ALBERT

Description: GTI-2040 is a 20-mer antisense to the R2 component of RNR mRNA. RNR is required for the conversion of ribonucleotides to deoxyribonucleotides, a crucial step during DNA synthesis and repair. Cytarabine (AraC) is a cytotoxic agent that is converted into AraC triphosphate (Ara-CTP) and is the backbone of several regimens in AML. Ara-CTP competes with deoxycytidine for DNA incorporation. We hypothesize that RNR downregulation by GTI-2040 results in decrease of levels of deoxycytidine thereby leading to a preferential DNA incorporation of ARA-CTP and increase in its cytotoxic activity. To test this hypothesis we undertook a CTEP-sponsored Phase I dose-escalation study of GTI-2040 plus HiDAC in patients (pts) with relapsed or refractory AML. Pts were stratified in 2 cohorts according to age. Cohort I (18–59 yrs) received escalating doses of GTI-2040 (dose level 1: 3.5 mg/m2/d) by continuous IV infusion (CIVI) on d 1–6 combined with escalating doses of cytarabine IV over 2 hrs q12 hrs on d 2, 4, and 6 (dose level 1: 2500 mg/m2/dose). Cohort II (≥60 yrs) received GTI CIVI on d 1–6 and cytarabine IV over 4 hrs on d 2 to 6 (dose level 1: 1500 mg/m2/d). To date, 30 pts were enrolled. Pts received median of 1 prior regimen (range 1–3). Cohort I included 8 pts with relapsed and 6 with refractory disease; 7 had intermediate risk cytogenetics (CyG) and 7 adverse CyG; 6 received prior HiDAC. Cohort II included 10 pts with relapsed and 6 with refractory disease; 8 pts had intermediate risk CyG and 8 high risk CyG; 5 pts received prior HiDAC. Toxicities were comparable to HiDAC therapy alone. Grade 3/4 non-hematologic toxicities included fatigue, fevers, anorexia, pneumonitis, and catheter related infections; a grade 3 reversible cerebellar toxicity (n=1) was observed at level 1/cohort I. An ELISA-based assay with a limit of quantification of 50 pMol was used to determine GTI2040 plasma and intracellular (IC) concentrations. Dose-dependent increase in plasma steady state concentration (Css) and area under the curve (AUC) of GTI2040 was observed in both cohorts, although higher AUC and longer t1/2 were demonstrated in the younger pts compared to the older ones. In cohort I, disease responses were seen at all dose levels Five of 14 pts achieved complete remission (CR) and one achieve incomplete CR (CRi; i.e., no marrow disease and incomplete blood count recovery). In cohort II, no disease response was observed. Median IC GTI2040 concentration in BM mononuclear cells at 120 hours following start of antisense infusion was higher in younger (i.e., 175 nM) than in older (i.e., 75 nM) pts. A median decrease in R2 protein levels of 50% (range 50–90%) detected by immunoblotting was noted in 5/9 and 5/10 pts in cohort I and II, respectively. In cohort I CR pts (n=4) had a median 50% decrease and non-responders (n=9) had a median 200% increase in R2 levels. In cohort II changes in R2 levels did not predict disease response. In summary, combination of GTI-2040/HiDAC is feasible. PK/PD studies demonstrate achievable plasma and IC levels of the antisense and target downregulation. Disease response was observed only with the dose/schedule administered to younger pts. Dose escalation in this group continues to establish a recommended dose for Phase II trials. [NCI U01 CA 76576-05].

Description: The TCL1 (T-cell leukemia/lymphoma1) oncogene is a coactivator of the AKT oncoprotein, an essential molecule in the transduction of antiapoptotic signals in T and B cells. Eμ-TCL transgenic mice with B cells with high TCL1 expression develop the aggressive phenotype of chronic lymphocytic leukemia (CLL). Studies in human CLL have found that expression of TCL1 correlates with high expression of ZAP-70 and use of unmutated IgVH genes. The expression of TCL1 may be regulated in part by microRNA, miR-29 and miR-181, which map to chromosome 11(11q). Because aberrations at 11q have been associated with poor prognosis in CLL, we interrogated the relationship between deletions at 11q and expression of TCL1 and ZAP-70. We used a direct immunophenotyping method to investigate the relative co-expression levels of TCL1 and ZAP-70 within CLL cells and examined the relationship between such levels and the proportion of leukemia cells within the CLL population bearing 11q deletions, as detected by FISH analysis. Direct staining of intracellular TCL1 protein was performed by using the monoclonal anti-TCL1 antibody (clone 1–21) labeled with Alexa647 in combination with the established ZAP-70 protocol (NEJM2004; 351:893) together with mAb directed against CD5 and B cell surface antigens. Negative staining levels were set using isotype control antibodies. FISH was performed on interphase nuclei by using uniform and cross-validated procedures at all CRC sites using the CLL-panel from Vysis. Chromosomal abnormalities were detected in 76% (520) of the 680 CLL samples analyzed. Sixteen percent of the patients had leukemia cells with monoalleleic deletions at 11q. We performed flow cytometry for intracellular co-expression TCL1 and ZAP-70 on cryopreserved samples obtained from 25 CLL patient samples with varying proportions of cells with the 11q deletion (10% to 98% abnormal cells with 11q deletion, mean 70%) and 30 CLL samples lacking any chromosomal abnormalities. We detected significantly higher levels of TCL1 in CLL cells that expressed ZAP-70 and/or unmutated IgVH genes. The ZAP-70pos cases (39/55) had a median percent of TCL1pos cells of 34% compared to 15% for the ZAP-70neg cases. The cases using unmutated IgVH genes (43/55) had a median percent of TCL1pos cells of 31%, which was greater than the 19% median observed for cases that used mutated IgVH genes. Multiparameter analyses revealed that the ZAP-70 positive fraction of each CLL clone had significantly higher levels of TCL1 than did the ZAP-70 negative cells (mean=45% versus 29%, respectively, p=0.002). We observed a significant difference between the expression levels of TCL1 for CLL cells that had deletions in 11q relative to that of CLL cells lacking any chromosomal abnormalities (mean=41% versus 18%, respectively p=0.0002). In addition, we observed a relationship between the levels of TCL1 expressed in leukemia cell populations and the relative proportion of leukemia cells with deletions at 11q. This study reveals a relationship between the levels of TCL1 expression in CLL leukemia-cell expression of ZAP-70, and the relative proportions of leukemia cells having deletions at 11q. Studies are in progress to define whether these relationships can be explained by altered expression of microRNA that map to 11q, which might also account for the noted adverse prognosis of CLL that has deletions at 11q.

