Effects of Deoxycytidine on Mycoplasma-Associated Inhibition of Thymidine Incorporation and Growth in Antifolate-Containing Media

1988 ◽  
Vol 1 (2) ◽  
pp. 123-137
Author(s):  
James W. Mier ◽  
John Przygoda ◽  
Mark Allegretta ◽  
Peeter A. Poldre ◽  
Ruth B. Kundsin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Culture Medium ◽  
Cellular Proliferation ◽  
Thymidine Incorporation ◽  
Cytidine Deaminase ◽  
Leukemia Cell ◽  
Lymphoid Cells ◽  
Mycoplasma Species ◽  
Cell Counts ◽  
Selection Medium

Several mycoplasma species markedly inhibit lymphokine- and mitogen-induced 3H-thymidine incorporation in cultured lymphoid cells, but have negligible short-term effects on cellular DNA synthesis as assessed by cytofluorography or by cell counts. The deoxyribonucleotide precursor deoxycytidine (dC) reverses this inhibition, but has little effect on isotope incorporation in uninfected cultures. Human lymphoblastoid leukemia cell lines contaminated with mycoplasma and hypoxanthine guanosine phosphoribosyl transferase (HGPRT)-deficient subclones do not grow in conventional HAT medium, but the unselected parent lines proliferate when dC is included in the culture medium. The beneficial effect of dC on the growth of contaminated cultures in selection medium is amplified by the addition of the cytidine deaminase inhibitor tetrahydrouridine (THU). These observations and corroborating nucleotide pool analysis suggest that dC may exert its beneficial effects on cellular proliferation and isotope utilization by inhibiting a mycoplasma-associated enzyme, thymidine phosphorylase. The data also suggest that the conversion of dC to dU by the cellular enzyme cytidine deaminase reduces the ability of dC to salvage contaminated cultures in the presence of an antifolate. The addition of dC to the culture medium in various 3H-thymidine incorporation assays makes possible the detection of stimulatory lymphokines despite the presence of mycoplasma contamination of the indicator cells. The normalization of nucleotide pools and cellular growth of mycoplasma-infected HGPRT (+) human leukemic cell lines with the addition of dC to HAT selection medium has made possible the use of infected HGPRT-deficient subclones as fusion partners in the generation of T-T hybridomas. Our studies also suggest that the ability of cells to grow in HAT medium only when dC is included is presumptive evidence for mycoplasma infection.

Dasatinib (BMS-354825), a Multi-Targeted Kinase Inhibitor, Inhibits the Kinase Activity of Wild-Type, Juxtamembrane and Activation Loop Mutant Kit Isoforms Associated with Human Malignancies.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3358-3358
Author(s):  
Marcus M. Schittenhelm ◽  
Sharon Shiraga ◽  
Arin Schroeder ◽  
Amie S. Corbin ◽  
Diana Griffith ◽  
...  
Keyword(s):  
Mast Cell ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Leukemia Cell ◽  
Mtor Inhibitors ◽  
Clinical Activity ◽  
Activation Loop ◽  
Proliferative Effect

