scholarly journals Molecular Mechanism for Synergistic Anti-Leukemic Activity of Tyrosine Kinase Inhibitors and Glucocorticoids Against Ph+ALL

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 1059-1059
Author(s):  
Meixian Huang ◽  
Takeshi Inukai ◽  
Keiko Kagami ◽  
Masako Abe ◽  
Tamao Shinohara ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tyrosine Kinase ◽  
Cell Lines ◽  
Induction Therapy ◽  
Kinase Inhibitors ◽  
Mapk Pathway ◽  
Gene Expression Level ◽  
Expression Level ◽  
Imatinib Resistant ◽  
T315i Mutation

Abstract The tyrosine kinase inhibitors (TKIs) have dramatically altered the management of patients with Philadelphia chromosome-positive ALL (Ph+ALL). In earlier trials of imatinib monotherapy for relapsed or refractory patients with lymphoid blast crisis of CML (CML-LC) and Ph+ALL, complete hematologic remission (CHR) was achieved only in 20%, and relapses occurred in most of the patients within several months. Thereafter, combination induction therapy of imatinib with conventional chemotherapeutic agents profoundly improved the therapeutic outcome in the patients with Ph+ALL. In elderly patients, to reduce the therapy-related toxicities, combination induction therapy of imatinib with glucocorticoids (GCs) followed by imatinib monotherapy was performed. Surprisingly, CHR was obtained in all of the patients with this simple therapy, and median survival from diagnosis was 20 months (Blood 2007). Similarly, induction therapy of dasatinib, a second-generation TKI, combined with GCs achieved CHR in all of the newly diagnosed Ph+ALL patients (Blood 2011). These clinical findings indicate a synergistic anti-leukemic activity of TKIs with GCs in Ph+ALL, but its underlying molecular mechanisms remain totally unknown. Thus, we analyzed synergistic effects of TKIs and dexamethasone (Dex) in a panel of leukemic cell lines derived from Ph+ALL. Indeed, in the presence of 0.5μM of imatinib, IC50 values of Dex were approximately 3-8 times lower than those in the absence of imatinib in the most of Dex-sensitive Ph+ALL cell lines. Since gene expression level of GC receptor (GR; NR3C1) was associated with Dex-sensitivity in Ph+ALL cell lines, we next analyzed the effects of imatinib on gene expression level of NR3C1. Of note, NR3C1 gene expression level was significantly upregulated in the presence of 0.5 μM of imatinib approximately 1.5-7-fold in all of 14 Ph+ALL cell lines except for SK9, which was an imatinib-resistant cell line having a T315I mutation of BCR-ABL, whereas it was unchanged in all of 11 Ph-negative ALL cell lines. Induction of GR was also confirmed by immuno-blotting in representative Ph+ALL cell lines. Moreover, treatment with either dasatinib or nilotinib clearly upregulated the NR3C1 gene expression in the representative Ph+ALL cell lines. Of importance, although gene expression level of NR3C1 was significantly upregulated in the presence of imatinib in the imatinib-sensitive Ph+ALL cell lines, SU-Ph2 and TCCY, it was unchanged in their imatinib-resistant sublines, SU/SR and TCCY/SR, respectively, in which T315I mutation was acquired after the culture with increasing concentrations of imatinib, indicating that upregulation of GR by TKIs in Ph+ALL cell lines was mediated by an inactivation of BCR-ABL. To further verify the downstream pathway of BCR-ABL that is critically involved in the TKI-induced GR upregulation, we treated imatinib-sensitive SU-Ph2 and its imatinib-resistant subline SU/SR with specific inhitors of PI3K (GDC0941, LY294002, and AS606240), JAK2(SD1029), and MAPK(UO126). Among five agents, only UO126 effectively upregulated the NR3C1 gene expression both in SU-Ph2 and in SU/SR, suggesting that TKIs upregulate GR in Ph+ALL cell lines mainly through an inactivation of the MAPK pathway. Previous reports revealed that three promoters, 1A, 1B, and 1C, are mainly involved in the NR3C1 gene expression in ALL cells. We therefore performed real time RT-PCR analysis of the NR3C1 gene using three sets of primers that are specific for exons 1A3, 1B, or 1C. The strongest induction by an imatinib-treatment was observed in the 1A promoter in Ph+ALL cell lines. Finally, since BIM, one of BH3-only pro-apoptotic members of BCL2 family, has been reported to be critically involved in the anti-leukemic activities of both TKIs and GCs, we analyzed BIM expression. Synergistic induction of BIM was confirmed both in mRNA and protein expression levels by a simultaneously treatment of Ph+ALL cell lines with imatinib and Dex. Taken together, these observations in Ph+ALL cell lines indicate that TKIs induce GR expression in Ph+ALL mainly through the MAPK pathway and the 1A promoter of NR3C1 gene by inactivating BCR-ABL and subsequently exert a synergistic anti-leukemic activity with GCs through the induction of BIM. Disclosures No relevant conflicts of interest to declare.

