scholarly journals Anti-Leukemia Effect of FF-10501-01, a Novel Inosine 5'-Monophosphate Dehydrogenase Inhibitor, in Advanced Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS), Including Hypomethylating Agent (HMA) Failures

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 3800-3800 ◽  
Author(s):  
Guillermo Garcia-Manero ◽  
Hui Yang ◽  
Zhihong Fang ◽  
Courtney DiNardo ◽  
Elias Jabbour ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
De Novo ◽  
Phase 1 ◽  
Leukemia Cell ◽  
Clinical Activity ◽  
Guanine Nucleotide ◽  
Leukemia Cell Lines ◽  
Induced Apoptosis

Abstract Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in AML cell lines and in a Phase 1 clinical study in advanced AML and MDS, including HMA failures. Thirteen leukemia cell lines were studied, including 5 parental AML cell lines and their HMA-resistant derivatives (MOLM13, SKM1, HL60, TF1, and U937), and 3 other AML cell lines (KG1, HEL, and OCI-AML3). Cell proliferation was determined using trypan blue analysis. Flow cytometry was performed to detect drug-induced apoptosis and cell cycle analysis. High-performance liquid chromatography (HPLC) was performed to detect the intracellular concentrations of guanine nucleotides. Mycophenolic acid-treated cells were used as positive control. Effect of guanosine supplement on FF-10501-01 treatment was evaluated. Within 72 hours of treatment, FF-10501-01 inhibited proliferation of all 13 AML cell lines. The IC50 of FF-10501-01 ranged between 4.3 and 144.5 µM. MOLM13 was the most sensitive leukemia cell line, whereas the decitabine-resistant TF1 cell line was the most resistant. FF-10501-01-induced apoptosis was observed in all cell lines. Increased numbers of cells in G1 phase and decreased numbers in S phase were observed in MOLM13, SKM1 and TF1 cell lines treated with <100 µM FF-10501-01. Decreased intracellular concentrations of guanine nucleotides were observed in MOLM13 and SKM1 cell lines treated with 3 to 30 µM of FF-10501-01 for 24 hours. Proliferation was partially rescued after 72 hours of treatment with 3 µM guanosine and FF-10501-01 in MOLM-13, HL60 cells and their HMA-resistant derivatives. No treatment synergy was observed with the combination of FF-10501-01 with HMAs in MOLM-14 and HL-60 or their HMA-resistant cell lines. In summary, FF-10501-01 produced potent anti-proliferative and apoptotic effects on AML cell lines through inhibition of de novo guanine nucleotide synthesis. In view of these pre-clinical findings, we performed a standard 3+3 dose-escalation Phase 1 trial to access the safety and clinical activity of FF-10501-01 in patients with advanced AML, MDS and chronic myelomonocytic leukemia (CMML). Eligibility criteria: age ³ 18 years, high risk MDS/CMML, AML with documented PD following previous therapy, AML ≥ 60 years of age and not a candidate for other therapy, adequate renal and hepatic function, and no known history of significant cardiac disease. Sixteen patients (12 AML, 4 MDS) have been enrolled in 5 dose cohorts (50 - 400 mg/m2 PO BID) for 14 days on/14 days off each 28-day cycle, including 8 M and 8 F. Median (range) values: age 75.3 yrs (59.1 - 88.6); bone marrow blasts for AML patients 40.5% (12 - 71), for MDS patients 10% (6 - 13), or 30% overall (6 - 71); and prior treatment regimens 2.5 (1 - 6). All patients relapsed from, or progressed on, prior HMAs. Mutations in FLT3, NPM1, GATA2, TET2, ASXL1, DNMT3A and/or MDM2 were present in 4/16 (25%) patients. The median number of FF-10501-01 cycles received to date is 1.5 (range 1 - 10). No DLTs or drug-related serious adverse events (AEs) have been observed and FF-10501-01 has been very well tolerated through 5 - 10 cycles. The most frequent drug-related AEs have been Gr 1-2 nausea, diarrhea and fatigue. Drug-related Gr 4 prolonged thrombocytopenia and Gr 4 prolonged neutropenia were reported in one patient at 200 mg/m2 BID. Two partial responses (PRs) have been achieved in 1 patient each at 50 and 100 mg/m2 BID after 3 cycles, 7 (50%) patients demonstrated long-term stable disease over 2 - 10 cycles, and 4 patients have remained on study drug through 5 - 10 cycles and are still ongoing. Updated safety and efficacy data, including PK/PD, will be presented at the meeting. FF-10501-01 is a promising new agent for the treatment of advanced AML and MDS. Preclinical activity was seen in multiple leukemia cell lines. In a Phase 1 trial, clinical activity with PRs, prolonged disease stabilization and a highly tolerable safety profile were observed. The Phase 2 expansion phase will be initiated soon. Disclosures DiNardo: Novartis: Research Funding. Pemmaraju:Stemline: Research Funding; Incyte: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; LFB: Consultancy, Honoraria. Smith:Westat Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Gipson:Strategia Therapeutics, Inc.: Employment. Rosner:Strategia Therapeutic, Inc.: Employment. Madden:Strategia Therapeutics, Inc.: Employment. Myers:Strategia Therapeutics, Inc.: Employment. Paradiso:Strategia Therapeutics, Inc.: Employment.