Abstract Activating mutations of the activation loop (AL) of KIT are associated with certain human neoplasms, including a subset of patients with AML,systemic mast cell disorders (SM), seminoma, and Gastrointestinal Stromal Tumors (GIST). KIT AL mutations such as D816V that are typically found in AML and SM are resistant to imatinib (IM, IC50 > 5–10 μM). Dasatinib (BMS-354825) is a novel, oral, multi-targeted kinase inhibitor that targets BCR-ABL and SRC. Due to its potent inhibition of these kinases, dasatinib is currently being tested in clinical trials of patients with imatinib resistant/intolerant CML/Ph+ ALL. Based on previous observations of the ability of certain SRC/ABL inhibitors to also inhibit KIT kinase, we hypothesized that dasatinib might inhibit the kinase activity of both WT and mutant KIT isoforms. The inhibitory potential of dasatinib against WT KIT, KIT mutant isoforms and KIT-dependent downstream pathways was evaluated by immunoblotting. In addition, we evaluated the effects of dasatinib on cellular proliferation and induction of apoptosis. Dasatinib potently inhibited WT, juxtamembrane- (JM) and AL-mutant KIT autophosphorylation. Based on the ability of dasatinib to bind to BCR-ABL irrespective of the ATP AL conformation (inactive versus active), dasatinib was expected to be insensitive to KIT AL conformation. In contrast, we found that the IC50 for KIT autophosphorylation varied significantly among the various KIT mutant isoforms: WT KIT, D816Y, V560G (JM mutation) [IC50 1–10 nM] <D816F [IC50 100 nM] <D816V [IC50 200–250 nM]. These results indicate that the conformation of the KIT AL does influence dasatinib potency. Inhibition of KIT kinase activity by dasatinib reduced cellular proliferation and induced apoptosis in mast cell/leukemia cell lines expressing mutant KIT isoforms. In these cell lines, KIT activates downstream pathways important for cell viability and cell survival such as RAS/MAPK, JAK/STAT and PI3K/AKT. Dasatinib potently blocked activation of MAPK1/2 and STAT3. Inhibition of KIT by dasatinib abrogated phosphorylation of AKT S473, but not AKT T308. This partial inhibition of AKT activation was insufficient to inhibit phosphorylation of p70S6K, a kinase downstream of AKT and mTOR. Combining dasatinib with rapamycin, a known mTOR inhibitor, had an additive to synergistic anti-proliferative effect on cells expressing KIT D816V. Our studies suggest that dastatinib may have clinical activity against human neoplasms that are associated with gain-of-function KIT mutations such as AML, SM, seminoma, and GIST. Combining dasatinib with mTOR inhibitors may further increase efficacy against KIT-driven malignancies.

Eltrombopag, but Not Romiplostim or Thrombopoietin, Inhibits Growth of Thrombopoietin Receptor Positive and Negative Human Leukemia Cell Lines

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4726-4726
Author(s):  
David W. Rusnak ◽  
Sharon K. Rudolph ◽  
Afshin Safavi ◽  
Connie L. Erickson-Miller
Keyword(s):  
Clinical Trials ◽  
Cell Line ◽  
Cell Lines ◽  
Dose Response ◽  
Leukemia Cell ◽  
Cell Counts ◽  
Thrombopoietin Receptor ◽  
Ic50 Values ◽  
Equity Ownership ◽  
The Impact

Abstract Abstract 4726 The thrombopoietin receptor agonists (TPO-RA), romiplostim and eltrombopag, are presently indicated for the treatment of certain patient groups with immune thrombocytopenia purpura. In a clinical study with romiplostim in patients with low-/intermediate-1 risk myelodysplastic syndromes (MDS), cases of transient increases in blast cell counts were observed and cases of MDS disease progression to acute myeloid leukemia (AML) were reported. In the present study, we evaluated the impact of romiplostim, eltrombopag, and recombinant human thrombopoietin (TPO) on the proliferation of 5 human AML and 1 TPO-dependent megakaryoblastic cell line. The cell lines evaluated include the TPO-dependent cell line, N2C TPO; the TPO-R positive AML lines, HEL92.1.7 and OCI-AML-3; and the TPO-R negative AML cell lines, HL60, THP-1, and NOMO-1. All cells were exposed to 11-point dose response curves of the 3 agents at concentrations sufficient to generate a full stimulatory response in the N2C TPO cell line. Cells were exposed to concentrations of romiplostim and eltrombopag that met or exceeded the reported Cmax achieved for each agent in high-dose clinical trials and were 3- (eltrombopag) to 30-fold (romiplostim) above trough levels from the same clinical trials. Neither romiplostim nor TPO treatment resulted in detectable stimulation or inhibition of leukemia cell growth at concentrations up to 10 μg/mL. Treatment with eltrombopag up to 40 μg/mL caused inhibition of all AML cell lines with mean IC50 values ranging from 6.4 to 13.5 μg/mL. These IC50 values reflect concentrations that are 3- to 6-fold below the Cmax of a 300 qd dose of eltrombopag (40.5 μg/mL) and at concentrations as low as 2-fold below Ctau levels (12.4 μg/mL). Cmax exceeded the IC90 for these AML cell lines, which ranged from 18.5 to 27.9 μg/mL. No stimulation of AML growth was evident through the range of the eltrombopag dose response curve on any of the cell lines evaluated. The results of this study confirm earlier in vitro studies (Will 2009, Erickson-Miller 2010) showing inhibitory effects of eltrombopag on leukemic cell lines and support clinical studies to evaluate a potential anti-leukemic effect of higher doses of eltrombopag in patients with AML. Disclosures: Rusnak: GlaxoSmithKline: Consultancy, Equity Ownership, Patents & Royalties. Off Label Use: Eltrombopag is an oral TPO agonist indicated for chronic ITP being studied in MDS/AML. Rudolph:GlaxoSmithKline: Consultancy, Equity Ownership. Erickson-Miller:GlaxoSmithKline: Employment, Equity Ownership, Patents & Royalties, Research Funding.

