ALBERT

Description: Abstract 564 Insertional oncogenesis poses a severe hurdle to current gene therapy for blood disorders. The semi-random insertion of retroviral vectors combined with the inability for prolonged ex vivo culture of hematopoietic stem cells (HSCs) prohibits prospective integration site selection. The advent of induced pluripotent stem cells (iPSCs) offers for the first time the possibility of generating patient-specific stem cells that can be extensively manipulated in vitro, thus creating a unique platform for precise genetic engineering. Here, we present a novel strategy for the genetic correction of ß-thalassemia major based on the identification and selection of patient-specific iPSC clones harboring a normal ß-globin gene integrated in genomic “safe harbor” sites that permit therapeutic levels of expression while minimizing the possibility of oncogenic risks. We generated a total of 20 iPSC lines from bone marrow stromal cells or skin fibroblasts from 4 patients with ß-thalassemia major of various genotypes using our traceable lentiviral vector system (Papapetrou et al., PNAS, 2009; Lee et al., Nature, 2009), as well as an excisable single polycistronic vector co-expressing OCT4, SOX2, KLF4 and cMYC. Additional transgene-free thal-iPSC lines were generated following Cre recombinase-mediated excision of the reprogramming vector. Seven thal-iPSC lines were selected for further characterization and were shown to fulfill multiple criteria of pluripotency, including teratoma formation. Four thal-iPSC lines were transduced with a lentiviral vector encoding the human ß-globin gene cis-linked to its hypersensitive site (HS) 2, HS3 and HS4 locus control region elements, derived from the TNS9 vector, previously shown to confer erythroid-specific ß-globin gene expression at therapeutic levels (May et al., Nature, 2000). Thal-iPSC clones harboring single vector copies were selected and vector integration sites were mapped to the human genome. To identify safe harbor sites we adopted a set of 5 criteria: (1) distance of at least 50 kb from the 5' end of any gene, (2) distance of at least 300 kb from any cancer-related gene, (3) distance of at least 300 kb from any miRNA, (4) location outside a transcription unit and (5) location outside ultraconserved regions. A survey of 5840 integration sites of the globin lentiviral vector that we mapped in thal-iPSCs revealed that 17.3% meet all five “safe harbor” criteria, supporting the feasibility of recovering thal-iPSC clones harboring vector integrations in “safe harbors” by screening a relatively small set of single-copy clones. Indeed, 3 “safe harbor” integrations were retrieved amongst 36 sites mapped in thal-iPSC clones. Among 13 clones randomly selected and thoroughly confirmed to harbor a single copy of the globin vector, we found one clone, thal5.10-2, with an integration that meets all five “safe harbor” criteria. Upon erythroid differentiation, 12 of the 13 single copy thal-iPSC clones expressed detectable vector-encoded ß-globin at levels ranging from 9% to 159% (mean 53%) of a normal endogenous ß-globin allele. 9 out of 13 clones expressed the ß-globin transgene at levels higher than 30%. Remarkably, the “safe harbor” clone 5.10-2, expressed 85% of a normal ß-globin allele. Microarray analysis of undifferentiated thal-iPSC clones and their erythroid progeny revealed that 3 out of 5 integrations eliminated by our “safe harbor” criteria result in perturbed expression of neighboring genes at a distance ranging between 9 and 275 kb from the vector insertion. Of note, the “safe harbor' integration site in clone 5.10-2 is in a genomic region with no genes within 300 kb on either side, while no significant differentially expressed genes were found elsewhere in the genome. These data demonstrate that the selection of iPSC clones, wherein therapeutic levels of transgene expression without perturbation of endogenous genes is obtained from selected chromosomal sites is feasible by screening a limited number of single-copy clones and applying five “safe harbor” criteria. This study provides a framework and a strategy combining bioinformatics and functional analyses for identifying “safe harbors” for transgene integration and expression in the human genome. This approach may be broadly applicable to introducing therapeutic, suicide or marker genes into patient-specific iPSCs towards the development of safer stem cell therapies. Disclosures: No relevant conflicts of interest to declare.