Phase 1 Results of FF-10501-01, a Novel Inosine 5'-Monophosphate Dehydrogenase Inhibitor, in Advanced Acute Myeloid Leukemia (AML) and Myelodysplastic Syndromes (MDS), Including Hypomethylating Agent (HMA) Failures

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1640-1640 ◽  
Author(s):  
Guillermo Garcia-Manero ◽  
Hui Yang ◽  
Zhihong Fang ◽  
Hagop M. Kantarjian ◽  
Courtney D. DiNardo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Disease Progression ◽  
Cell Lines ◽  
Dose Level ◽  
Research Funding ◽  
De Novo ◽  
Phase 1 ◽  
Clinical Activity ◽  
Guanine Nucleotide ◽  
Bone Marrow Blast

Abstract Inosine 5'- monophosphate dehydrogenase (IMPDH) is a rate-limiting enzyme that catalyzes de novo synthesis of the guanine nucleotide and is overexpressed in both hematologic and solid tumors. FF-10501-01 is a potent new competitive IMPDH inhibitor. We investigated the anti-leukemia effect of FF-10501-01 in a Phase 1 clinical study in advanced AML and MDS, including HMA failures. Previous preclinical studies demonstrated potent anti-proliferative and apoptotic effects of FF-10501-01 on AML cell lines, including HMA-resistant derivatives, through inhibition of de novo guanine nucleotide synthesis. Therefore, we performed a standard 3+3 dose-escalation Phase 1 trial to access the safety and clinical activity of FF-10501-01 in patients with advanced AML, MDS and chronic myelomonocytic leukemia (CMML). Eligibility criteria: age ≥ 18 years, high risk MDS/CMML, AML with documented PD following previous therapy, AML ≥ 60 years of age and not a candidate for other therapy, adequate renal and hepatic function, and no known history of significant cardiac disease. A total of 29 patients, 15M and 14F (23 AML, 6 MDS) have been treated in 7 dose cohorts (50 - 500 mg/m2 PO BID) for 14 days on and 14 days off, and 400 mg/m2 for 21 days on and 7 days off, each 28-day cycle. Median (range) values: age 75 yrs (59 - 88); baseline bone marrow blast counts for AML 34% (12 - 82), for MDS 10% (5 - 16), and overall 30% (5 - 82); and prior treatment regimens 2 (1 - 7). All patients relapsed from, or progressed on, prior HMAs. At baseline, mutations in FLT3, NPM1, GATA2, TET2, ASXL1, DNMT3A, NOTCH1, JAK2, IDH2, PTPN11, KRA, TP53, RUNX1, EZH2 and/or MDM2 were present in 13 of 29 (45%) of patients. Atrial fibrillation (Gr 2) was reported in 2 subjects at a dose of 500 mg/m2 BID. This met the definition of dose-limiting toxicity (DLT) and no further enrollment was made at this dose level. The maximally tolerated dose (MTD) was declared at 1 dose level lower, 400 mg/m2 BID, and this cohort was expanded to 6 subjects. No DLTs have been observed in N=7 total subjects treated at 400 mg/m2 BID x 14 days. FF-10501-01 has been very well tolerated through 24 cycles. The most frequent drug-related AEs have been Gr 1-2 nausea, diarrhea and fatigue. Drug-related thrombocytopenia, neutropenia and bone marrow aplasia (all Gr 4) were reported in 1 patient at 200 mg/m2 BID. The median number of FF-10501-01 cycles received to date is 2 (range 1 - 24). Partial remissions have occurred in 2 AML patients (50 and 100 mg/m2 BID) after 3 cycles, lasting for 5 and 24 cycles, respectively, with the higher dose patient still on study after 24 cycles. A total of 8/23 (34.8%) AML patients, including the 2 PRs, have attained stable disease (SD) control with no disease progression over 3 - 24 cycles. Three AML patients remain on study through 3, 23 and 24 cycles, respectively. A bone marrow complete response was achieved in 1 MDS patient treated at 400 mg/m2 BID after 1 cycle. Although the bone marrow blast counts have increased since, this patient remains stable and is still on therapy through 14 cycles. Three of 6 MDS patients (50%), including the marrow CR, attained SD control with no disease progression over 3, 14 and 14 cycles, and 2 remain on study through 3 and 14 cycles, respectively. FF-10501-01 was rapidly absorbed with mean Tmax of 2.74 hours and mean t1/2 of 4.05 hours. Drug exposure (AUC0-24 and AUCcourse) increased with dose in a near linear manner. Potent suppression of circulating xanthine monophosphate (XMP), a marker of IMPDH activity, has been observed following FF-10501-01 administration on Day 1 of Cycles 1 and 2 at dose levels of 50 mg/m2 BID and above. FF-10501-01 is a promising new agent for the treatment of advanced AML and MDS in patients who have failed or progressed on HMAs and with one or more baseline mutations in pathways known to be affected in AML and MDS. Preclinical activity was seen in multiple leukemia cell lines, including HMA-resistant derivatives. In a Phase 1 trial, clinical activity with a marrow CR, PRs, long-term disease stabilization (≥ 5 cycles) and a highly tolerable safety profile were observed. The Phase 2a expansion phase of the study is soon to begin. Disclosures DiNardo: Agios: Research Funding; Daiichi Sankyo: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Abbvie: Research Funding. Jabbour:ARIAD: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Research Funding; BMS: Consultancy. Daver:BMS: Research Funding; Kiromic: Research Funding; Pfizer: Consultancy, Research Funding; Otsuka: Consultancy, Honoraria; Ariad: Research Funding; Karyopharm: Honoraria, Research Funding; Sunesis: Consultancy, Research Funding. Denton:Westat Corporation: Employment. Smith:Westat Corporation: Employment. Tiefenwerth:Westat Corporation: Employment. Iwamura:FUJIFILM Corporation: Employment. Gipson:Strategia Therapeutics, Inc.: Employment. Rosner:Strategia Therapeutics, Inc.: Employment. Myers:Strategia Therapeutics, Inc.: Employment. Paradiso:Strategia Therapeutics, Inc.: Employment.