ASPP2 Attenuation Is an Early Step in Leukemogenesis Facilitating Acquisition of Mutations

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2221-2221
Author(s):  
Kerstin M Kampa-Schittenhelm ◽  
Figen Akmut ◽  
Barbara Illing ◽  
Charles Lopez ◽  
Marcus M Schittenhelm
Keyword(s):  
Malignant Transformation ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Conflicts Of Interest ◽  
Leukemia Cell ◽  
Population Doubling ◽  
Acute Leukemias ◽  
P53 Pathway ◽  
Leukemia Cell Lines

Abstract Inactivation of the p53 pathway is an universal event in human cancers. As p53 mutations are rare in de novo acute leukemias, the p53-pathway must be inactivated by other mechanisms instead. The Apoptosis stimulating Protein of p53-2 (ASPP2) is a haploinsufficient tumor suppressor, which we have shown to be attenuated in acute leukemia. We now provide evidence that ASPP2 regulates pathways involved in early hematopoiesis, and attenuated expression perturbs malignant transformation. Gene-chip microarrays were performed on ASPP2+/+ versus +/- mouse embryonic fibroblasts (MEFs) (-/- knockouts are not viable) and pathway analysis was performed using Affimetrix software. ASPP2 expression was silenced in IL3-dependent Ba/F3 pro-B cells and leukemia cell lines using standard siRNA protocols. Cellular proliferation was assessed using XTT-based assays. To evaluate for malignant transformation, long term cell cultures with stably retrovirally silenced Ba/F3 cells (+/- stress inforcement using gamma irradiation, 4x 5Gy) were employed. IL3 weaning was performed as an indicator of autoactivated cellular proliferation, and analysis of chromosomal defects or gene mutations were performed. Microarray mRNA analysis revealed that attenuated ASPP2 expression resulted in significant alterations of pathways involved in mediating cellular growth, proliferation and tissue differentiation. Subanalysis proofed involvement of genes playing a major role in embryogenesis, the development of the hematopoietic system and leukemogenesis (homeobox (HOX) family members, NOTCH, BCL, IRF7 and EGR1). Population doubling times were significantly increased in ASPP2+/- MEFs compared to the parental cells. Modeling ASPP2 attenuation via specific siRNA knockdown in leukemia cell lines (K562 and Kasumi1) resulted in significantly increased cellular proliferation. Similarly, ASPP2-interference in pro-B Ba/F3 cells lead to perturbed proliferation and an increase of polyploid cells with a dramatic change in morphology with megaloid cells, indicating mitotic failure. This effect was even more pronounced after stress induction with daunorubicin. Lentiviral shRNA-ASPP2 stable knock-out was performed in Ba/F3 cells to follow cells in long term cultures for malignant transformation. Again, we confirmed signs of mitotic failure as observed in the siRNA approach. Even more, gamma irradiated ASPP2-lacking strains were successfully IL3 weaned, which indicates autoactivated cellular proliferation. A screen for acquired oncogenic mutations was performed and analyses are ongoing. In summary, we provide evidence that ASPP2 has a pivotal role in early hematopoiesis, and loss of ASPP2 is a driver mechanism to fuel leukemogenesis. Disclosures No relevant conflicts of interest to declare.

scholarly journals CREB: A Key Regulator of Normal and Neoplastic Hematopoiesis

2009 ◽  
Vol 2009 ◽  
pp. 1-8 ◽  
Author(s):  
Salemiz Sandoval ◽  
Martina Pigazzi ◽  
Kathleen M. Sakamoto
Keyword(s):  
Bone Marrow ◽  
Transgenic Mice ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Cellular Proliferation ◽  
Leukemia Cell ◽  
Cellular Transformation ◽  
Camp Response Element Binding ◽  
Lymphoid Leukemia ◽  
Leukemia Cell Lines