Description: Recovery of bone marrow cellularity, CFU-C, and CFU-S were studied sequentially over 90 days time after syngeneic bone marrow transplantation in mice. A minimal cell dose of 2 X 10(5) bone marrow cells was given. At day 28 after transplantation, CFU-C reached more than 50% of the normal range whereas the CFU-S concentration was less than 15%. Normalization of CFU-S occurred at day 90. The effect of the addition of peripheral blood nucleated cells on bone marrow hemopoietic recovery was studied at day 28. The augmentation of CFU-C and CFU-S recoverey were dose dependent. Optimal enhancement was seen with bone marrow to blood ratios of 1:1 and 1:2.5. This enhancement effect was lost when nucleated blood cells in a ratio of 1:10 were administered.

Description: Recovery of bone marrow cellularity, CFU-C, and CFU-S were studied sequentially over 90 days time after syngeneic bone marrow transplantation in mice. A minimal cell dose of 2 X 10(5) bone marrow cells was given. At day 28 after transplantation, CFU-C reached more than 50% of the normal range whereas the CFU-S concentration was less than 15%. Normalization of CFU-S occurred at day 90. The effect of the addition of peripheral blood nucleated cells on bone marrow hemopoietic recovery was studied at day 28. The augmentation of CFU-C and CFU-S recoverey were dose dependent. Optimal enhancement was seen with bone marrow to blood ratios of 1:1 and 1:2.5. This enhancement effect was lost when nucleated blood cells in a ratio of 1:10 were administered.

Description: In t(8;21) AML, the AML1/ETO fusion protein promotes leukemogenesis by recruiting histone deacetylase (HDAC) and silencing target genes important for hematopoiesis. In addition to its anticonvulsant properties, valproic acid (VPA), an eight-carbon branched-chain fatty acid, has shown a significant antitumor activity mediated, at least in part, by inhibition of the HDAC enzymatic activity. The molecular mechanisms through which VPA inhibits HDACs and restore gene expression in cancer cells remain, however, to be fully elucidated. Herein, we showed that in AML1/ETO-positive cells, VPA modulates HDAC activity by releasing HDAC1 and AML1/ETO from the DNA binding sites at concentrations attainable in pts. Nuclear and whole cells extracts were obtained from AML1/ETO-positive Kasumi-1 cells untreated or treated with VPA (0.3 to 3 mM) for 24 hours and subjected to immunoblotting with AML1, ETO or HDAC1 antibody. Decreased levels of HDAC1 was noticed in the nuclear extract, but not in the unfractionated cell lysates following VPA treatment. These changes correlated with a dose-dependent inhibition of HDAC enzymatic activity and global hyperacetylation of histone H3 and H4. Decrease of HDAC1 and AML1/ETO in the nucleus was likely due to dissociation of the repressor complex proteins from DNA and relocation in the cytoplasm as indicated by a “band-depletion” assay. Untreated or VPA treated Kasumi-1 cells were cross-linked with 1% formaldehyde to stabilize the binding of proteins to their target DNA sequences, and subjected to immublotting with HADC1, AML1 or ETO antibody. While free protein can be resolved on a polyacrylamide gel by immunoblotting, high-molecular weight DNA-protein complexes cannot. Following formaldehyde cross-linking, in untreated cells, the AML1/ETO-HDAC1 complex was stabilized onto the DNA binding sites thereby forming a low-mobility DNA-protein complex which resulted in the absence of the corresponding gel band. In contrast, following VPA treatment, high-intensity AML1/ETO and HDAC1 bands were visualized, suggesting a decrease in DNA binding and an increase in the free-pools of the AML1/ETO and HDAC1 proteins. No changes were observed in HDAC1 and AML1/ETO RNA or protein levels in cells not cross-linked with formaldehyde, thereby excluding upregulation of the corresponding genes following VPA treatment. Similar results were confirmed at the promoter of specific AML1/ETO-target genes (i.e., IL-3). Following VPA exposure, decreased levels of HDAC1 and AML1/ETO, and histone H3 and H4 hyperacetylation were detected on the IL-3 promoter by chromatin immunoprecipitation. The functional relevance of these changes was supported by detection of mRNA expression of IL-3, which was otherwise silenced in untreated cells. Notably, the activity of VPA to HDAC1 and AML1/ETO appeared quite specific, as levels of other factors that initiate histone acetylation and/or gene transcription such as HAT1 and Pol II did not decrease, rather increased on the IL-3 promoter. Further, following VPA exposure, we observed posttranslational changes of specific H3 and H4 lysine residues associated with gene transcription (H3K9 and H4K16 acetylation and H3K4 methylation), partial cell differentiation, cell cycle arrest via upregulation of p21 and caspase-induced apoptosis. Taken together, these data suggest VPA as a promising compound for targeting molecular subgroups of AML, especially those in which leukemogenesis is promoted by aberrant HDAC activity.