Arsenic Trioxide and Melarsoprol Induce Programmed Cell Death in Myeloid Leukemia Cell Lines and Function in a PML and PML-RAR Independent Manner

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1497-1504 ◽  
Author(s):  
Zhu-Gang Wang ◽  
Roberta Rivi ◽  
Laurent Delva ◽  
Andrea König ◽  
David A. Scheinberg ◽  
...  
Keyword(s):  
Cell Line ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Nuclear Bodies ◽  
Independent Manner ◽  
Leukemia Cell Lines ◽  
Induced Apoptosis ◽  
Myeloid Leukemia Cell

Abstract Inorganic arsenic trioxide (As2O3) and the organic arsenical, melarsoprol, were recently shown to inhibit growth and induce apoptosis in NB4 acute promyelocytic leukemia (APL) and chronic B-cell leukemia cell lines, respectively. As2O3 has been proposed to principally target PML and PML-RAR proteins in APL cells. We investigated the activity of As2O3 and melarsoprol in a broader context encompassing various myeloid leukemia cell lines, including the APL cell line NB4-306 (a retinoic acid–resistant cell line derived from NB4 that no longer expresses the intact PML-RAR fusion protein), HL60, KG-1, and the myelomonocytic cell line U937. To examine the role of PML in mediating arsenical activity, we also tested these agents using murine embryonic fibroblasts (MEFs) and bone marrow (BM) progenitors in which the PML gene had been inactivated by homologous recombination. Unexpectedly, we found that both compounds inhibited cell growth, induced apoptosis, and downregulated bcl-2 protein in all cell lines tested. Melarsoprol was more potent than As2O3 at equimolar concentrations ranging from 10−7 to 10−5 mol/L. As2O3 relocalized PML and PML-RAR onto nuclear bodies, which was followed by PML degradation in NB4 as well as in HL60 and U937 cell lines. Although melarsoprol was more potent in inhibiting growth and inducing apoptosis, it did not affect PML and/or PML-RAR nuclear localization. Moreover, both As2O3 and melarsoprol comparably inhibited growth and induced apoptosis of PML+/+ and PML−/− MEFs, and inhibited colony-forming unit erythroid (CFU-E) and CFU granulocyte-monocyte formation in BM cultures of PML+/+ and PML−/− progenitors. Together, these results show that As2O3 and melarsoprol inhibit growth and induce apoptosis independent of both PML and PML-RAR expression in a variety of myeloid leukemia cell lines, and suggest that these agents may be more broadly used for treatment of leukemias other than APL. © 1998 by The American Society of Hematology.

Arsenic Trioxide and Melarsoprol Induce Programmed Cell Death in Myeloid Leukemia Cell Lines and Function in a PML and PML-RAR Independent Manner

Blood ◽  
1998 ◽  
Vol 92 (5) ◽  
pp. 1497-1504 ◽  
Author(s):  
Zhu-Gang Wang ◽  
Roberta Rivi ◽  
Laurent Delva ◽  
Andrea König ◽  
David A. Scheinberg ◽  
...  
Keyword(s):  
Cell Line ◽  
Arsenic Trioxide ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Leukemia Cell ◽  
Nuclear Bodies ◽  
Independent Manner ◽  
Leukemia Cell Lines ◽  
Induced Apoptosis ◽  
Myeloid Leukemia Cell

