

Description: An obstacle with continued clinical development of CAR T cells is the limited understanding of their biology and mechanisms of anti-tumor immunity. We and others have shown that CARs with a CD28 co-stimulatory domain drive high levels of T cell activation that also lead to exhaustion and shortened persistence. The CD28 domain includes 3 intracellular subdomains (YMNM, PRRP, and PYAP) that regulate signaling pathways post TCR-stimulation, but it is unknown how they modulate activation and/or exhaustion of CAR T cells. A detailed understanding of the mechanism of CD28-dependent exhaustion in CAR T cells will allow the design of a CAR less prone to exhaustion and reduce relapse rates. This led us to hypothesize that by incorporating null mutations of CD28 subdomains (Fig 1A) we could optimize CAR T cell signaling and reduce exhaustion. In vitro, we found mutated CAR T cells with only a functional PYAP (mut06) subdomain secrete significantly less IFNγ, IL6, and TNFα after 24hr stimulation compared to non-mutated CD28 CAR T cells, but greater than the 1st generation m19z CAR. Also, cytotoxicity was enhanced compared to non-mutated CARs (Fig 1B). Using a pre-clinical immunocompetent mouse tumor model, we found the mut06 CAR T cell treated mice had a significant survival advantage compared to non-mutated CD28 CAR T cells (Fig 1C). To examine exhaustion, we ex vivo stimulated CAR T cells with target cells expressing CD19 and PDL1 and found mut06 CAR T cells had increased IFNγ (42%), TNFα (62%) and IL2 (73%) secretion compared to exhausted non-mutated CD28 CAR T cells. This suggests that mut06 CAR T cells are more resistant to exhaustion. To find a mechanistic explanation for this observation we examined CAR T cell signaling. After 24hr stimulation with CD19 target cells mut06 CAR T cells had a significant reduction in pAkt compared to m1928z CAR T cells, which is a critical signaling mediator in the NFAT and NR4A1 transcription factor pathways. Additionally, mut06 had decreased p-NFAT compared to m1928z when examined by western blot. To determine how optimized CAR signaling affected T cell exhaustion we looked at 22 genes that are upregulated when NFAT is constitutively active and overlap with genes identified as important for T cell exhaustion. We found that most of the exhaustion related genes were upregulated in m1928z CAR T cells while they were decreased in m19hBBz. The mut06 CAR T cell gene expression pattern was more similar to m19hBBz with exhaustion related genes downregulated compared to m1928z (Fig 1D). To examine differences in the accessibility of exhaustion related genes we performed ATAC-seq and found NFAT (Nfatc1) and NR4A2 (Nr4a2) had lower chromatin accessibility profiles in mut06 compared to m1928z (Fig 1E). We also found that exhaustion related genes Havcr2 (TIM3), Pdcd1 (PD1), and Lag3 (LAG3) all had greatly reduced chromatin accessibility in mut06 CAR T cells compared m1928z. Overall, these genomic studies support our findings that mut06 optimizes CAR T cell signaling by lowering transcription factors that regulate exhaustion. Figure 1 Disclosures Li: ImmuneBro Therapeutics: Other: sole shareholder . Davila:Atara: Research Funding; Celgene: Research Funding; GlaxoSmithKline: Consultancy; Novartis: Research Funding; Anixa: Consultancy; Bellicum: Consultancy; Adaptive: Consultancy; Precision Biosciences: Consultancy.