Association between the Polycomb Group (PcG) BMI-1 Gene Expression and Outcome in Chronic Myeloid Leukemia (CML) Patients Receiving Allogeneic Stem Cell Transplantation (allo-SCT).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 464-464
Author(s):  
Mohamad Mohty ◽  
Agnes S.M. Yong ◽  
Richard M. Szydlo ◽  
Jane F. Apperley ◽  
John M. Goldman ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Tyrosine Kinase ◽  
Acute Gvhd ◽  
Kinase Inhibitors ◽  
The Other ◽  
Expression Level ◽  
Low Bmi ◽  
Grade 3 ◽  
High Bmi

Abstract Despite a consistent molecular abnormality, the BCR-ABL oncogene, and the success of tyrosine kinase inhibitors, CML still exhibits a marked heterogeneity in prognosis. The PcG gene BMI-1 plays an essential role in regulating the proliferative activity of both normal and leukemic stem cells. BMI-1 correlation with outcome was described in different malignancies. We have previously shown that levels of BMI-1 RNA were significantly higher in advanced phase than in chronic phase (CP) CML. In addition, the level of BMI-1 at diagnosis correlated with time to transformation to blast crisis and, in patients treated with hydroxyurea and IFN-a, low BMI-1 expression was associated with an improved overall survival (OS), suggesting that BMI-1 may be a biomarker for the intrinsic heterogeneity of CML (Mohty et al., Blood 2007). Here, we investigated whether BMI-1 and the other previously established prognostic genes (CD7, PR-3 and ELA-2) are implicated in the prognosis of CML in the context of allo-SCT. For this purpose, we studied 84 CP-CML patients who received allo-SCT from HLA-identical related donors. CD7, PR-3, ELA-2 and BMI-1 expression was assessed by Q-RT/PCR in the recipient’s PBMCs collected before allo-SCT. The median expression level for each gene was used to segregate the patients into 2 groups (“low”: gene expression <median, and “high”: gene expression >median). The median follow-up post-allo-SCT was 9.9 (range, 1.7–23.9) years. The median EBMT-Gratwohl score was 3. None of the 4 tested genes showed any significant association with neutrophil or platelet engraftment, or with graft rejection. CD7, PR-3 and ELA-2 expression was not associated with OS. However, in sharp contrast to our previous findings in the non-allo-SCT setting, patients displaying a “high” BMI-1 expression level prior to allo-SCT had significantly better OS than those with “low” expression (P=0.005). When BMI-1 was included in a multivariate survival model and adjusted for the other prognostic variables (EBMT-Gratwohl score, allo-SCT era, and other relevant parameters), a high expression was found to be an independent marker associated with better survival (RR=2.72, 95%CI; 1.1–6.9; P=0.034). Given the impact of BMI-1 expression level on OS, without a significant association with relapse, and since neither BMI-1, nor the other genes showed any significant association with leukemia-free survival, we assessed their impact on transplant-related mortality (TRM). There was a striking and significant association between acute GVHD and BMI-1 expression, not only in overall incidence (“low” BMI-1: grade 0–1 (n=21), grade 2 (n=10), grade 3–4 (n=9); “high” BMI-1: grade 0–1 (n=32), grade 2 (n=9), grade 3–4 (n=1); P=0.005), but also in cumulative incidence at day 100 (48% vs. 24%, P=0.016). In multivariate analysis, a “low” BMI-1 expression level was associated with an increased risk of grade 2–4 acute GVHD (RR=2.85, 95%CI; 1.3–6.4; P=0.011). These results suggest that BMI-1 measured prior to allo-SCT can serve as a biomarker for predicting outcome in CP-CML patients receiving allo-SCT. The presence of a potential donor immune response against BMI-1, which is a genuine tumor-associated antigen, may abrogate any neoplastic proliferative advantage within the leukemia stem cell pool conferred by higher expression of BMI-1. Such measurement allows for tailored therapeutic intervention, including informed recommendation for allo-SCT in patients failing tyrosine-kinase inhibitors.

