Activity Of Omacetaxine Mepesuccinate Against Ponatinib Resistant Philadelphia Chromosome Positive Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3840-3840
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Yuko Tanaka ◽  
Seiichiro Katagiri ◽  
Toshihiko Kitahara ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Myeloid Leukemia ◽  
Philadelphia Chromosome ◽  
Point Mutations ◽  
Kinase Domain ◽  
Leukemia Cells ◽  
Primary Cells ◽  
Abl Kinase ◽  
Omacetaxine Mepesuccinate ◽  
High Level

Abstract Chronic myeloid leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric tyrosine kinase BCR-ABL. ABL tyrosine kinase inhibitor (TKI) therapy (e.g. imatinib, nilotinib and dasatinib) has improved the survival of Ph-positive leukemia patients. However, despite the impressive efficacy of these agents, disease relapse has been observed in clinically. Mutations in the BCR-ABL kinase domain can cause of ABL TKI resistance. In particular, one of the BCR-ABL kinase domain mutations (e.g. T315I) is associated with a high level of resistance to all available ABL TKIs. Ponatinib (formally, AP24534) is a multi-target TKI. Recently, in the PACE (Ponatinib Ph+ acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML) Evaluation) trial, ponatinib showed significant efficacy against Ph-positive leukemia in patients with multi-resistant T315I mutations. However, in some patients, especially those with Ph-positive ALL, ponatinib resistant clones were identified. Omacetaxine mepesuccinate, formally known as homoharringtonine is a natural alkaloid obtained from various Cephalotaxus species. Omacetaxine is a first-in-class cephalotaxine in clinical development as anti-leukemic therapy. Omacetaxine acts by binding to the A-site cleft of ribosomes and thereby transiently inhibits protein synthesis. Omacetaxine was approved for the treatment adult patients with chronic or the accelerated phase of CML resistant to other therapies. We investigated the efficacy of omacetaxine against ponatinib resistant Ph-positive cells. Ba/F3 ponatinib resistant cells (Ba/F3 ponatinib-R) have three BCR-ABL point mutations (Y253H, E255K and T315I: data not shown). With 72 h omacetaxine treatment, the cell growth of Ba/F3 ponatinib-R and Ph-positive ALL cell line was significantly reduced even at a low concentration and it is also effective to the other hematological malignancies such as acute myeloid leukemia. In contrast, Ba/F3 ponatinib-R was resistant to ponatinib. With 48 h treatment, omacetaxine dependent apoptosis was increased. Although anti-apoptotic proteins were not increased in this cell line compared to parental cells, as compound mutations such as E255V/T315I confer high-level resistance to ponatinib, these three point mutant was associated with vitro resistance to ponatinib. We also examined intracellular signaling. The phosphorylations of BCR-ABL and a down-stream molecule, Crk-L, were decreased. Protein expressions of BCR-ABL and Crk-L were also decreased. However, caspase-3 and cleaved Poly (ADP-ribose) polymerase (PARP) levels were significantly increased in low concentration. In a previous study, omacetaxine was shown to induce apoptosis in leukemic cells due to a selective decrease in short-lived proteins. We found that omacetaxine reduced the expression of BCR-ABL and heat shock protein 90(HSP90) which is stabilize BCR-ABL protein. We also found that omacetaxine reduced the expression of anti-apoptotic protein, Bcl-2. The protein expression of c-myc was also reduced. We next examined a ponatinib resistant primary Ph+ ALL and chronic phase CML samples. The ponatinib resistant primary cells have several BCR-ABL point mutations (e.g. Q252H, E255K/V, and T315I). We found the growth of primary cells was resistant to ponatinib but to be reduced after omacetaxine treatment and similar signaling events were occurred in OM-treated primary ALL cells. Omacetaxine is an inhibitor of protein synthesis. Because omacetaxine inhibits the BCR-ABL, Bcl-2 and HSP90 pathways in BCR-ABL positive leukemia cells through reduced the levels of these proteins, Omacetaxine has anti-tumor activity and promotes apoptosis. Our findings suggest that omacetaxine may benefit patients with leukemic BCR-ABL mutant cells, possibly allowing ponatinib resistant clones to be overcome. Disclosures: Ohyashiki: Novartis: Honoraria, Research Funding.