Description: Epstein-Barr Virus (EBV)+ lymphomas are an important subgroup of aggressive malignant lymphomas which include lymphomas in the post-transplantation setting, Burkitt’s lymphomas (BL), AIDS-related lymphomas (ARL), and some forms of Hodgkin, T-cell, and natural killer (NK) cell lymphomas. EBV is a member of the herpes virus family, characterized by their ability to support two different life cycles: the productive “lytic” cycle leading to the production and release of new virions and the non-productive “latent” cycle. Most lymphoma cells are infected with latent EBV, and only few viral genes are expressed at low levels. Several groups of broad-acting chemical agents are able to reactivate EBV and induce herpes thymidine kinase (TK) expression in vitro and in vivo. NF-kB has been described to play a critical role in regulating the balance between latency and lytic replication of EBV. Therefore, we hypothesized that the proteasome inhibitor Bortezomib can be used to initiate EBV lytic antigen expression in EBV-related malignancies enabling the antiviral drug Ganciclovir to kill EBV+ lymphoma cells. The human cell line HR-1, derived from a Burkitt’s lymphoma and latently infected with EBV, was cultured in the presence of 50nM bortezomib for 24 hrs. The immediate early lytic phase EBV antigens ZEBRA and RTA were induced and expressed as measured by flow cytometry. The EBV-VCA and EBV-R antigens were not expressed in untreated controls but were induced as demonstrated by western blot analysis, indicating the switch to the lytic life-cycle of EBV. These results were successfully repeated using the EBV+ Akata cell line. Induction of viral thymidine kinase (vTK) was shown by QRT-PCR as well. Histone deacetylase inhibitors are a well known group of broad-acting chemical agents able to reactivate EBV. In combination experiments, we found that Bortezomib plus sodium butyrate or SAHA act at least additive in inducing the EBV lytic life cycle in HR-1 or RAJI cells. Bortezomib sensitizes the EBV+ Akata cell line 2A8-1 to growth inhibitory effects of ganciclovir as shown by MTT assays. The cells were treated with two different non-toxic drug concentrations which were chosen low enough not to induce apoptosis (bortezomib: 1nM and 2nM; ganciclovir:15 and 30μM). Bortezomib induces the lytic EBV life cycle in vivo. In murine xenograft models growing the Akata A.15 line subcutaneously bortezomib induces the immediate-early protein ZEBRA as shown by immunohistochemistry and vTK as shown by QRT-PCR. Experiments to induce lytic phase EBV in murine xenograft models using the Akata cell line and to combine EBV induction with the nucleoside analogue ganciclovir are in progress. Murine studies with EBV-transformed lymphoblastoid cell line (LCL) xenograft models, combination bortezomib plus ganciclovir, and molecular imaging with FHBG specific PET probes are in progress. Reactivating EBV with proteasome inhibitors alone or in combinations with low concentrations of histone deacetylase inhibitors may be a less toxic therapeutic strategy for EBV-associated lymphomas.

Description: Enhanced angiogenesis is a hallmark of cancer. Pleiotrophin (PTN) is an angiogenic factor that is produced by many different human cancers and stimulates tumor blood vessel formation when it is expressed in malignant cancer cells. Recent studies show that monocytes may give rise to vascular endothelium. In these studies, we show that PTN combined with macrophage colony-stimulating factor (M-CSF) induces expression of vascular endothelial cell (VEC) genes and proteins in human monocyte cell lines and monocytes from human peripheral blood (PB). Monocytes induce VEC gene expression and develop tube-like structures when they are exposed to serum or cultured with bone marrow (BM) from patients with multiple myeloma (MM) that express PTN, effects specifically blocked with antiPTN antibodies. When coinjected with human MM cells into severe combined immunodeficient (SCID) mice, green fluorescent protein (GFP)–marked human monocytes were found incorporated into tumor blood vessels and expressed human VEC protein markers and genes that were blocked by anti-PTN antibody. Our results suggest that vasculogenesis in human MM may develop from tumoral production of PTN, which orchestrates the transdifferentiation of monocytes into VECs.