The cAMP response element-binding protein (CREB) is a nuclear transcription factor downstream of cell surface receptors and mitogens that is critical for normal and neoplastic hematopoiesis. Previous work from our laboratory demonstrated that a majority of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) overexpress CREB in the bone marrow. To understand the role of CREB in leukemogenesis, we examined the biological effect of CREB overexpression on primary leukemia cells, leukemia cell lines, and CREB overexpressing transgenic mice. Our results demonstrated that CREB overexpression leads to an increase in cellular proliferation and survival. Furthermore, CREB transgenic mice develop a myeloproliferative disorder with aberrant myelopoiesis in both the bone marrow and spleen. Additional research from other groups has shown that the expression of the cAMP early inducible repressor (ICER), a CREB repressor, is also deregulated in leukemias. And, miR-34b, a microRNA that negative regulates CREB expression, is expressed at lower levels in myeloid leukemia cell lines compared to that of healthy bone marrow. Taken together, these data suggest that CREB plays a role in cellular transformation. The data also suggest that CREB-specific signaling pathways could possibly serve as potential targets for therapeutic intervention.

scholarly journals Treatment of Mycoplasma Contamination in Cell Cultures with Plasmocin

2012 ◽  
Vol 2012 ◽  
pp. 1-8 ◽  
Author(s):  
Cord C. Uphoff ◽  
Sabine-A. Denkmann ◽  
Hans G. Drexler
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Culture Medium ◽  
Detection Method ◽  
Mycoplasma Species ◽  
Mycoplasma Contamination ◽  
Cure Rate ◽  
Mycoplasma Detection ◽  
Additional Cell ◽  
Limited Period

A high percentage of cell lines are chronically infected with various mycoplasma species. The addition of antibiotics that are particularly effective against these contaminants to the culture medium during a limited period of time is a simple, inexpensive, and very practical approach for decontaminating cell cultures. Here, we examined the effectiveness of the new antimycoplasma compound Plasmocin that has been employed routinely to cleanse chronically infected cell lines. In a first round of treatment 45 out of 58 (78%) mycoplasma-positive cell lines could be cured. In a second attempt using back-up cryopreserved original cells, four additional cell lines were cured; thus, the overall cure rate was 84%. Even if the mycoplasma contamination was not eradicated by Plasmocin, the parallel treatment with several other antibiotics (Baytril, BM-Cyclin, Ciprobay, MRA, or MycoZap) led to the cure of all 58 cell lines. The successful decontamination was permanent as mycoplasmas were no longer detected at day +14 posttreatment and at later time points as examined by PCR which is the most sensitive and specific mycoplasma detection method. Collectively, our results highlight certain antibiotics as effective antimycoplasma reagents and support the therapeutic rationale for their use in the eradication of this notorious cell culture contaminant.

MEIS1 Is a Required Transcription Factor for AML Cell Proliferation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1944-1944
Author(s):  
Stefan Heinrichs ◽  
Chad J. Brenner ◽  
Rima V. Kulkarni ◽  
A. Thomas Look
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Leukemia Cell ◽  
Tyrosine Kinase Receptor ◽  
Mouse Bone Marrow ◽  
Human Leukemia ◽  
Knock Down ◽  
Low Levels