ShRNA Targeting p190BCR-ABL Successfully Eliminates Ph-ALL Cells with or without ABL Kinase Domain Mutation.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1838-1838
Author(s):  
Muneyoshi Futami ◽  
Toshiyuki Hatano ◽  
Yasushi Soda ◽  
Seiichiro Kobayashi ◽  
Makoto Miyagishi ◽  
...  
Keyword(s):  
Lentiviral Vector ◽  
High Titer ◽  
Therapeutic Option ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Kinase Domain ◽  
Leukemia Cells ◽  
Specific Cell ◽  
Dependent Manner ◽  
Abl Kinase

Abstract In the majority of Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (Ph-ALL) cases, the resulting BCR-ABL gene generates 190 kD active tyrosine kinase (p190) which is responsible for leukemogenesis and can be a molecular target for therapy. Although a series of ABL kinase inhibitors including imatinib, nilotinib and dasatinib reveal potent activities against Ph-ALL, acquired resistance caused by point mutations in the kinase domain such as T315I still remains to be overcome. That is why a novel strategy is desired in the treatment of Ph-ALL. We previously reported that lentiviral delivery of maxizyme targeting p190 specifically induced apoptosis of Ph-ALL cells (Blood 104:356, 2004). Since RNA interference proved to be a more powerful tool in selective gene silencing, we applied this technology to test whether specific and efficient killing of Ph-ALL cells could be achieved by down-regulation of p190. We designed a series of 21-mer and 27-mer small hairpin RNA (shRNA) targeting p190 mRNA and constructed plasmid vectors expressing these shRNA, which were screened by transfection of 293T/p190 cells to determine optimal target sites. As a result, three candidate sequences were identified; junctional 27-mer, junctional 21-mer and ABL 21-mer. Then, we inserted each of the shRNA expression cassettes into the lentiviral vector (HIV-U6/shRNA) and prepared high titer virus stock for infection of leukemia cells. shBCR-ABL/21, but not shBCR-ABL/27, induced significant and specific cell death of p190+ Ph-ALL cells in a time-dependent manner. shABL was more potent than shBCR-ABL/21 and also active against p210+ CML cells as well as 293 cells, but did not substantially affect Ph-negative leukemia cells. Both shABL and shBCR-ABL/21 completely inhibited growth of Ba/F3 cells harboring either wild-type or mutant p190 which renders those resistant to imatinib. Furthermore, both shRNA at low multiplicity of infection additively cooperated with imatinib in growth inhibition of Ba/F3-p190 cells. These data suggest that shRNA targeting p190 may become a therapeutic option in Ph-ALL by improvement of its delivery system like liposome. Growth of BA/F3-p190BCR-ABL Cells transduced with shRNA Targeting p190BCR-ABL Growth of BA/F3-p190BCR-ABL Cells transduced with shRNA Targeting p190BCR-ABL

Activities and Mechanism Based Combinations Of Omacetaxine In Acute Myeloid Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1288-1288
Author(s):  
Rong Chen ◽  
Bonnie Leung ◽  
Yuling Chen ◽  
William Plunkett
Keyword(s):  
Acute Myeloid Leukemia ◽  
Protein Synthesis ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Lymphocytic Leukemia ◽  
Leukemia Cells ◽  
Bh3 Mimetics ◽  
Abl Kinase ◽  
Acute Myeloid

