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

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.

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.

scholarly journals Early Risk Prediction of BCR-ABL Kinase Domain Acquired Mutations in Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia

2021 ◽  
Author(s):  
Rongrong Chen ◽  
Lulu Wang ◽  
Ting Shi ◽  
Lixia Zhu ◽  
Xiujin Ye
Keyword(s):  
Logistic Regression ◽  
Acute Lymphoblastic Leukemia ◽  
Poor Prognosis ◽  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Multivariable Logistic Regression ◽  
Significant Difference ◽  
Philadelphia Chromosome Positive

Abstract Tyrosine kinase inhibitors (TKI) resistance is a predominant cause of therapy failure of Philadelphia chromosome-positive acute lymphoblastic leukemia (Ph+ ALL). The mutation in the BCR-ABL kinase domain, as the indicator of drug resistance, suggested recurrence and poor prognosis. Our data aimed to evaluate the factors having significant roles in the prognostic value of the acquired BCR-ABL mutations. Three hundred and thirteen Ph+ ALL patients (64 patients with BCR-ABL mutations and 171 patients without BCR-ABL mutations) were enrolled in this study. Univariate and multivariable logistic regression analysis was used to evaluate the relationship between clinical features and BCR-ABL KD mutation. Univariate analysis showed that the patients with higher WBC (P=0.005), MMR after first induced chemotherapy (P=0.053), and Age≥45 years (P=0.072) were more likely to occur BCR-ABL mutations during the treatments. Finally, multivariable logistic regression indicated that higher WBC (≥22×109/L) was an independent risk of resistance to TKI and raise the possibility of KD mutations in Ph+ ALL patients. The happen of BCR-ABL mutations mean poor prognosis with shorter OS (P=0.000) and allogeneic hematopoietic stem-cell transplantation (allo-HSCT) would improve the long-term survival (OS: P=0.000). Conversely, for the defined low-risk populations, no significant difference was found between the transplant group and the non-transplant group in subgroup analysis, providing a rationale to potentially avoid allo-HSCT in this subgroup of patients.

Correlation of Clinical Response to Dasatinib (BMS-354825) with BCR-ABL Mutation Status in Imatinib- Resistant Patients (pts) with Chronic Myeloid Leukemia (CML) Treated at MD Anderson Cancer Center (MDACC).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1091-1091 ◽  
Author(s):  
Elias Jabbour ◽  
Jorge Cortes ◽  
Moshe Talpaz ◽  
Dan Jones ◽  
Susan O’Brien ◽  
...  
Keyword(s):  
Lymphoblastic Leukemia ◽  
Philadelphia Chromosome ◽  
Chronic Phase ◽  
Cytogenetic Response ◽  
Kinase Domain ◽  
Cancer Center ◽  
Abl Kinase ◽  
Hematologic Response ◽  
Imatinib Resistant ◽  
Wide Range

Abstract Resistance to imatinib in CML occurs most frequently through mutations of the BCR-ABL kinase domain. Dasatinib is an orally available, dual SRC/ABL kinase inhibitor with 300-fold greater potency than imatinib, and with preclinical activity against all but one (T315I) type imatinib-resistant Bcr-Abl mutants. Dasatinib is currently used to treat pts with CML in chronic phase (CP), accelerated phase (AP), or blast phase (BP) or with Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL). We analyzed the response to dasatinib among pts with Bcr-Abl kinase mutations. Prior to therapy, peripheral blood samples were analyzed for mutation by DNA sequencing. 26 pts with 13 different imatinib-resistant point mutations in the Bcr-Abl kinase domain were treated (CP n=10, AP n=11, BP n=4, ALL n=1). The most common mutations were G250E/A (n=7, 27%), T315I (n=3, 12%), F317L (n=3, 12%), and E355G/A (n=3, 12%). 20 (77%) pts responded to therapy. Responses were: major molecular remission (MMR) in one pt (4%), complete cytogenetic response (CGCR) in 3 (12%), partial cytogenetic response (CGPR) in 5 (19%), complete hematologic response (CHR) in 6 (23%), partial hematologic response (PHR) in 3 (12%), and return to CP in 2 (8%). 6 pts did not respond: 3 of them were in AP (T315I, L364I, and G250E) and 3 in CP (2 T315I and F317L). Three pts (1 CP [F317L], 2 BP [M351T and E355G]) have lost their response (CGPR, CHR, and PHR) after a median of 3 months (range, 2–4 months) without developing any detectable new mutations. The median duration of response for the other 19 pts was 5+ months (range 1+–14+); 4 pts (1 CP [F486S], 3 AP [E255V and 2 G250E]) have a sustained response (1 MMR, 1 CGCR, and 2 CHR) beyond 6 months. Among 12 pts with P-loop mutations (2 in CP, 8 in AP, 2 in BP) 11 (92%) responded to therapy and their median survival since the start of therapy is 5+ months (range, 1+-11+ months). In 2 pts (CHR) subsequent analysis revealed persistence of mutations (E355G and E255V). In one pt G250E mutation occurred while patient was on treatment, but has remained in CHR. We conclude that clinical activity of dasatinib in imatinib-resistant CML is observed in pts with a wide range of imatinib-resistant Bcr-Abl kinase domain mutations. Pts harboring the T315I mutations are resistant to BMS-354825.

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