scholarly journals Involvement of Allele-Specific Methylation of Asparagine Synthetase Gene in Asparaginase Sensitivity of BCP-ALL

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3966-3966
Author(s):  
Atsushi Watanabe ◽  
Takeshi Inukai ◽  
Minori Tamai ◽  
Tamao Shinohara ◽  
Shinpei Somazu ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Cpg Island ◽  
Methylation Status ◽  
Gene Expression Level ◽  
Expression Level ◽  
Ic50 Value ◽  
Allele Specific ◽  
Specific Restriction ◽  
Allele Specific Methylation

Abstract Asparaginase is one of the most important components for the treatment of ALL. ALL cells are supposed to be unable to synthesize adequate amounts of Asparagine (Asn), and, therefore, depend on extracellular source of Asn to survive. Asparaginase therapy induces the depletion of serum Asn by catalyzing the deamination of Asn and leads to cell death of ALL cells. Asparagine synthetase (ASNS) is an enzyme that produces Asn from Aspartic acid. Thus, silencing of the ASNS gene in ALL cells could be crucial for complete starving ALL cells of the Asn. Considering that the ASNS gene has a CpG island in its promotor, aberrant methylation of CpG island could be one of epigenetic mechanisms for silencing of ASNS gene in ALL cells. Previous qualitative analysis of ALL samples using methylation-specific restriction enzyme revealed frequent methylation of CpG island in the ASNS gene. However, associations of methylation status of ASNS gene with its expression level and sensitivity to asparaginase in ALL cells remain unknown. Moreover, little is known about mechanisms for leukemia-specific ASNS gene silencing by methylation. To shed light on these issues, we analyzed a large panel of BCP-ALL cell lines. We quantified ASNS gene expression level by real time RT-PCR in 79 BCP-ALL cell lines cultured in the presence or the absence of L-asparaginase (L-asp), and determined IC50 values of L-asp using alamar blue assay. In the majority of cell lines, although degree of the induction was highly variable, ASNS gene expression level was upregulated in the presence of L-asp. IC50 value of L-asp showed significant correlation with ASNS gene expression level cultured in the presence of L-asp (r=0.222, p=0.049) rather than that in the absence of L-asp (r=0.193, p=0.089). We next analyzed methylation status of the ASNS gene in 79 BCP-ALL cell lines by bisulfite PCR sequencing using a next-generation sequencer (NGS). Strong correlation was confirmed between mean % methylation by NGS and Sanger sequencing in representative cell lines. Of importance, mean % methylation in 79 BCP-ALL cell lines showed significant negative correlation with ASNS gene expression level cultured in the presence of L-asp (r=-0.482, p=6.73x10-6) and, subsequently, IC50 value of L-asp (r=-0.39, p=3.86x10-4). Unexpectedly, % methylation of 79 cell lines distributed in three clusters; 15 cell lines (19%) were highly methylated (>66%, median; 89%), 26 cell lines (32.9%) were moderately methylated (33-66%, median; 40%), and 38 cell lines (48.1%) were weakly methylated (<33%, median; 3.7%). In the majority of moderately methylated cell lines, histograms of % methylation in each read of NGS showed two peaks of high and low methylation, suggesting an allele-specific methylation. In the middle of CpG island, tandem repeat polymorphism of 14bp nucleotides is located adjacent to methylation-specific restriction enzyme site of Aor13HI. Of note, in 7 out of 8 moderately methylated cell lines with heterozygous tandem repeat genotype, only single PCR product was detectable when PCR was performed after Aor13HI treatment, whereas two PCR products derived from two- and three-repeat alleles was detectable when PCR was performed without treatment, indicating an allele-specific methylation. We next analyzed a possible one-allele-loss of the ASNS gene in highly methylated (>66%; 8 cell lines) and weakly methylated (<20%; 12 cell lines) cell lines. We directly sequenced genotype in a portion of introns 2 and 4 and exon 5 based on the imputated SNP genotypes, and confirmed heterozygous genotype in every cell lines at least in one of eight SNPs analyzed, demonstrating that loss-of-heterozygosity is not the mechanism for high or low methylation of the ASNS gene. Similar pattern of methylation was observed in 52 BCP-ALL samples. Taken together, these observations indicate that stepwise allele-specific methylation of ASNSgene is critically involved in the sensitivity to L-asp of BCP-ALL. Disclosures No relevant conflicts of interest to declare.