Description: It has been recognized that dysfunction of CB immune system is in part due to the immaturity of CB cellular immunity (Cairo, Blood,1997). The molecular mechanisms associated with the immaturity of CB cellular immunity including DC subset remain to be defined. The maturation status of DC greatly influences its antigen presentation capacity. Recently, we have utilized oligonucleotide microarray to demonstrate differential gene expression profiles of CB vs APB Mo (Jiang/Cairo, JI, 2004). In the current study, differential expressed genes and proteins were examined in Mo-derived CB vs. APB DC during DC developmental stages: Mo, immature DC (iDC) and mDC, by utilizing oligonucleotide microarray and proteomics. Briefly, Mo isolated from CB or APB and cultured for 8 days with GM-CSF/IL-4 (iDC) and further stimulated with LPS (mDC). Oligonucleotide microarray was carried out using U133A gene chip (Affymetrix). The representative differentially expressed genes resulted from microarray analysis were selected and analyzed by quantitative RT-PCR (Roche). The proteomic technique was conducted by liquid chromatography (LC) and mass spectrometry (MS) (Lim, Mol Cell Proteomics, 2006). The differentially expressed proteins were compared in CB vs. APB for iDC and mDC. We identified different gene expression patterns that were significantly lower in CB vs. APB in different stages during DC differentiation: Mo, iDC and mDC. These differentially expressed genes included RELA (5F), JUNB (6F), IRF-1 (3F) in Mo; CREB5 (3F), MAP7 (5F), IL1R2 (6F) in iDC; and HLA-DQA1 (4F), CD80 (3F), IRF-5 (3F) in mDC. The proteomic studies demonstrated Tyrosine Kinase Fer (12.5F), Actin regulator 3 (2.5F), Rap guanine nucleotide exchange factor 1 (2.4F) and Myeloid cell nuclear differentiation antigen (1.5F) were expressed higher in APB vs.CB iDC, while MAX binding protein MNT (5.5F), IRS2 (2.2F) and Zinc-Finger Proteins (514, 212, 462) (3–14F) were expressed higher in CB vs. APB iDC. Further, the proteomic results also indicated other Zinc-Finger Proteins (292, 221, 474) (2–5F), Fibrillin 1 precursor (2.5F) and interleukin-4 (7.7F) were expressed higher in APB vs. CB mDC. In contrast, cyclin I (3F), Rb-like protein 2 (4.35 F) and PKC theta (2F) were significantly lower in APB vs. CB DC. Moreover, the comparison of CB vs. APB DC antigen presenting activity by ELISPOT was performed and the influenza-peptide loaded CB-mDC demonstrated weaker ability to induce T cells to produce IFNg compared with APB-mDC. In summary, these differentially expressed genes in Mo (RELA, JUN) may play key roles in initiating Mo differentiation toward DC. The increased expression of genes in APB vs. CB iDC, like CREB5, IL1R2, may be involved in mediating maturation process of iDC to mDC. Lastly, the elevated expression of genes in APB vs. CB mDC, such as HLA-DQA1, CD80, IRF5 among others, may be likely to control mDC functionality as demonstrated by weaker antigen presenting activity of CB vs. APB mDC. We postulate that decreased expression of specific genes in CB vs. APB DC during DC developmental stages may in part be responsible for the lack of maturity of CB, and ultimately may partially be responsible for differential CB vs. APB innate and adaptive immunity.