Inorganic arsenic trioxide (As2O3) and the organic arsenical, melarsoprol, were recently shown to inhibit growth and induce apoptosis in NB4 acute promyelocytic leukemia (APL) and chronic B-cell leukemia cell lines, respectively. As2O3 has been proposed to principally target PML and PML-RAR proteins in APL cells. We investigated the activity of As2O3 and melarsoprol in a broader context encompassing various myeloid leukemia cell lines, including the APL cell line NB4-306 (a retinoic acid–resistant cell line derived from NB4 that no longer expresses the intact PML-RAR fusion protein), HL60, KG-1, and the myelomonocytic cell line U937. To examine the role of PML in mediating arsenical activity, we also tested these agents using murine embryonic fibroblasts (MEFs) and bone marrow (BM) progenitors in which the PML gene had been inactivated by homologous recombination. Unexpectedly, we found that both compounds inhibited cell growth, induced apoptosis, and downregulated bcl-2 protein in all cell lines tested. Melarsoprol was more potent than As2O3 at equimolar concentrations ranging from 10−7 to 10−5 mol/L. As2O3 relocalized PML and PML-RAR onto nuclear bodies, which was followed by PML degradation in NB4 as well as in HL60 and U937 cell lines. Although melarsoprol was more potent in inhibiting growth and inducing apoptosis, it did not affect PML and/or PML-RAR nuclear localization. Moreover, both As2O3 and melarsoprol comparably inhibited growth and induced apoptosis of PML+/+ and PML−/− MEFs, and inhibited colony-forming unit erythroid (CFU-E) and CFU granulocyte-monocyte formation in BM cultures of PML+/+ and PML−/− progenitors. Together, these results show that As2O3 and melarsoprol inhibit growth and induce apoptosis independent of both PML and PML-RAR expression in a variety of myeloid leukemia cell lines, and suggest that these agents may be more broadly used for treatment of leukemias other than APL. © 1998 by The American Society of Hematology.

RNAi Screening Identifies BCL-XL As An Erythroid Lineage-Specific 5-Azacytidine Sensitizer While the BCL-2/BCL-XL/BCL-W Inhibitor ABT-737 Results in More Universal Sensitization in Leukemia Cells,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3513-3513 ◽  
Author(s):  
James M Bogenberger ◽  
Chang-Xin Shi ◽  
Irma Gonzales ◽  
Rodger E. Tiedemann ◽  
Pierre Noel ◽  
...  
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Ex Vivo ◽  
Leukemia Cell ◽  
Myeloid Cell ◽  
Myeloid Malignancies ◽  
Rnai Screening ◽  
Leukemia Cell Lines ◽  
Rational Combination

Abstract Abstract 3513 To identify targets for rational combination therapies with 5-Azacytidine (5-Aza) in myeloid malignancies, we utilized high-throughput RNA-interference (RNAi) viability screening. A siRNA library targeting 572 kinases and a custom collection of 289 putative cancer targets, including cell cycle and apoptosis regulatory genes, were screened alone and in combination with 5-Aza in TF-1 and ML-2 myeloid leukemia cell lines to identify synergistic interactions in reducing cell viability. Of the 572 kinases that were individually silenced, less than 1% significantly increased sensitivity to 5-Aza. The kinase library was also screened in combination with 5-Aza in a third myeloid cell line, THP-1, confirming that few kinases sensitize to 5-Aza when inhibited. While few kinases sensitized to 5-Aza, the anti-apoptotic Bcl-2 family of genes emerged as potent sensitizers to 5-Aza from RNAi screens. Therefore, silencing by siRNA of BCL-XL, BCL-2, BCL-W, MCL-1 and BFL-1 was evaluated in combination with 5-Aza treatment in an expanded panel of myeloid cell lines including TF-1, HEL, THP-1, ML-2 and MDS-L. BCL-XL validated as a vulnerability and potent sensitizer to 5-Aza in erythroid leukemia cell lines TF-1 and HEL, whereas MCL-1 was a strong vulnerability in the monocytic leukemia cell line THP-1 and also a moderate sensitizer to 5-Aza in ML-2, THP-1, TF-1 and HEL. Published proteomics data from our group indicate that M6 and M7 leukemias exhibit higher levels of BCL-XL, while additional unpublished data suggest elevated levels of MCL-1 in M4 and M5 leukemias, supporting our functional observations. Additionally, data from the public database Oncomine suggest that BCL-XL expression is elevated in M6 and M7 leukemias while MCL-1 shows a trend towards elevation in M4 and M5 leukemias. Based on RNAi screening results, siRNA validation experiments and proteomic/mRNA expression data, we evaluated the BCL-2/BCL-XL/BCL-W inhibitor ABT-737 in combination with 5-Aza. ABT-737 resulted in dose-dependent sensitization to 5-Aza in all AML-derived cell lines examined (including M7, M6, M5, M4 and M2 FAB subtypes) and in the MDS cell line MDS-L; however, no sensitization was observed in the CML cell line K562. In extensive ex vivo experiments with 17 primary specimens, potent synergy between 5-Aza and ABT-737 was observed in AML, MDS and MPN samples, but not in most CML samples examined. Calculations with CalcuSyn software demonstrate synergy, with combination index values as low as 0.2, between 5-Aza and ABT-737 both ex vivo and in vitro. The combination of 5-Aza with ABT-737 resulted in substantial induction of apoptosis, measured by the induction of cleaved caspase 3 in TF-1 and HL-60 cells, as compared to either compound alone. Interestingly, although siRNA silencing of MCL-1 in combination with 5-Aza was potent across several cell lines, and silencing of BCL-XL preferentially in an erythroid differentiation background, ABT-737 with 5-Aza sensitized across a variety of cell lines and all myeloid primary specimens ex vivo. We suggest that inhibition of anti-apoptotic Bcl-2 family members is a most promising rational combination strategy with 5-Aza for the treatment of leukemias. Our results also highlight the potential utility of more specific anti-apoptotic Bcl-2 family inhibitors in the lineage-specific treatment of myeloid malignancies. Disclosures: Off Label Use: AraC in AML. Experimental Agent MK1775. Mesa:Incyte: Research Funding; Lilly: Research Funding; SBio: Research Funding; Astra Zeneca: Research Funding; NS Pharma: Research Funding; Celgene: Research Funding.