Tyrosine Kinase Inhibitors as Potential Therapeutic Agents in the Treatment of Granulosa Cell Tumors of the Ovary

2015 ◽  
Vol 25 (7) ◽  
pp. 1224-1231 ◽  
Author(s):  
Stacey Jamieson ◽  
Peter J. Fuller
Keyword(s):  
Tyrosine Kinase ◽  
Cell Viability ◽  
Tyrosine Kinase Inhibitors ◽  
Granulosa Cell ◽  
Cell Lines ◽  
Cellular Proliferation ◽  
Kinase Inhibitors ◽  
Mapk Pathway ◽  
Granulosa Cell Tumors ◽  
Specific Subset

ObjectiveGranulosa cell tumors of the ovary (GCTs) represent a specific subset of malignant ovarian tumors, of which there are 2 distinct subtypes, the juvenile and the adult form. Aside from surgery, no reliable therapeutic options currently exist for patients with GCT. This study sought to investigate the potential role of small molecule tyrosine kinase inhibitors (TKIs) as novel therapeutics in the clinical management of GCT.Materials and MethodsUsing TKI with distinct but overlapping multitargeted specificities, cellular proliferation, viability, and apoptosis were evaluated in 2 human GCT-derived cell lines, COV434 and KGN.ResultsSunitinib, which targets the imatinib-inhibited tyrosine kinases of VEGFR, KIT, PDGFR, and FLT-3, was without effect in COV434 and KGN cell lines. Sorafenib, which has a high affinity for RAF1 and BRAF, dose dependently inhibited cellular proliferation and viability in both cell lines at concentrations equivalent to that seen in other systems. A RAF1 kinase inhibitor was without effect, suggesting that sorafenib is acting via inhibition of BRAF, or that aberrant signaling originates upstream of BRAF in the MAPK pathway. In the presence of a selective Src family inhibitor (SU6656), cell proliferation and cell viability responses dissociated; that is, although SU6656 dose dependently inhibited cell viability, it had limited effect on proliferation and apoptosis.ConclusionsThese findings implicate BRAF in the activated signaling responsible for the growth and viability of GCT and suggest that TKI already in clinical use may be a therapeutic option in the treatment of GCT.