Abstract MEIS proteins constitute an important subgroup of three amino acid loop extension (TALE) class transcription factors, which are characterized by an atypical homeodomain. MEIS1 has been shown to act as an important cofactor in HOXA9-mediated leukemogenesis in bone marrow transduction experiments. In human leukemia, MEIS1 was identified as a gene consistently upregulated in leukemias with MLL translocations. However, MEIS1 overexpression is also frequently found in AMLs with normal or complex karyotypes. To investigate the role of MEIS1 in AML pathogenesis, we measured the expression of MEIS1 and MEIS2 in 26 different established human AML cell lines, and documented high levels of expression of one or both genes in 20 of these lines. Despite the prevalence of MEIS1 expression in AML blasts, the downstream pathways and their contribution to the proliferation or self-renewal of the malignant clone are not well understood. To identify gene expression programs controlled by MEIS1, we targeted MEIS1 expression by RNAi in two AML cell lines that express HOXA9 and MEIS1, but only low levels of MEIS2. In comparison to a control shRNA, cells expressing two different MEIS1-specific shRNAs were markedly deficient in thymidine incorporation indicating very low levels of cellular proliferation. The RNAi-mediated knock-down of MEIS1 expression resulted in down-regulation of the tyrosine kinase receptor FLT3 by quantitative RT-PCR. Thus our data in human AML is consistent with results of Wang et al. (Blood, 106(1):254–64) implicating MEIS1 in the regulation of FLT3 tyrosine kinase expression, based on overexpression studies in mouse bone marrow progenitors. Our results in human AML suggest that MEIS1 is required for high levels of FLT3 expression, which is significant because FLT3 is mutated and activated in these two myeloid leukemia cell lines, suggesting the hypothesis that FLT3 is an important component of the pathway through which MEIS1 promotes proliferation. Programmed re-expression of activated FLT3 in AML lines with MEIS knock-down is underway to test whether FLT3 is the essential component acting downstream of MEIS1 expression in AML pathogenesis.

Genetic Mechanisms of Cytarabine Resistance in Leukemia: Cell Culture and Mouse Transplantation Models.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4374-4374
Author(s):  
Bin Yin ◽  
Miechaleen D. Diers ◽  
Susan K. Rathe ◽  
Scott W. Lowe ◽  
David A. Largaespada
Keyword(s):  
Cell Lines ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Leukemia Cell ◽  
Microarray Gene Expression ◽  
Major Barrier ◽  
Primary Tumors ◽  
Cell Counts ◽  
Leukemia Cell Lines ◽  
Myeloid Leukemia Cell

Abstract Chemoresistance remains a major barrier to successful clinical treatment of acute myeloid leukemia (AML). However, the underlying molecular mechanisms of chemoresistance in AML are largely unknown. Previously, we reported that loss of tumor suppressor genes, Nf1 and Trp53, can confer upon myeloid leukemia cells partial resistance to cytarabine (Ara-C), a mainstay of AML chemotherapy. Microarray gene expression profiling revealed that a group of lysosome function-related genes, and a major regulator of apoptosis, Bcl-xL, were upregulated in in vitro Ara-C resistant myeloid leukemia cell lines. We now report that the lysosome number and size is increased in resistant cells. Also, overexpression of Bcl-xL in both murine and human myeloid leukemia cell lines increased Ara-C resistance to a variant extent in different lines. Furthermore, we found that the key enzyme involved in intracellular activation of Ara-C, Cda, can do so, too, but to a less extent than Bcl-xL. Interestingly, in our Ara-C resistant AML cell lines which were retrovirally induced in BXH-2 stain of mice, we were able to detected new proviral insertion sites which may cause mutations associated with the leukemia resistance feature. We have now examined the possibility of modeling AML chemoresistance in mouse, and through this model, to identify and characterize AML relapse-related genetic abnormalities. During establishing such a model, treatment dosage, intervals, courses, routes of administration, the number of injection, food preparation and supplements have been considered for testing. Ara-C treatment caused a decrease in body weights and peripheral blood cell counts. One major lethal toxicity response, intestine deterioration, appeared to be correlated with high doses of Ara-C. We arrived at a regimen comprised of two 10-day treatment courses at 90 mg/kg/day by one intraperitoneal injection, with a 5-day break between treatment courses. This treatment course was found to give rise to 10~30% lethality due to Ara-C toxicity but it can also suppress the onset of transplanted AML disease in recipient mice for nearly two weeks compared to saline-treated mice as a control. Interestingly, the tumors collected from Ara-C treated transplants displayed higher resistance than saline treated tumors in a methylcellulose colony-forming assay. Testing of apoptosis regulatory genes for Ara-C response in primary tumors is in process.

scholarly journals The Role of Cytidine Deaminase and GATA1 Mutations in the Increased Cytosine Arabinoside Sensitivity of Down Syndrome Myeloblasts and Leukemia Cell Lines

2004 ◽  
Vol 64 (2) ◽  
pp. 728-735 ◽  
Author(s):  
Yubin Ge ◽  
Tanya L. Jensen ◽  
Mark L. Stout ◽  
Robin M. Flatley ◽  
Patrick J. Grohar ◽  
...  
Keyword(s):  
Down Syndrome ◽  
Cell Lines ◽  
Cytosine Arabinoside ◽  
Cytidine Deaminase ◽  
Leukemia Cell ◽  
Leukemia Cell Lines