Abstract Omacetaxine, an inhibitor of translation, was recently granted accelerated approval for the treatment of chronic myeloid leukemia (CML). Omacetaxine blocks translation elongation by competing with the incoming aminoacyl-tRNAs for binding to the A-site cleft in the peptidyl-transferase center. Our previous studies showed that by transiently inhibiting translation, omacetaxine reduced the expression of the key, short-lived oncoproteins Bcr/Abl and Mcl-1, leading to cell death in the CML cells. This action sensitized the cells to the Abl kinase inhibitor and killed the CML cells synergistically. Further, as omacetaxine acts in a different mechanism than the Abl kinase inhibitors, it overcame resistance to TKI that was associated with kinase domain mutations. These studies paved the foundation for the clinical development of omacetaxine in CML. We also demonstrated that omacetaxine was active in chronic lymphocytic leukemia by translational inhibition of Mcl-1 expression. In contrast to normal tissues, the fact that the leukemia cells are critically dependent on the oncogene activity for survival provided a biologic context for a positive therapeutic index. As the biological features of acute myeloid leukemia (AML) rely largely on the overexpressed oncoproteins or constitutively activated kinases, we hypothesized that omacetaxine would have therapeutic benefit in AML either alone or in mechanism based combinations. To test this hypothesis, first, we compared omacetaxine to AC220, a potent FLT3 inhibitor, in AML cell lines OCI-AML3 and MV4-11. OCI-AML3 cells harbor the signature mutation of NPM1, whereas MV4-11 is a widely used model for the internal tandem duplications of FLT3 (FLT3-ITD), a common FLT3 mutation that constitutively activates the receptor tyrosine kinase. AC220 was selectively toxic to the MV4-11 cells, but had no effect on the viability of OCI-AML3. This is consistent with the biological context of MV4-11, but not OCI-AML3, that is addicted to the sustained activity of FLT3 for survival. In contrast, omacetaxine induced apoptosis in both cell lines with IC50s less than 100 nM. Protein synthesis was inhibited in both lines, measured by the incorporation of tritiated leucine. Apoptosis was induced rapidly within 24 h by omacetaxine, whereas AC220 required 72 h to kill the leukemia cells. These results indicated a common dependence on the continued protein synthesis in the AML lines, suggesting a potentially broad application of omacetaxine in AML patients with diverse genetic backgrounds. Over-expression of the anti-apoptotic protein Mcl-1 is associated with AML disease maintenance and resistant to therapy. Both Mcl-1 and FLT3 turn-over rapidly and are vulnerable targets of transient translation inhibition. Immunoblots showed that omacetaxine reduced the levels of both FLT3 and Mcl-1 in the MV4-11 cells. This activity augmented the effect of AC220 on FLT3 kinase, and induced synergistic apoptosis. Same synergistic combination was observed with omacetaxine and sunitinib, an inhibitor of FLT3, KIT and PDGF-R. Dose reduction index derived from these analyses showed that omacetaxine greatly potentiated the activity of both AC220 and sunitinib, resulting in profound apoptosis. Both Bcl-2 and Mcl-1 are pro-survival proteins that regulate apoptosis by interacting with the BH3 motifs of their pro-apoptotic partners. BH3 mimetics, such as ABT-199, bind with high affinity to Bcl-2 and block this interaction, but not to Mcl-1. Resistance to BH3 mimetics in AML cells is associated with upregulation of Mcl-1. Since ABT-199 inhibits Bcl-2 but spares Mcl-1, and omacetaxine reduces Mcl-1 without affecting Bcl-2 expression, we hypothesized that their combination would target the two parallel arms of apoptosis control and kill the AML cells synergistically. Indeed, omacetaxine reduced Mcl-1 in the OCI-AML3 cells, leading to loss of mitochondrial membrane potential and apoptosis. ABT-199 blocked Bcl-2 function and also induced the intrinsic pathway of apoptosis. Their combination induced greater mitochondrial damage and apoptosis than either drug alone. The median effect analysis showed that they potentiate each other and exhibited strong synergy. Taken together, these results demonstrated that omacetaxine is active in AML cells alone and in mechanism based combinations. These actions provide rationale that warrants investigation in the clinic. Disclosures: No relevant conflicts of interest to declare.

Characteristics of BCR–ABL kinase domain point mutations in Chinese imatinib-resistant chronic myeloid leukemia patients

2010 ◽  
Vol 90 (1) ◽  
pp. 47-52 ◽  
Author(s):  
YaZhen Qin ◽  
ShanShan Chen ◽  
Bin Jiang ◽  
Qian Jiang ◽  
Hao Jiang ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Point Mutations ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Imatinib Resistant

Mutations in the BCR/ABL Kinase Encoding the Resistance to Imatinib Mesylate Do Not Modify the Sensitivity to Genotoxic Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2194-2194
Author(s):  
Milene Bargaoanu ◽  
Tomasz Skorski
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Point Mutations ◽  
Kinase Domain ◽  
Dna Lesions ◽  
Leukemia Cells ◽  
Activation Loop ◽  
Γ Irradiation ◽  
Abl Kinase