Description: CD56+ NK subsets exhibit differential receptor profiles including killer-Ig-like receptors (KIR), C-lectin (NKG2) and natural cytotoxicity receptors involved with tumor target recognition which may play a role in ACI for malignancies (Farag et al, Blood 2002). UCB is limited by the absence of available donor effector cells (NK, CTL, LAK and NKT cells) for infusion after transplantation for the treatment of minimal residual resistant hematological relapse and/or PTLD (Barker et al, Blood 2001; Locatelli, et al Blood 1999). We have demonstrated the ability to ex-vivo expand (EVE) CB in short term culture (48hrs) with IL-2, IL-7, IL-12 and anti-CD3 (AB/CY) cryopreserved, thawed, recryopreserved, rethawed and ex vivo expanded (CTCTE) with a significant increase in CD3−/16+/56+ bright/dim subsets expressing KIR3DL1, KIR2DL1/S1, KIR2/DL2, CD94/NKG2A (Ayello/Cairo et al, BBMT 25a 2004). Timeus et al (Hematologica 2003) demonstrated the ability to sequentially freeze-thaw UCB twice without a detrimental effect on UCB clonogenic potential or viability (Hematologica 2003). In this study we compared previous short term (48hr) cultures with prolonged cultures (48–240hrs) on expansion, maturation, NK and LAK cytolytic ability, KIR inhibitory and C-lectin NK subsets in CTCTE UCB utilizing the same AB/CY cocktail. UCB were cryopreserevd and thawed by the NHBLI/COBLT method (Fraser/Cairo et al, J Hemat 1998). After rethawing, nonadherent UCB cells were cultured in serum-free media alone or with anti-CD3 (50 ng/ml), IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) [ABCY] for 4 time periods: 48, 96, 168 and 240 hrs. NK subsets (CD94+, CD16+, CD56+) and NK receptor (KIR3DL1, KIR2DL1/S1, KIR2DL2 and NKG2a) expression were analyzed by flow cytometry using CD3, CD16, CD56, NKBL1, CD158a, CD158b, CD94 and NKG2a mAbs. Additionally, NK and LAK cytotoxicity was measured by europium release assay. There was a significant increase in NKT (CD3+/16+/56+) UCB with AB/CY from 48–240 hrs vs media (2.97±.3 vs 66.7±7.1 vs 35.8±5.9%, p

Description: Abstract 3928 Background: NK cells play a role in reducing relapse in hematological malignancy following AlloSCT (Dunbar et al, Haematologica, 2008). NK cell limitations include lack of tumor recognition and/or limited numbers of viable and functional NK cells (Shereck/Cairo et al, Ped Bld Can, 2007). NK ACI provide safe and effective therapy against tumor relapse; yet NK cells are limited to specific cancer types and not all patients demonstrate optimal response (Ruggieri et al. Science, 2002; Ljunggren et al. Nat Rev Immuno, 2007). To circumvent these limitations, methods to expand and activate PBMNCs with genetically engineered K562 cells expressing membrane bound IL-15 and 41BB ligand (K562-mbIL15-41BBL [modK562]; Imai/Campana et al, Blood, 2005) have shown to significantly increase NK cells in number and maintain heterogeneous KIR expression (Fusaki/Campana et al BJH, 2009). We have shown that CB NK cells can be activated/expanded and exhibit enhanced cytolytic activity when cultured in a cytokines/antibody cocktail (Ayello/Cairo et al, BBMT, 2006; Exp Heme, 2009). Objective: To evaluate CBNK expansion, activation, cytolytic mechanism and function against Burkitt lymphoma (BL) tumor target and its influence on NK cell mediated in-vitro and in-vivo cytotoxicity in NOD-SCID mice following stimulation with modK562 cells (generously supplied by D.Campana, St Jude's Children's Hospital, Memphis, Tx). Methods: Following 100GY irradiation, modK562cells were incubated 1:1 with CBMNCs in RPMI+IL-2 (10IU/ml) for 7 days in 5%CO2, 37°C. NK activation marker (LAMP-1), perforin and granzyme B were determined by flow cytometry. Cytotoxicty was determined via europium assay at 20:1 E:T ratio with Ramos (BL) tumor targets (ATCC). The mammalian expression construct (ffLucZeo-pcDNA (generously supplied by L.Cooper, MD, PhD) was transfected to BL cells using lipofectin and selected by zeocin for stable transfection. Six week old NOD-SCID mice received 5×106 BL cells subcutaneously. Upon engraftment, xenografted NOD-SCID mice were divided in 5 groups: injected with PBS (control), BL only, 5×106 wildtype (WT) K562 expanded (E) CBNK cells, modK562 expanded (E) CB NK cells (5×106) and modK562 expanded (E) CBNK cells (5×107). Ex-vivo ECBNK cells were injected weekly for 5 weeks and xenografted NOD-SCID mice were monitored by volumetric measurement of tumor size (Tomayko/Reynolds, Can Chemother Pharmac, 1989), bioluminescent imaging (Inoue et al Exp Heme, 2007) and survival. The survival distribution for each group was estimated using the Fisher exact test. Results: On Day 0, NK cells (CD56+/3-) population was 3.9±1.3%. After 7 days, modK562 expanded CBNK cells was significantly increased compared to WTK562 and media alone (72±3.9 vs 43±5.9 vs 9±2.4%, p