Phase 1 Dose Escalation Study of TLK199 Tablets (Telintra), a Novel Glutathione Analog, in Myelodysplastic Syndrome.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1454-1454 ◽  
Author(s):  
Azra Raza ◽  
Magda Melcert ◽  
Naomi Galili ◽  
Scott E. Smith ◽  
John E. Godwin ◽  
...  
Keyword(s):  
Cell Lines ◽  
Dose Escalation ◽  
Cellular Proliferation ◽  
Clinical Symptoms ◽  
Phase 1 ◽  
Dose Range ◽  
Leukemia Cell ◽  
Human Leukemia ◽  
Dose Escalation Study ◽  
Dose Levels

Abstract Introduction: Glutathione S-transferase (GST) P1-1 binds to and inhibits Jun kinase (JNK), a key regulator of cellular proliferation, differentiation and apoptosis. TLK199, a glutathione analog, binds selectively to GSTP1-1 fostering dissociation from JNK, kinase activation and the promotion of growth and maturation of hematopoietic progenitors in preclinical models, while promoting apoptosis in human leukemia cell lines. The intravenous study with liposomal TLK199 resulted in hematologic improvement (HI) in MDS patients (pts); this trial utilizes an oral formulation of TLK199 in MDS pts. Methods: The objectives of this study were to determine the safety and pharmacokinetics (PK) of TLK199 Tablets given b.i.d. at total daily doses ranging from 200 mg to 6000 mg for the first 7 days of each 3-week cycle. The design was a standard 3 by 3 (3 pts per dose level) dose escalation. Patients were treated until MDS progression or unacceptable toxicity up to a maximum of 8 cycles. Six pts underwent fed-fast PK analysis to determine the effect of food absorption. The PK was evaluated over the dose range for TLK199, and metabolites TLK236, TLK235, and TLK117. Results: 44 MDS pts (32 M/12 F), (9 RA, 11 RARS, 3 RAEB/RAEB-1, 1 RAEB-II, 7 RCMD, 2 RCMD-Rs, 2 CMML, 5 MDS-U, 4 Unknown); IPSS risk-low 14 (32%), INT-1 27 (61%), and INT-2 3 (7%); median age 72 years (range 53–84), received total 206 cycles, median 4.5 (range 1–9) cycles/pt. Ten pts (23%) completed the intended 8 cycles of therapy. Two pts had dose reductions and 4 pts had dose delays (2 due to adverse event (AE) and 2 for scheduling difficulty) at single cycle. Twenty-seven pts (61%) were red cell transfusion (tx) dependent and 5 pts (11%) were platelet tx dependent. Sixteen pts (36%) had abnormal karyotypes. Most common treatment-related AEs were non-hematologic: There were no Grade 3 or 4 toxicities; Grade 2 toxicities were diarrhea and nausea in 2 pts each (5%). Grade 1 toxicities were nausea (43%), diarrhea (25%), vomiting (18%), abdominal pain (7%) and constipation (7%). Three pts (7%) experienced pill-induced dysphagia and reflux esophagitis. Two pts (5%) had Grade 4 neutropenia and 1 pt had febrile neutropenia. There were no DLTs reported. The plasma concentration of the primary active metabolite, TLK236, increases with TLK199 Tablets dose with a mean t1/2 = 2.5 h (range 1.5–4); Cmax = 4.5 uM (range 0.4–6.3). There was no difference seen in the fed vs. fasted patients. By IWG criteria, 15 individual cell line HI responses were observed at the various dose levels of 1000–6000 mg/day with 9 HI responses at dose levels 4000–6000 mg/day. These HI responses were characterized as 1 HI-E major and 4 HI-E minor, 1 HI-N major and 2 HI - N minor, 1 HI-P major and 6 HI-P minor. One bilineage response was reported at 5000 mg/day and 2 trilineage responses at 6000 mg/day. These responses were accompanied by clinical symptoms improvement. Conclusions: TLK199 Tablets are well tolerated and HI responses in all three cell lines were observed with oral TLK199 short-course schedule. These data support the Phase 2 development of extended schedules of oral TLK199 in MDS.

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