Abstract Imatinib mesylate (IM), a selective inhibitor of ABL kinase activity, revolutionized the treatment of BCR/ABL-positive leukemias. Unfortunately, clinical and experimental observations reveal that resistance to IM is a rising problem, which obscures an otherwise very successful oncogene-targeted therapy. IM resistance can be achieved by point mutations in the kinase domain of BCR/ABL and have been detected in 50–90% of patients with acquired resistance to IM, including ~23% of the IM-naive patients. Strategies to enhance the effect of IM and eventually overcome the resistance are dose escalation, addition of a growth factor, and combinations with novel tyrosine kinase inhibitors like AMN107 and dasatinib, or with inhibitors targeting downstream BCR/ABL effectors, e.g. PI-3k. Unfortunately, resistance to other small molecule inhibitors is likely to appear, as well. Therefore, we tested the sensitivity of leukemia cells expressing IM-resistant BCR/ABL mutants to genotoxic agents. Baf3 cells expressing similar levels of p210BCR/ABL wild-type (WT) and Y253F, Y253H, E255K, E255V, T315I, M351T, and H396P mutants were established. These mutants were selected since they represent several functionally distinct ABL kinase domain regions, including P-loop (Y253F/H and E255K/V), the site of a hydrogen bond with IM (T315I), the activation loop hinge (M351T), and the activation loop (H396P). In addition, they exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to IM. Western analysis demonstrated similarity, but also differences in the patterns of tyrosine phosphorylated proteins in total cell lysates. As expected, these clones displayed different level of sensitivity to IM: T315I>Y253H>E255V>E255K=Y253F>M251T=H396P>WT. However, leukemia cells expressing the WT and IM-resistant BCR/ABL kinase mutants demonstrated similar sensitivity to cytotoxic drugs such as hydroxyurea (HU), mitomycin C (MMC), and N-Methyl N’-nitro-N-nitrosoguanidine (MNNG). Surprisingly, clones expressing IM-resistant BCR/ABL kinase mutants were more sensitive to γ-irradiation than their WT counterparts. γ-H2AX (histone H2AX phosphorylated on S139) nuclear foci, which serve as marker of DNA double-strand breaks (DSBs), the most lethal DNA lesions, were detected by immunofluorescence. Leukemia cells expressing WT and IM-resistant p210BCR/ABL proteins accumulated similar numbers of DSBs after MMC treatment and γ-irradiation. This observation suggests that leukemia cells expressing IM-resistant p210BCR/ABL mutants may be eliminated by genotoxic treatment, especially γ-irradiation. In addition, IM-resistant BCR/ABL kinase mutants, in contrast to the WT kinase may not be able to modulate the mechanisms protecting from apoptosis induced by γ-irradiation.

Kinase Domain Point Mutations in Ph+ Acute Lymphoblastic Leukemia (ALL) and Lymphoid Blast Crisis of Chronic Myeloid Leukemia (CML) and Their Emergence Following Therapy with Bcr-Abl Kinase Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1831-1831
Author(s):  
Dan Jones ◽  
Rayjalakshmi Luthra ◽  
Hagop M. Kantarjian ◽  
Megan Breeden ◽  
Susan O’Brien ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Lymphoblastic Leukemia ◽  
Blast Crisis ◽  
Philadelphia Chromosome ◽  
Point Mutations ◽  
Kinase Domain ◽  
Current Therapy ◽  
Abl Kinase ◽  
T315i Mutation

Abstract Bcr-Abl kinase domain (KD) point mutations are detected in the dominant clone(s) in approximately 45% of CML at the time of disease resistance, developing after an average of 20–35 months of imatinib therapy. However, low numbers of Philadelphia chromosome (Ph)+ tumor cells with KD mutations could be present at earlier timepoints providing a pool of potential resistant subclones. Since current therapy of Ph+ ALL relies on imatinib maintenance therapy, the pattern of Bcr-Abl KD mutations in this tumor is an important and understudied phenomenon. We assessed the frequency and levels of Bcr-Abl KD mutations at different points in ALL, including at diagnosis, upon relapse and following salvage therapy with kinase inhibitors. We performed Bcr-Abl KD mutational analysis by direct sequencing in 25 cases of Ph+ ALL at the time of diagnosis and 25 cases upon disease persistence/relapse. For comparison, we analyzed 22 cases of lymphoid blast crisis of CML (LyBC), most of which transformed following long-term imatinib monotherapy. To track the emergence of mutated clones, we also performed more sensitive analysis for the T315I mutation by pyrosequencing (5% sensitivity) and allele-specific oligonucleotide probe (ASO) PCR (1:500 sensitivity). KD mutations were not seen by direct sequencing in ALL cases at diagnosis. The T315I mutation was also not detected by pyrosequencing (n =25) or ASO-PCR (n = 10) in newly diagnosed ALL. In contrast, Bcr-Abl KD mutations (Y253H in 3, Q252H, T315I, F317L, E355Q, H396R in 1 each) were seen in 8 of 25 (32%) relapsed/persistent ALL, occurring in patients who had been receiving imatinib for a median of 14 months (range 2–26). An additional 3 patients treated with dasatinib or nilotinib for relapse subsequently developed KD mutations (T315I and Y253H, and F317L) after 1, 4 and 9 months of second therapy. KD mutations were seen in 16 of 22 (73%) patients with lymphoid blast crisis, including T315I in 7, E255K and M244V in 2 each, and Y253H, V299L, F311I, E355G, F359V in 1 each. All KD mutations in LyBC developed following imatinib or nilotinib therapy. As with CML, kinase inhibitor therapy particularly in the relapse/salvage setting is the primary risk factor for emergence of Bcr-Abl KD mutations in Ph+ ALL. There is a high frequency of Bcr-Abl KD mutations associated the lymphoid transformation of CML. However, Bcr-Abl KD mutations develop more rapidly in persistent or relapsed Ph+ ALL than in CML and there is a higher frequency of Y253H mutations noted. These findings will likely have consequences for the timing and dosages of imatinib and other kinase inhibitors in maintenance and relapsed ALL regimens.