Breakpoint junctions of chromosome 9p deletions in two human glioma cell lines

1994 ◽  
Vol 14 (11) ◽  
pp. 7604-7610
Author(s):  
H M Pomykala ◽  
S K Bohlander ◽  
P L Broeker ◽  
O I Olopade ◽  
M O Díaz
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Repetitive Sequences ◽  
Leukemia Cell ◽  
Chromosome 9 ◽  
Breakpoint Junction ◽  
Leukemia Cell Lines ◽  
Long Interspersed Nuclear Elements

Interstitial deletions of the short arm of chromosome 9 are associated with glioma, acute lymphoblastic leukemia, melanoma, mesothelioma, lung cancer, and bladder cancer. The distal breakpoints of the deletions (in relation to the centromere) in 14 glioma and leukemia cell lines have been mapped within the 400 kb IFN gene cluster located at band 9p21. To obtain information about the mechanism of these deletions, we have isolated and analyzed the nucleotide sequences at the breakpoint junctions in two glioma-derived cell lines. The A1235 cell line has a complex rearrangement of chromosome 9, including a deletion and an inversion that results in two breakpoint junctions. Both breakpoints of the distal inversion junction occurred within AT-rich regions. In the A172 cell line, a tandem heptamer repeat was found on either side of the deletion breakpoint junction. The distal breakpoint occurred 5' of IFNA2; the 256 bp sequenced from the proximal side of the breakpoint revealed 95% homology to long interspersed nuclear elements. One- and two-base-pair overlaps were observed at these junctions. The possible role of sequence overlaps, and repetitive sequences, in the rearrangement is discussed.

Effects of rmhTRAIL Combined with Daunorubicin in Inducing Apoptosis of Leukemia Cell Lines and Its Mechanism.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1986-1986
Author(s):  
Xuejun Zhang ◽  
Li Wen ◽  
Fuxu Wang ◽  
Ling Pan ◽  
Jianmin Luo ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Mrna Level ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Expression Level ◽  
U937 Cells ◽  
Leukemia Cell Lines ◽  
Before And After

Abstract Tumor Necrosis factor (TNF)-related apoptosis- inducing ligand (TRAIL) is a new member of TNF superfamily discovered recently. Several studies showed that TRAIL can preferentially induce apoptosis in a variety of tumor cells, while most normal cells tested do not appear to be sensitive to TRAIL. In the present study, we treated K562 and U937 leukemia cell lines with recombinant mutant human TRAIL (rmhTRAIL) alone or together with daunorubicin (DNR) to investigate the apoptosis of the treated cells and the synergistic reaction of rmhTRAIL and DNR. The normal cell line MRC-5 was used as control. The expression of four TRAIL receptors mRNA (death receptor DR4 and DR5, decoy receptor DcR1 and DcR2) in the cells lines were detected before and after the treatment by DNR. (1) AO-EB double staining and TUNEL staining were used to evaluate the morphological change of leukemia cell lines before and after the treatment. The results showed that rmhTRAIL could induce the apoptosis of leukemia cell lines and a dose-dependent manner was found in leukemia cell lines but not in MRC-5 cell lines. (2) The growth inhibition rate of leukemia cell lines induced by rmhTRAIL alone or combined with DNR was examined with MTT assays. Different concentrations of rmhTRAIL(8, 40, 200, 1000ng/mL)alone or combined with DNR(8, 40, 200, 1000ng/mL) was used. The result showed a dose-dependent growth inhibition by rmhTRAIL alone for K562- and U937-cell line (P<0.05) also, but not for MRC-5 cell line (P>0.05). The IC50 for K562 cells and for U937 cells had no statistic difference (538.80 vs 301.56ng/mL, P>0.05). In leukemia cell lines, the growth inhibition rates in combination groups were much higher than in rmhTRAIL or DNR alone groups (P<0.05), and no synergistic killing effects was found in MRC-5 cells (P<0.05). It was concluded that rmhTRAIL had synergistic effects with DNR in the growth inhibition of K562 and U937 cells. (3). To explore the antitumor mechanisms of rmhTRAIL combined with DNR, the expression level of the DR4, DR5 and DcR1, DcR2 mRNA in these three cell lines was examined by semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) before and after the treatment with DNR. The high expression of DR4,DR5 mRNA in the tested cells were observed before the treatment of DNR, while very low or even undetectable expression level of DcR1 and DcR2 mRNA were observed in U937 and K562 cells, and a high expression level of DcR1 and DcR2 mRNA in MRC-5 cells were observed. After 24 hours treatment of three cell lines with DNR (200ng/ml), the expression level of DR5 mRNA increased in K562 and U937 cells (P<0.05). DR4 mRNA also increased in K562 cells but not in U937 cells. There was no change in DcR1 and DcR2 mRNA level in three cell lines. The four receptors’ mRNA level in MRC-5 cells was not influenced by DNR. Our results indicated that rmhTRAIL could induce the apoptosis of leukemia cell lines, and DNR could enhance significantly the sensitivity of K562 and U937 cells to apoptosis induced by rmhTRAIL through up-regulation of death receptors. Therefore, we presumed TRAIL might be act as a new agent for biological therapy in leukemia.