Description: BACKGROUND: LGL leukemia is a neoplasm arising from either CD3+ T-cells or CD3− NK-cells. Autoimmune-mediated anemia, neutropenia, and rheumatoid arthritis occur frequently in these patients and immunosuppressive agents are used for these associated clinical syndromes. In our previous studies, we found that patients display a constitutively activated Ras and MAPK/ERK signaling cascade that may drive leukemia survival. A multicenter phase 2 clinical trial was initiated to treat LGL leukemia patients with the farnysltransferase-inhibitor R115777 (tipifarnib, Zarnestra®, Johnson & Johnson) that inhibits Ras and other farnesylated proteins. One of the goals of this study was to determine the shifts in cytokine production during therapy. We found that LGL cells treated with tipifarnib in vitro displayed a switch to Th2 (IL-4 and IL-10)-polarized differentiation. After tipifarnib treatment of LGL patients, antigen-activated T-cells produced greater amounts of Th2 (IL4/IL-10) cytokines but less Th1 (IFNγ/TNFα). In this study, we determined the mechanism governing tipifarnib-mediated Th2 polarization in T-cells. METHODS: PBMCs were isolated from 10 healthy donors and from seven patients with T-LGL leukemia before and after treatment with tipifarnib 300 mg twice daily for 21 days of a 28- day cycle. LGL leukemia patients had increased numbers of αβ T lymphocytes and evidence of clonality in association with either neutropenia or transfusion-dependent anemia. Th1 and Th2 cytokines were determined by intracellular staining and flow cytometry after activation with anti-CD3 plus anti-CD28. In some experiments, Th1 polarization was induced by IL-12; whereas, Th2 was induced by IL-4. Expression of T-bet and GATA-3 transcription factors that regulate Th1 and Th2 polarization, respectively, phosphorylated (active) MAPK (ERK1 and ERK2), and total MAPK were measured by Western blots. FTI2153, tipifarnib, and geranylgeranyl transferase inhibitor(GGTI)-2417 were used compared to DMSO control. RESULTS: PBMCs from patients with T-LGL leukemia displayed a dose and time-dependent increase in IL-4 and IL-10 production after drug treatment (average increase to 100% and 43%, respectively). A dose-dependent increase in these Th2 cytokines in T-cells from healthy donors showed that the farnesylated protein targeted by tipifarnib was not selectively expressed in LGL leukemia. Culture with IL-12 induced Th1 differentiation associated with ERK phosphorylation and increased T-bet expression. Pre-treatment with tipifarnib and FTI2153 but not GGTI2417 prior to IL-12 inhibited T-bet induction with no change in anti-CD3-induced MAPK leading to enhanced IL-4 signaling and greater Th2 polarization. CONCLUSIONS: Our data reveal unique, previously unreported effects of FTIs on cytokine signaling in T-cells by inhibiting the induction of T-bet and blocking Th1 differentiation. These results are critical to determine the mechanism of action of tipifarnib in LGL leukemia and suggest that FTIs may be useful for autoimmune or lymphocyte-mediated disorders associated with excessive Th1 cytokine production.

Description: Patients with deficiency in ferrochelatase (FECH), the last enzyme of the heme biosynthetic pathway, experience a painful type of skin photosensitivity called erythropoietic protoporphyria (EPP), which is caused by the excessive production of protoporphyrin IX (PPIX) by erythrocytes. Controversial results have been reported regarding hematologic status and iron status of patients with EPP. We thoroughly explored these parameters in Fechm1Pas mutant mice of 3 different genetic backgrounds. FECH deficiency induced microcytic hypochromic anemia without ringed sideroblasts, little or no hemolysis, and no erythroid hyperplasia. Serum iron, ferritin, hepcidin mRNA, and Dcytb levels were normal. The homozygous Fechm1Pas mutant involved no tissue iron deficiency but showed a clear-cut redistribution of iron stores from peripheral tissues to the spleen, with a concomitant 2- to 3-fold increase in transferrin expression at the mRNA and the protein levels. Erythrocyte PPIX levels strongly correlated with serum transferrin levels. At all stages of differentiation in our study, transferrin receptor expression in bone marrow erythroid cells in Fechm1Pas was normal in mutant mice but not in patients with iron-deficiency anemia. Based on these observations, we suggest that oral iron therapy is not the therapy of choice for patients with EPP and that the PPIX–liver transferrin pathway plays a role in the orchestration of iron distribution between peripheral iron stores, the spleen, and the bone marrow.