Description: The BET (bromodomain and extraterminal) protein family members including BRD4 bind to acetylated lysines on the histone proteins, help assemble transcriptional regulators at the target gene promoters and enhancers, and regulate the expression of important oncogenes, e.g., MYC and BCL-2. Here we determined the effect of histone deacetylase (HDAC) inhibitor panobinostat (PS) on sensitizing human AML blast progenitor cells (BPCs) to the BET protein inhibitor JQ1. Treatment with JQ1, but not its inactive enantiomer (R-JQ1), dose-dependently increased the % of cells in the G1 phase and reduced the % of S phase cells, while concomitantly inducing apoptosis in the AML BPCs expressing mutant NPM1c+ (OCI-AML3 cells) or with the ectopic co-expression of FLT3-ITD (OCI-AML3/FLT3-ITD cells), or in AML cells expressing MLL fusion oncoprotein (MV4-11 and MOLM13 cells). JQ1 was equally effective in inducing apoptosis of OCI-AML3 and OCI-AML3/FLT3-ITD cells. JQ1 treatment inhibited the clonogenic survival of OCI-AML3 more than of MOLM13 cells. Treatment with JQ1 also dose-dependently exerted lethal anti-leukemia effects against 10 primary CD34+ AML cell samples. JQ1 treatment reduced binding of BRD4 and RNA polymerase II to the DNA of MYC and BCL2. The heat map from the gene expression microarray profile in OCI-AML3 cells demonstrated that JQ1 treatment downregulated the mRNA expression of more genes, as compared to the number of genes whose mRNA expression was up regulated by JQ1 treatment. Total RNA from the untreated and JQ1-treated cells used for the microarray analysis was also used for quantitative PCR analysis, which confirmed that JQ1 treatment markedly decreased the mRNA expression of the MYC and BCL2 genes. Depletion of BRD4 by shRNA phenocopied the effects of JQ1 treatment in OCI-AML3 cells. While it had no effect on BRD4 and NPM1, JQ1 treatment dose-dependently depleted the protein levels of MYC, BCL2, CDK6 and pSer2 RNA POL II, as well as induced the levels of p21, p27, BIM and cleaved PARP in OCI-AML3. Similar effects were observed in a representative primary AML BPC sample that expressed mutant NPM1c+ and FLT3-ITD. As compared to each agent alone, co-treatment with JQ1 (but not its inactive enantiomer, R-JQ1) and panobinostat (PS) synergistically induced apoptosis of the AML BPCs, especially the stem cell sub-population of CD34+CD38-Lin- BPCs, but not of normal CD34+ hematopoietic progenitor cells. This was associated with greater attenuation of MYC and BCL2, while increasing the levels of p21, BIM and cleaved PARP levels in the AML BPCs. In the cells treated with BRD4 shRNA, but not with the non-targeted shRNA, PS treatment induced significantly more apoptosis of OCI-AML3 cells (p 〈 0.05). As compared to treatment with the vehicle alone, daily intra-peritoneal (IP) treatment x 5 days, for three weeks, with either JQ1 (50 mg/kg formulated in 10% 2-hydroxypropyl-β-cyclodextrin) or panobinostat (5 mg/kg by IP injection 3 times per week) significantly improved the survival of the NOD/SCID mice engrafted with OCI-AML3 AML xenografts in the bone marrow, without inducing any toxicity (p 〈 0.05). Notably, combined treatment with PS and JQ1 further significantly improved survival of the mice, as compared to treatment with JQ1 or PS alone (p 〈 0.001). In a Kaplan Meier plot depicting survival, this translated into a plateau in the survival curve. Co-treatment with PS and JQ1 was also associated with the most reduction in the levels of MYC, BCL-2 and CDK6 proteins in the AML xenograft. Collectively, these pre-clinical findings highlight a potential, therapeutically efficacious, combination of BRD4 antagonist and HDAC inhibitor for further development as a therapy of AML. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2608 Introduction: HUCB has been described to contain hematopoietic multi-lineage progenitor cells that contribute to the success in treating malignant and non-malignant diseases (Cairo et al, Blood, 1997). We demonstrated that multi-lineage progenitor cells derived from HUCB can differentiate into cells representative of the 3 germ layers in vitro (Vandeven/Cairo et al, Exp Hem, 2007). Zaehres et al (Exp Hem, 2010) have described another population of primitive stem cells, USSCs, as a new potential source to generate iPS cells following retroviral transduction. This suggests that USSCs are a strong candidate as a source for developing patient specific or HLA matched iPSC banks. Since using retroviral iPS cells are challenged with the possibility of oncogene reactivation; gene integration free methods of generating iPS cells such as small molecule treatment to activate ES transcription factor genes are needed. Furthermore, the epigenetic modification of the USSCs at the key ES transcription factors has not been described and this information will provide insights on the differentiation potential of USSCs and their capacity to reprogramming. Objective: To determine the DNA methylation patterns in the core regulatory regions, including both enhancer and promoter of ES transcription factors Oct4 and Nanog in USSCs prior to and following gene transcription effects of DNMT1 inhibition by treatment with 5-azacytidine. Methods: USSCs were derived from HUCB mononuclear cells in 30% FBS and 10-7M dexamethasone (Kogler, J Exp Med, 2004). The USSC population was confirmed by flow cytometry and their fibroblastic morphology. The cells were passaged in the same medium without dexamethasone. Bisulfite sequencing was performed to characterize the CpG island methylation in the regulatory regions of Oct4 (from 2563 bp upstream to 250 bp downstream of Oct4 transcription start site) and Nanog (from 1449 bp to 82 bp upstream of the Nanog transcription start site). RT-PCR and qPCR were conducted to determine the expression levels of Nanog, Oct4 and Sox2, using isoform specific and intron spanning primers. 3mM 5-azacytidine was used to treat USSCs for demethylation studies and the RNA was collected 24 hours following treatment. The results were compared to human embryonic stem cells and human foreskin fibroblasts as positive and negative controls, respectively. Results: USSCs were derived from 50% (n=25) of HUCB; with 1–10 colonies from each successfully derived cord blood. Flow cytometry indicated that USSCs were lineage negative and express overlapping but distinct cell surface markers with MSCs; positive for CD73, CD90, CD146, CD50, but negative for CD106. Bisulfite sequencing of USSCs demonstrated a mosaic methylation pattern of CpG islands at the regulatory sites of both Oct4 and Nanog. An average of 65% and 47% of the CpGs were unmethylated in the enhancer and promoter regions of Nanog, respectively. 56% were unmethylated at the enhancer of Oct4 while the promoter was heavily methylated, except for the 400 bp region that spans the Oct4 transcription start site, which was 80% unmethylated. This is consistent with the RT-PCR results showing a low but consistent level of Nanog, Oct4 and Sox2 (Figure 1). Based on q-PCR using isoform-specific and intro-spanning primers, we determined that USSCs have about 20-and 400- fold higher levels of Nanog and Oct4 expression, respectively,compared to fibroblasts. Following a 24hr exposure to a DNMT1 inhibitor, 5-azacytidine, to the USSCs, there was a 10-fold increase in the mRNA expression of Oct4, Nanog, and Sox2. Conclusions: HUCB derived USSCs have a mosaic pattern of CpG island methylations in the distal and proximal regions of key ES transcription factors, Oct4 and Nanog. This is supported by a consistent low expression level of these genes. The mosaic pattern of CpG islands seems to be more malleable by small molecule perturbation; 3mM of 5-azacytidine appeared to significantly increase the Oct4 and Nanog expression. We hypothesize that due to their semi-permissive chromatin structure at the core regulatory regions in key ES transcription factors, HUCB derived USSCs are likely to be a more optimal choice of small molecule derived induced pluripotent stem cells compared to other cell types. Disclosures: No relevant conflicts of interest to declare.