Gain-of-Function KIT Mutations Sensitize the Mutant Isoform to the Type I Tyrosine Kinase Inhibitor Crenolanib: A Rationale for the Therapeutic Use in Systemic Mastocytosis (SM) and Core Binding Factor Leukemias (CBFL)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 2230-2230 ◽  
Author(s):  
Marcus M Schittenhelm ◽  
Figen Akmut ◽  
Barbara Illing ◽  
Julia Frey ◽  
Katja Schuster ◽  
...  
Keyword(s):  
Mast Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Ex Vivo ◽  
Systemic Mastocytosis ◽  
Leukemia Cell ◽  
High Sensitivity ◽  
Hematologic Malignancies ◽  
Leukemia Cell Lines ◽  
Nanomolar Range

Abstract Activating mutations of the class III receptor tyrosine kinases FLT3 and KIT are associated with certain human neoplasms, including hematologic malignancies, i.e. the majority of patients with systemic mast cell disorders (KIT) and subsets of patients with acute myelogenous leukemia (FLT3 and KIT). Crenolanib is a potent selective FLT3 inhibitor with high efficacy against internal tandem dupliction mutations (ITD) – but also secondary kinase domain mutations conferring resistance towards other TKI. Interestingly, crenolanib does not target the wildtype KIT isoform, which is believed to reduce clinical side effects such as prolonged myelosuppression observed with other TKI. Clinical studies are currently enrolling. We now show that gain-of-function mutations of KIT, including codon D816 alterations as the most prevalent mutation in SM and CBFL, sensitize the mutant isoform to crenolanib. Several mast cell and leukemia cell lines harboring autoactivating KIT or FLT3 isoforms were treated with crenolanib in dose dilution series (MOLM14, MV4;11, HMC1.1/1.2, p815). To minimize cell-type specific off-target effects, an isogenic cell model was established. The murine pro B-cell line Ba/F3 was retrovirally transduced with either a FLT3 ITD or a KIT D816 isoform. Apoptosis induction was analyzed by annexin V-based assays. FLT3/KIT tyrosine phosphorylation was assessed by western immunoblots. As previously described, the FLT3 ITD positive cell line MOLM14 revealed high sensitivity towards crenolanib with IC50s in the lowest nanomolar range. We also confirmed high sensitivity towards crenolanib ex vivo in the low nanomolar range in a native sample of a heavily pretreated patient. This patient relapsed with FLT3 ITD positive leukemia harboring a secondary D835H mutation in a subclone. Interestingly, leukemia cells in the relapse situation were much more oncogene-addicted than cells at primary diagnosis, which is in line with previous findings by others. Due to the structural homology of FLT3 D835 and KIT D816 mutations, we extended our studies to mutant-KIT mastocytosis and leukemia cell models and confirm clinically relevant antiproliferative as well as proapoptotic sensitivities towards crenolanib: for HMC mastocytosis cells harboring a KIT V560G and/or a D816V mutation, potent induction of apoptosis was observed with IC50s of 100-250nM. The murine p815 mastocytosis cell line (harboring a D814Y mutation corresponding to D816Y in humans) demonstrated a proapoptotic effect of crenolanib with an IC50 of 60 nM. Treatment of corresponding KIT or FLT3 isoform-transduced Ba/F3 cells confirmed similar IC50s in the leukemia cell lines. Parental Ba/F3cells did not show any sensitivity towards crenolanib up to concentrations of 1000 nM. Additionally, potent dephosphorylation at 100 nM of KIT D816V in Ba/F3 and HMC cells after exposure to crenolanib confirmed mutant-KIT as a target of the drug. Evaluation of a broader range of native mast cell and leukemia patient samples as well as additional leukemia cell lines and isogenic Ba/F3 KIT or FLT3 transfectants is ongoing. First results demonstrate activity of crenolanib in native cells of a subset of patient samples with SM or CBFL treated ex vivo. Even more, combination of crenolanib with anthracyclines revealed additive to superadditive proapoptotic effects. Moreover, combination of crenolanib with cladribine, a hallmark agent in the treatment of systemic mastocytosis, resulted in potent induction of apoptosis already at doses that did not display any proapoptotic effects when administered as single agents, thereby providing a rationale for combinatorial therapeutic approaches. In summary, crenolanib is effective against the KIT D816V isoform associated with several hematologic malignancies. Notably, while not as effective towards mutant-KIT compared to the FLT3 ITD isoform, the observed estimated IC50 of crenolanib is well in the range of achievable plasma concentrations and in the range of the potent KIT inhibitor dasatinib, which is successfully under clinical investigation in CBFL. Our data provide a rationale to test crenolanib as a potent inhibitor of mutant-KIT isoforms in KIT-associated neoplasms. Disclosures Schuster: AROG Pharmaceuticals: Employment. Ramachandran:AROG: Employment.