Description: Background: Biologic prognostic markers in diffuse large B-cell lymphoma (DLBCL) have focused on putative cell of origin (germinal center (GC) vs. activated B-cell (ABC)), apoptosis, and proliferation. Such markers, shown to be predictive in CHOP-treated patients, are being validated with rituximab(R)-CHOP. CALGB 50103 is a phase 2 trial of dose adjusted etoposide, vincristine, doxorubicin, cyclophosphamide, prednisone, R (DA-EPOCH-R) therapy for DLBCL. We studied CD10, BCL6, MUM1, LMO2, BCL2, and Ki67 in CALGB 50103 to determine which markers had prognostic significance. Methods: Prospectively procured slides were stained with appropriate primary antibodies at the Pathology Coordinating Office of the CALGB (CD10, BCL6, MUM1, BCL2, Ki67) or the Cleveland Clinic (LMO2). Slides were scored independently in 10% increments by two pathologists, with a third review in case of disagreement of 〉20% for all stains (mean score used as final value) except Ki67, for which image analysis (IA, Aperio, Scanscope) and a visual estimate (quartiles) was used. A 30% cutoff was used for CD10, BCL6, and MUM1. 40% was used for BCL2 and 60% for Ki67. Progression-free and event-free survival (PFS, EFS) served as the endpoints. Results: Data for at least one of the markers were available for 53 of the 75 treated patients. The median age was 56 years (range 23 – 80) and the median follow-up for the 45 patients who are still alive is 4.5 years (range 3.2–6.0). The international prognostic index (IPI) was available in 51 patients and 33 had an IPI score of 2 while 18 had scores of 3 or 4. 11 patients had progressive disease and there were 14 treatment failures. 8 patie0nts have died. Table 1 shows the significant predictors of outcome. Table 1: Predictors of PFS and EFS in CALGB 50103 PFS EFS Variable No. (%) 2 yr survival prob (95% CI) 2-sided p-value 2 yr survival prob (95% CI) 2-sided p-value CI = confidence interval CD10 0.057 0.030 〈 30% 39 (76.5) 0.76 (0.59 – 0.87) 0.74 (0.58 –0.85) ≥ 30% 12 (23.5) 1.00 (---) 1.00 (---) Ki67 IA 0.028 0.045 〈 60% 32 (64.0) 0.87 (0.69 – 0.95 ) 0.84 (0.66 – 0.93) ≥ 60% 18 (36.0) 0.67 (0.40 – 0.83) 0.67 (0.40 – 0.83) IPI 0.008 0.003 2 33 (64.7) 0.91 (0.74 – 0.97) 0.87 (0.71 – 0.95) 3–4 18 (35.3) 0.65 (0.38 –0.82) 0.61 (0.35 – 0.79) Of the cell of origin markers (CD10, BCL6, MUM1, LMO2) only CD10 negativity was associated with an inferior PFS and EFS. GC vs. non-GC phenotype (as per Hans et al Blood 2004) and LMO2 were not associated with outcome. High Ki67% by IA was also associated with inferior PFS and EFS. Similar results were seen with Ki67 visual estimates (〉75% cutoff, P60% hazard ratio [HR] 7.1, P=.007; IPI 3/4 HR 7.2, P=.006) and EFS (Ki67〉60% HR 5.3, P=.007; IPI 3/4 HR 7.1, P=.002). The multivariate analysis with CD10 and IPI was not possible because there are no events in CD10+ cases. Conclusions: Lack of CD10 (suggesting non-GC origin) and high Ki67 are associated with poor outcome in DA-EPOCH-R treated patients, further validating the concept of cell of origin and proliferation as predictors of outcome in DLBCL. BCL2 remains an important predictor of outcome in patients that lack the GC marker CD10, even in rituximab treated patients.

Description: Programmed Death Ligand 1 (PD-L1) is expressed on antigen presenting cells and serves to inhibit activation of T cells through its receptor, Programmed Death 1 (PD-1). PD-L1 is aberrantly expressed on epithelial malignancies and may prevent an effective host antitumor immune response. However, the pattern of expression and function of PD-L1 in non-Hodgkin lymphoma (NHL) is largely unknown. We examined 77 primary NHL tissue specimens and 16 NHL cell lines for expression of PD-L1. PD-L1 was detected by immunohistochemical staining in all 14 anaplastic large cell lymphoma (ALCL) specimens (both ALK+ and ALK-), and in 18 of 22 diffuse large B cell lymphomas (DLBCL). PD-L1 was expressed in all cases of Hodgkin’s, mediastinal B cell, and grey zone lymphoma, 50% of peripheral T cell lymphomas, 15% of follicular lymphomas, and 33% of mantle cell lymphomas. Among DLBCL cases subtyped into germinal-center B (GCB) and non-GCB using CD10, BCL-6, and MUM-1 (Hans et al, Blood2004;103:275), 8 of 12 (66%) GCB cases expressed PD-L1, compared to 10 of 11 (91%) of non-GCB cases (p=0.31). Among NHL cell lines, all 9 B cell lines were negative for PD-L1, as measured by flow cytometry, but all 5 ALCL cell lines were strongly positive. Therefore, we chose to focus on ALCL to study the role of PD-L1 in modulating anti-lymphoma T cell activity. PD-L1 expressed by ALCL was biologically active in that proliferation of allogeneic T cells co-cultured with Karpas 299 ALCL cells was significantly enhanced by blocking PD-L1 with monoclonal antibodies. To further study tumor-T cell interactions, malignant ascites from a patient with newly diagnosed ALK+ ALCL, containing approximately equivalent proportions of PD-L1-expressing tumor cells and tumor-associated T cells, was used as an autologous system. Secretion of interferon-γ after 3- or 5-day incubation of the T cell/tumor mixture with phytohemagglutinin (a polyclonal T cell activator) was increased four-fold in the presence of anti-PD-L1 antibody, as compared with control antibody or media alone. Secretion of other inflammatory cytokines, including