Description: Introduction: The Sokal and Hasford (Euro) scores were developed in the chemotherapy and interferon era and are widely used as prognostic indicators in patients with chronic myeloid leukemia (CML).Recently, European Treatment and Outcome Study (EUTOS) scoring system was introduced. Data on risk stratification in pediatric CML population was lacking due to its rarity [

Description: Deficiency of clotting factors VIII (Hemophilia A) and IX (Hemophilia B) represent one of the most debilitating inherited groups of bleeding disorders. According to the most recent survey report from the World Hemophilia Federation, an estimated 178,500 patients suffer from hemophilia globally (143,000 Hemophilia A, 28,000 with Hemophilia B approximately). By most recent estimates from 2014 India has surpassed every country in the global database with a total of 17,470 patients with hemophilia ahead of USA with its 17,131 reported cases. Infectious disease and perinatal mortality take precedence for resource allocation in evolving economies like India, presenting an ongoing challenge for "rare" diseases like hemophilia. This study assesses the quality of hemophilia care at the SMS Medical School which is the flagship medical care center in the northwestern Indian city of Jaipur, the capital of the largest Indian state with an area of 0.34 million square kilometers and population of 68.55 million. Since the introduction of the free factor distribution in the fall of 2012, the program grew from a modest patient population size of 60 patients to a robust 700 plus patients within 4 years aided by the World Hemophilia Twinning program grant. A standardized self-administered questionnaire was administered to all the hemophilia patients seen at the outpatient comprehensive hemophilia program (CHP) at the SMS Medical College between February 1, 2016 and May 5, 2016. Two hundred patients met the inclusion criteria and participated in the study. One hundred eighty-seven (94%) and 13 (6) patients were diagnosed with hemophilia A and B respectively; 126 (63) had severe disease while 65 (33) had moderate and 9 (5) had mild hemophilia. In an expected male predominant sample (n=198 males [99.5]), the median age at the time of study was 12 years (range=1-53 years), and 144 (72) patients were from rural outskirts of the resource poor state visiting the tertiary care center for hemophilia care. Contrary to popular belief, only a minority 11% and 1% of patients identified themselves of the Muslim and Sindhi faith respectively, versus a majority 87% who followed Hinduism. Tenets of consanguinity and family size have often led to misunderstanding and stigmatization of the disease in the Asian subcontinent prior to this study thus far. Despite a majority 63% patients suffering from severe hemophilia, nearly half (44%) reported a delay of more than 6 months in diagnosis time from the first bleed. Remarkably, 96 % reported to know their diagnosis fully and 93% reported understanding that hemophilia is a genetically transmitted disorder but approximately 82.5 % did not know if they ever underwent testing for viral infections (H.I.V, Hepatitis B, C ) since their diagnosis . Nearly 45 % were offered genetic counseling services at some point during their care, a remarkable feat for a hemophilia program in a resource strapped environment. Inpatient stay for bleeds and complications in this predominantly severe mix of patients was encouragingly less than 1-5 days in 93% of the patients but despite free drug delivery program 79% families reported an out of pocket expense of more than 10,000 Indian rupees (INR; approximately 147 USD) during the hospital stay. This in a state where 65% of patients reported per capital household income less than 100,000 INR (1,485 USD) was particularly concerning. Nearly 18 % of the respondents identified themselves as below poverty (BPL) and enjoyed the benefits of free transportation under the governmental subsidies through a BPL card. Household income and below poverty status were not associated with hemophilia care outcomes (p-values