Preclinical Evaluation of the BET-Bromodomain (BET-BRD) Inhibitor OTX015 in Leukemia Cell Lines Harboring the JAK2 V617F Mutation

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 873-873
Author(s):  
Maria Eugenia Riveiro ◽  
Lucile Astorgues-Xerri ◽  
Charlotte Canet-jourdan ◽  
Mohamed Bekradda ◽  
Esteban Cvitkovic ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Line ◽  
Cell Lines ◽  
Research Funding ◽  
Leukemia Cell ◽  
Jak2 V617f ◽  
Jak2 V617f Mutation ◽  
Mrna Levels ◽  
Protein Levels ◽  
V617f Mutation

Abstract Background: Exposure of cancer cells to BET-BRD protein inhibitors has been associated with a significant downregulation of C-MYC expression, leading to suppression of the transcriptional program linked to proliferation and survival. C-MYC mRNA expression, mediated by STAT5 activation, is induced by the JAK2 (V617F) mutation (JAK2mu) in transfected BA/F3 cells (Funakoshi-Tago, et al. 2013). We selected JAK2mu leukemia-derived cell lines for preclinical evaluation of OTX015 (Oncoethix, Switzerland), a selective orally-bioavailable inhibitor of BET-BRD proteins with promising early results in an ongoing phase I study in hematologic malignancies (Herait et al, AACR 2014, NCT01713582). Material and Methods: Antiproliferative effects of OTX015 and JQ1 were evaluated in three established JAK2mu human myeloid leukemia cell lines (SET2, MUTZ8, HEL 92.1.7). GI50 (OTX015 concentration inducing 50% growth inhibition) and Emax (% cell proliferation at 6 µM OTX015) values were determined by MTT assay after 72h exposure. Protein levels were analyzed by Western blot, and RT-PCR was performed with Fast SYBR Green Master Mix on a StepOnePlus Real-Time PCR System. For cell cycle analysis, cells were stained with propidium iodide and analyzed with a FACScan flow cytometer. Induction of apoptosis was evaluated by Annexin-V. Simultaneous schedules of OTX015 combined with ruxolitinib, a JAK2 inhibitor, were evaluated. Combination index (CI) was determined using the Chou & Talalay method; CI<1 reflects synergy, CI=1 additivity and CI>1 antagonism. Results: After 72h exposure, SET2 was the most sensitive cell line (GI50=0.12 µM and Emax=15%), and HEL92.1.7 cells had a GI50=1.9 µM with an Emax=23%. MUTZ8 was the most resistant cell line with an Emax=61%. Similar GI50 and Emax values are observed with JQ1. A significant increase in the fraction of apoptotic cells was observed in SET2 cells after 72h 500 nM OTX015 exposure. Non-significant increases in Annexin-positive cells were seen in HEL92.1.7 and MUTZ8 cells. Cell cycle analysis revealed a significant increase in the percentage of SET2 cells in subG0/G1 after 24, 48, and 72h 500 nM OTX015, correlating with the increase in apoptosis. Conversely, an increase in the percent cells in the G1 phase was observed in HEL 92.1.7 cells. After 4h 500 nM OTX015, BRD2 mRNA levels were significantly increased in all three cell lines, whereas BRD3 levels were not modified. BRD4 mRNA levels increased significantly after 48h in SET2 cells. OTX015 treatment induced a transitory reduction of C-MYC mRNA levels after 4h with an increase at 24h in all cell lines. At the protein level, C-MYC decreased substantially in SET2 cells after 4h, with complete disappearance after 48h without recovery, while in the less sensitive MUTZ8 cell line, the decrease in C-MYC protein levels was transitory. Conversely, this proto-oncogene was not modified in HEL92.1.7 cells. In addition, p-STAT5 protein was downregulated by OTX015 in SET2 cells, but was increased in MUTZ8 cells after longer exposure time. Furthermore, BCL2 mRNA and protein levels decreased in SET2 cells, correlating with the apoptosis induction seen with OTX015 treatment. In HEL92.1.7 cells, P21 mRNA levels and cyclin D1 protein levels increased after 4h and 48h OTX015 treatment, respectively. Moreover, concomitant combination of OTX015 with ruxolitinib showed a highly antagonist effect (CI>7) in SET2 cells, the most sensitive cell line to both agents. On the other hand, very strong synergy was observed in HEL92.1.7 (CI=0.19) and MUTZ8 (CI=0.41), despite their low sensitivity to single agent OTX015. Conclusions. Our findings demonstrate that OTX015 exhibits potent activity against cultured leukemic cells expressing the JAK2 V617F mutation, inducing apoptosis or cell cycle arrest at submicromolar concentrations. This activity correlates with modulation of C-MYC, p-STAT5, BCL2, P21 and cyclin D1 mRNA and protein levels following OTX015 treatment. Our study highlights the novel and synergistic activity of the combination of a BRD antagonist and a JAK inhibitor in human leukemic cells harboring the JAK2 V617 F mutation, supporting the rationale for in vivo testing of OTX015 in combination with JAK inhibitors in leukemic JAK2mu models. Disclosures Riveiro: Oncoethix SA: Research Funding. Astorgues-Xerri:Oncoethix SA: Research Funding. Canet-jourdan:Oncoethix SA: Research Funding. Bekradda:Oncoethix SA: Research Funding. Cvitkovic:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Shareholder and CSO Other. Herait:Oncoethix SA: CMO and Shareholder Other. Raymond:Oncoethix SA: Membership on an entity's Board of Directors or advisory committees, Research Funding.