Description: Oblimersen sodium (Ob, Genasense™) is an 18 mer phosphorothioate antisense directed against bcl-2, an antiapoptotic protein that mediates chemoresistance in malignant cells. In order to assess whether detectable intracellular concentrations (ICs) of Ob are achievable in vivo, we have developed a sensitive and specific ELISA-based assay. This assay involves Ob hybridization to a 5′-end overhang of a 3′-biotinylated capture probe ligated to a digoxigenin (Dig)-labeled probe, and detection by an anti-Dig-alkaline phosphatase system. In K562 cell extract, the assay was linear within 50–2000 pM range with a limit of quantification (LOQ) of 50 pM (equivalent to 5.0 fmol/100 μL). The withtin-run coefficients of variation (CVs) in 4 spiked concentrations were between 3–7% in 6 replicates with accuracy values between 93–109%. The between-run CVs were between 6–12% and accuracy values between 97–102%. The specificity of the assay was demonstrated by low cross reactivity with mismatched oligonucleotides and putative 3′-end metabolites shortened by 1, 2 or 3 nucleotides. Validation of IC measurement was performed in vitro in K562 cells treated with fluorescent labeled Ob conjugated to oligofectamine. At Ob concentrations between 0.1 – 10 μM, Bcl-2 mRNA downregulation measured by real-time RT-PCR occurred efficiently. Nonlinear regression analysis of a dose-response curve showed that 50% Bcl-2 downregulation (IC50) occurred at approximately 0.29 μM, corresponding to an Ob IC concentration of 37 pmole/mg protein. Cellular uptake was confirmed by microscopy and flow cytometry. To validate these results in vivo, we measured Ob IC in bone marrow samples from untreated AML pts aged 〉 60 yrs enrolled on the phase I study OSU 0164. These pts were induced with Ob 7 mg/kg/d CIVI on days 1–10, cytarabine 100 mg/m2/d CIVI on days 4–10 and daunorubicin administered iv at two dose levels (45 mg/m2/d IV and 60 mg/m2/d) on days 4–6. Among 21 pts assessable for clinical response and Bcl-2 levels, at pretreatment, Bcl-2 copy numbers (normalized to ABL) were higher among 12 pts who achieved a CR (median 85,325; range 19,120–149,100) than among 9 non-responsive (NR) pts (32,100 bcl-2 /abl copies; range 1,488–163,500) (P=.04; Mann-Whitney test). Following 72 hr Ob infusion, a decrease (−38%) in median Bcl-2/ABL mRNA copies in CR patients and an increase (+115%) in Bcl-2/ABL copies in NR pts (P=.002; Mann-Whitney test) were observed by real time RT-PCR. A trend in higher median IC of Ob was observed in CR pts (17.0 pmole/mg protein; range 1.5–30.0) as compared to NR pts (4.4 pmole/mg protein; range 0.33–28.0) (P=.06; Mann-Whitney test). Six of 7 pts with IC above the median obtained a CR. No differences were observed in the Ob plasma PKs between the CR pts [median steady state concentration (Css) 2.8 μg/mL, area-under-the-curve (AUC) 772 μg*hr/mL and clearance (Cl) 9.6 L/hr) and the NR pts (median Css 3.4 μg/mL, AUC 752 μg*hr/mL, Cl 6.4 L/hr). Although the number of samples analyzed was small, our data suggest that, despite interpatient variability of both Bcl-2 mRNA expression and Ob uptake, this antisense can be successfully delivered to pts and result in clinically relevant target downregulation. A Cancer and Leukemia Group B phase III AML study to characterize prospectively the interplay of IC levels of the Ob and Bcl-2 downregulation is in progress.