Description: CB use is limited by the absence of available donor effector cells (NK, and NKT cells) for SCT (Cairo, et al Transfusion, 2005). The immaturity of CB, characterized by reduced expression and production of IL-15, IL-12 and IL-18 in activated CB (Qian et al, Blood, 1997; Lee at al, Blood, 1996; Satwani et al. Br J Hem, 2005), may contribute to reduced CB cellular immunity and delayed immune reconstitution after UCBT. In innate immunity, NK cells (CD3−/56+) are regulated by various NK receptor (NKR) expression. NK(CD56dim) cells are cytotoxic, constitute 90% of the NK population while 10% (CD56bright) are cytokine producing (Shereck/Cairo, Ped Bld Can, 2007). NKT cells, (CD3+/CD56+), may play a role in allograft and tumor cytotoxicity. We demonstrated the ability to EvE (48 hrs; with anti-CD3 (50ng/ml), IL-2 (5 ng/ml), IL-7 (10 ng/ml) and IL-12 (10 ng/ml) [ABCY]) c ryopreserved, t hawed, re c ryopreserved, re t hawed, (CTCTE) CB with increases in NKbright/dim subsets expressing NKRs and enhanced NK in-vitro and in-vivo cytotoxicity (Ayello et al. BBMT, 2006). We compared day 2 vs 7 EvE CTCTE CB expansion and activation (LAMP-1) of NK and NKT cells expressing NKRs, IL-15, IL-18 and IFN-g protein levels and mechanisms of cytotoxicity. Rethawed nonadherent CB cells were EvE with ABCY. NKRs (CD3, CD16, CD56, CD94, NKG2A, NKG2D, NKp46, KIR2DS4, KIR2DL2), intracellular perforin and LAMP-1 expression were determined by flow cytometry. IL-18, IL-15 and IFN-g protein measured by ELISA. Non-adherent total cell number were significantly increased at day 7vs2 (6.28±214.7×107 vs 5.87±56.9×106, p

Description: R115777 is a nonpeptidomimetic enzyme-specific inhibitor of farnesyl protein transferase (FT) that was developed as a potential inhibitor of Ras protein signaling, with antitumor activity in preclinical models. This study was a phase 1 trial of orally administered R115777 in 35 adults with poor-risk acute leukemias. Cohorts of patients received R115777 at doses ranging from 100 mg twice daily (bid) to 1200 mg bid for up to 21 days. Dose-limiting toxicity occurred at 1200 mg bid, with central neurotoxicity evidenced by ataxia, confusion, and dysarthria. Non–dose-limiting toxicities included reversible nausea, renal insufficiency, polydipsia, paresthesias, and myelosuppression. R115777 inhibited FT activity at 300 mg bid and farnesylation of FT substrates lamin A and HDJ-2 at 600 mg bid. Extracellular signal-regulated kinase (ERK), an effector enzyme of Ras-mediated signaling, was detected in its phosphorylated (activated) form in 8 (36.4%) of 22 pretreatment marrows and became undetectable in 4 of those 8 after one cycle of treatment. Pharmacokinetics revealed a linear relationship between dose and maximum plasma concentration or area under the curve over 12 hours at all dose levels. Weekly marrow samples demonstrated that R115777 accumulated in bone marrow in a dose-dependent fashion, with large increases in marrow drug levels beginning at 600 mg bid and with sustained levels throughout drug administration. Clinical responses occurred in 10 (29%) of the 34 evaluable patients, including 2 complete remissions. Genomic analyses failed to detect N-ras gene mutations in any of the 35 leukemias. The results of this first clinical trial of a signal transduction inhibitor in patients with acute leukemias suggest that inhibitors of FT may have important clinical antileukemic activity.