scholarly journals The 3-Drug Combination Treatment ART838, ABT199 Plus Sorafenib Is Effective Against AML Xenografts, Possibly Via Cooperative Targeting of MCL1

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 4044-4044
Author(s):  
Blake S Moses ◽  
Jennifer Fox ◽  
Xiaochun Chen ◽  
Samantha McCullough ◽  
Sang Ngoc Tran ◽  
...  
Keyword(s):  
Acute Leukemia ◽  
Board Of Directors ◽  
Cell Lines ◽  
Drug Combination ◽  
Research Funding ◽  
Leukemia Cell ◽  
Advisory Committees ◽  
Leukemia Cell Lines ◽  
Equity Ownership

Abstract Antimalarial artemisinins have broad antineoplastic activity in vitro, are well tolerated and inexpensive, and can be parenterally or orally administered in humans. Artemisinin-derived trioxane diphenylphosphate dimer 838 (ART838; a potent artemisinin-derivative) inhibited acute leukemia growth in vivo and in vitro, at doses where normal human CD34+ hematopoietic stem-progenitor cell clonogenicity was essentially unaffected (Fox et al, Oncotarget 2016, PMID: 26771236). In our focused drug combination screen for drugs that synergize with ART838, the only BCL2 inhibitors in the screen library of 111 emerging antineoplastic compounds, navitoclax (ABT737) and venetoclax (ABT199; FDA-approved), were identified as 2 of the top 3 candidates. Synergies between ART838 and BCL2 inhibitors were validated in multiple acute leukemia cell lines and primary cases. This ART838-BCL2 inhibitor synergy may be due to reduced levels of MCL1 protein that we and others have observed in multiple acute leukemia cell lines and primary cases treated with artemisinins (Budhraja et al, Clin Cancer Res 2017, PMID: 28974549). Treatment of acute leukemia xenografts with the ART838 plus ABT199 combination reduced leukemia growth rates and prolonged survivals, compared to vehicle or either ART838 or ABT199 alone. To add to the efficacy of this ART838 plus ABT199 treatment regimen, we sought to rationally add a third low-toxicity active antileukemic agent. Sorafenib (SOR; FDA-approved) inhibits multiple kinases which may mediate its antileukemic activity, with the importance of the targets varying from case to case; e.g. FLT3 is an important target in many AMLs. In addition, several reports have found that SOR reduces MCL1 protein stability and translation through inhibition of the ERK and PI3K pathways (Wang et al, Clin Cancer Res 2016, PMID: 26459180; Huber et al, Leukemia 2011, PMID: 21293487). In all acute leukemia cell lines tested, we observed large reductions in MCL1 protein levels with SOR treatment, which may further rationalize the addition of SOR to our ART838 plus ABT199 antileukemic regimen. We had previously observed strong in vitro synergy between ART838 and SOR (PMID: 26771236). Treatment of acute leukemia xenografts with the ART838 plus SOR combination reduced leukemia xenograft growth rates and prolonged survivals, compared to single drugs. Mice bearing luciferase-labelled acute leukemia xenografts were treated (PO daily x5) with single drug or 2-drug or 3-drug combinations of ART838, ABT199, and SOR, each at their individual maximally tolerated doses. Treatment with this 3-drug combination caused rapid regression of luciferase-labelled MV4;11 AML xenografts (Fig 1A). The 5-day treatment cycles were repeated every other week, and mice receiving this 3-drug combination survived >4 times longer than vehicle-treated mice (Fig 1B). Mouse body weights were stable during treatment. Although myelosuppression is the human clinical dose-limiting toxicity of each of these 3 drugs, mouse blood cell counts during 3-drug combination treatment were in the normal range. Treatment of a luciferase-labelled primary AML leukemia xenograft with this 3-drug combination reduced leukemia growth more than the single drugs or 2-drug combinations (Fig 1C). Assessment of efficacy and pharmacokinetics-pharmacodynamics against diverse acute leukemia xenografts will test this combination of ART838, ABT199 plus SOR as a rational low-toxicity drug triad for treatment of acute leukemias and potentially other cancers. Disclosures Fox: Intrexon Corporation: Employment. Tyner:Genentech: Research Funding; Janssen: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Constellation: Research Funding; Array: Research Funding; Takeda: Research Funding; Vivid Biosciences: Membership on an entity's Board of Directors or advisory committees; Aptose: Research Funding. Civin:ConverGene LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Research Funding; GPB Scientific LLC: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; 3DBioWorks Inc: Consultancy, Equity Ownership, Membership on an entity's Board of Directors or advisory committees; BD (Becton Dickinson): Honoraria.

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