IL-1, TNFα, IL-5, IL-13, and MIP1α, was also markedly increased with the addition of anti-PD-L1 antibody to the mixture of tumor cells and lymphocytes. In conclusion, PD-L1 is expressed in a broad array of non-Hodgkin lymphomas, particularly in ALCL. Blockade of tumor-associated PD-L1 promoted activation of adjacent T cells. PD-L1 may play a role in thwarting an effective antitumor immune response, and thus represents an attractive target for lymphoma immunotherapy using anti-PD-L1 or anti-PD-1 monoclonal antibodies. Heterogeneity of PD-L1 expression among DLBCLs may be related to GCB subtype or other biological properties, and these associations are being explored in a larger set of DLBCL cases. Table 1. PD-L1 expression by lymphoma subtype. Subtype Number PD-L1+ % Anaplastic large cell lymphoma 14 14 100% Diffuse large B cell lymphoma 22 18 82% T cell rich DLBCL 7 4 57% Follicular lymphoma 13 2 15% Mantle cell lymphoma 6 2 33% Mediastinal DLBCL 2 2 100% Hodgkin lymphoma 5 5 100% Grey zone lymphoma 1 1 100% Peripheral T cell lymphoma 7 4 57%

Description: Combination therapy utilizing 2 novel agents with independent mechanisms of action and non-overlapping toxicities may be useful in the setting of advanced cancers. Tipifarnib (T) is an orally bioavailable farnesyltranferase inhibitor with documented single-agent activity in acute myeloid leukemia (AML). Bortezomib (B) is a broad inhibitor of proteasomal function, approved for treatment in multiple myeloma and mantle cell lymphoma. Preclinical studies indicated synergistic activity between these 2 agents for eliciting apoptosis within leukemia and myeloma cell lines and ex-vivo cells adhered to fibronectin. In this phase I combination trial, we studied the effect of combined effect of T plus B in patients with advanced acute leukemias. Objectives: The primary endpoint was toxicity assessment. Secondary endpoints included effect of combined therapy on signaling intermediates, including p-AKT, Bim, Bax, and NF-kB, as well as effects on farnesyltransferase (FT) and the proteasome activity. Eligibility: Patients with AML, ALL, or CML-BC who had received 〈 3 cycles of prior therapy were eligible. Methods: Patients received T on days 1–14 and B on days 1, 4, 8, and 11. Cycles were repeated every 21 days. Dose escalation occurred using cohorts of 3–6 patients. The starting dose was T: 300 mg/m2 and B: 1.0 mg/m2 Bone marrow aspirate was obtained at baseline, day 8, and between day 15 and the start of the next cycle. Measurement of signaling intermediates were performed in Ficoll-enriched leukemic marrow blasts using Western Blot (p-AKT, Bax, Bim) and ELISA (NF-kB). FT and proteasomal activity were directly measured within peripheral blood mononuclear cells (PBMC) using previously described methods. Results: To date, 27 patients have been enrolled at 3 centers. Four patients were ineligible after screening, and 23 patients have been treated. Median age was 69 years (range 48–84) Diagnosis: AML=25, ALL=1, MDS=1. Accrual to the 4th and final dosing cohort has occurred, without maximum tolerated dose being reached at the 4th and final planned dosing cohort (T: 600 mg/m2 and B: 1.3 mg/m2). Six patients received ≥ 2 cycles of treatment. Dose-limiting toxicities to date have included: nausea/diarrhea (1 patient), sensory neuropathy (1 patient), and fatigue (1 patient). Common drug-related (〉 10%) non dose-limiting toxicities included: infection/febrile neutropenia, diarrhea, nausea, vomiting, sensory neuropathy, and fatigue, most of which were grade 1 or 2. FTase inhibition within peripheral blood mononuclear cells (PBMC) was measured serially in 8 patients to date, with a median of 70% inhibition by day 8, and with 5 out of 6 evaluable patients having sustained inhibition at day 22. Proteasome function within PBMCs was reduced by a median of 44.3% in 7 patient samples pre-infusion and 1 hour post-infusion on day 8. Proteasome activity within PBMCs at day 22 was decreased from baseline in 5 out of 7 patient samples tested. Compared to baseline, NF-kB binding activity within leukemic blasts at day 8 was decreased by a median of 39% at in 10 out of 14 paired samples. No significant change in expression of p-AKT, Bax, or Bim, as measured by Western Blot, was detected at day 8. Two patients achieved clinical response; 1 patient had a complete response and another patient had complete response with incomplete count recovery. Four others had stable disease following cycle 1. Conclusion: combined therapy with T + B was well tolerated and demonstrated inhibition of several relevant target signals within leukemic blasts and PBMCs. In addition, clinical activity was seen in 2 patients to date. Accrual to the trial is ongoing and updated clinical and pharmacodynamic data will be presented.