Examination of Point Mutations within the ABL Kinase Domain of Bcr-Abl Fusion Gene in Chinese Patients with Chronic Myeloid Leukemia Who Develop Imatinib Resistance.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4573-4573
Author(s):  
Dongguang Yang ◽  
Ri Zhang ◽  
Honglin Gang ◽  
Jian Gu ◽  
Jian Nong Cen
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Point Mutation ◽  
Mutation Rate ◽  
Myeloid Leukemia ◽  
Fusion Gene ◽  
Point Mutations ◽  
Kinase Domain ◽  
Chinese Patients ◽  
Single Mutation ◽  
Abl Kinase

Abstract Objective: To investigate the point mutations within the ABL kinase domain (KD) of Bcr-Abl fusion gene in Chinese patients with chronic myeloid leukemia who develop IM resistance and investigate the mutation. Method: We collect a total of 17 bone marrow samples obtained from 11 patients who showed hematology resistance(n=7) or cytogenetic refractoriness(n=4). A long semi-nest PCR method was used to amplify the ABL KD of the Bcr-Abl fusion gene. After two cycles of PCR reaction, we get a fragment including 863 bases, purified and sequenced it. Result: The ABL point mutation was detected in 45.45% of patients. The mutation rate of hematology resistance is 57.14% and the mutation rate of cytogenetic refractoriness is 25%. In total, we find three point mutation in all of our patients tested, ie, G250E(2), E255K(1) and T315I(2). In patients who develop hematology resistance, the earliest time to detect mutation is 4 months later after the therapy of IM and the longest time to detect mutation is 9 months later after the therapy of IM. In patients who develop cytogenetic refractoriness, the time to detect mutation is 12 months later after the therapy of IM .For the patients whose samples are available, no single mutation happens before IM thearpy. Conclusion: There is high frequency of point mutations clustered within the adenosine triphosphate-binding region of Bcr-Abl fusion gene in Chinese patients with chronic myeloid leukemia, and the mutation is related to IM therapy. It’s good for patients to switch to another therapeutic strategy when the mutations were detected earlier. Figure Figure

BCR-ABL kinase domain mutation analysis in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors: recommendations from an expert panel on behalf of European LeukemiaNet

Blood ◽  
2011 ◽  
Vol 118 (5) ◽  
pp. 1208-1215 ◽  
Author(s):  
Simona Soverini ◽  
Andreas Hochhaus ◽  
Franck E. Nicolini ◽  
Franz Gruber ◽  
Thoralf Lange ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Mutation Analysis ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Direct Sequencing ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Abl Kinase

AbstractMutations in the Bcr-Abl kinase domain may cause, or contribute to, resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia patients. Recommendations aimed to rationalize the use of BCR-ABL mutation testing in chronic myeloid leukemia have been compiled by a panel of experts appointed by the European LeukemiaNet (ELN) and European Treatment and Outcome Study and are here reported. Based on a critical review of the literature and, whenever necessary, on panelists' experience, key issues were identified and discussed concerning: (1) when to perform mutation analysis, (2) how to perform it, and (3) how to translate results into clinical practice. In chronic phase patients receiving imatinib first-line, mutation analysis is recommended only in case of failure or suboptimal response according to the ELN criteria. In imatinib-resistant patients receiving an alternative TKI, mutation analysis is recommended in case of hematologic or cytogenetic failure as provisionally defined by the ELN. The recommended methodology is direct sequencing, although it may be preceded by screening with other techniques, such as denaturing-high performance liquid chromatography. In all the cases outlined within this abstract, a positive result is an indication for therapeutic change. Some specific mutations weigh on TKI selection.

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