Correlation Between c-MYC Gene Expression and Response after Induction Therapy Among Patients with Newly Diagnosed Multiple Myeloma and Monoclonal Gammopathy Undetermined Significance

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 5324-5324
Author(s):  
Maria Nareyko ◽  
Larisa P. Mendeleeva ◽  
Vadim Surin ◽  
Ekaterina Demidova ◽  
Olga S. Pokrovskaya ◽  
...  
Keyword(s):  
Gene Expression ◽  
Induction Therapy ◽  
Gene Expression Level ◽  
Expression Level ◽  
Newly Diagnosed ◽  
Antitumor Response ◽  
Qrt Pcr ◽  
Healthy Donors ◽  
Myc Gene ◽  
The Moment

Abstract Introduction. Patients (pts) with oncopathology who have hyperexpression of c-MYC demonstrate low response to chemotherapy and poor prognosis. In our study we investigated the level of c-MYC gene expression in pts withmultiple myeloma (MM) and/or monoclonal gammopathy undetermined significance (MGUS) at the moment of MM/MGUS diagnosis. Patients and Methods. 40 patients with newly diagnosed MM/MGUS and 9 healthy bone marrow donors (as a control group) were included in our study. The median age of patients with MM/MGUS was 56 years (29-77), donors - 32 years (24-41). The bone marrow collection for examination was done at the moment of diagnosis. The investigated material was divided into 2 samples: all mononuclears and separated CD138+ cells. Level of c-MYC expression was identified by quantitative real time PCR (qRT-PCR). The whole number of qRT-PCR analysis was 98. Induction therapy (VCD/PAD) was initiated for 36 patients with MM, 4 patients with MGUS remained under observation. Antitumor response was evaluated after induction therapy according to the IMGG criteria. Results. From 98 samples among pts reliable results qRT-PCR and evaluated c-MYC expression were in 36 samples from total mononuclears and 17 from separated CD138+ cells. In all 18 samples from healthy donors: 9 from total mononuclear, and 9 from CD138+ cells results of qRT-PCR were reliable. Mean c-MYC expression level among MM/MGUS pts was 55.014 (2ΔCt) in CD138+ cells and in samples from total mononuclears - 17.585 (2ΔCt). In healthy donors the results were 7.183(2ΔCt) and 1.907 (2ΔCt) respectively. After induction therapy complete response (CR) was demonstrated 6 patients, very good partial response (VGPR) - in 7 patients, partial response (PR) - in 15 and other 5 pts were refractory. Mean c-MYC expression level in samples from separated CD138+ cells in pts with CR was-14.259 (2ΔCt), with VGPR-38.314 (2ΔCt), with PR-50.506 (2ΔCt), and in refractory pts-181.271 (2ΔCt), among patients with MGUS the level was 10.928 (2ΔCt). In samples from mononuclears: 5.138, 30.355, 11.877, 38.911, 7.323 (2ΔCt) respectively. Thus, response after induction therapy was worse among patients with high gene c-MYC expression (Figure 1 a). Such a correlation was not revealed in analysis of data from samples of total mononuclears (Figure 1 b). Conclusion. Mean c-MYC gene expression level in CD138+ cells among MM/MGUS pts was higher than among healthy donors (p<0.05). Correlation between antitumor response and c-MYC gene expression level in CD138+ cells was found. Separation of cells to the membrane receptor of syndecan-1 (CD138 antigen) is necessary if we want to use the c-MYC gene expression level as a predictor of antitumor response. Disclosures No relevant conflicts of interest to declare.

Subcutaneous omacetaxine mepesuccinate in imatinib-resistant chronic myeloid leukemia (CML) patients (Pts) with the T315I mutation: Data from an ongoing phase II/III trial

2009 ◽  
Vol 27 (15_suppl) ◽  
pp. 7008-7008
Author(s):  
J. E. Cortes ◽  
H. J. Khoury ◽  
S. Corm ◽  
F. Nicolini ◽  
T. Schenk ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Kinase Inhibitors ◽  
Progression Free Survival ◽  
Median Number ◽  
Clinical Activity ◽  
Imatinib Resistant ◽  
T315i Mutation ◽  
Grade 3 ◽  
Induction Cycle ◽  
Number Of Cycles

7008 Background: Omacetaxine (OM), a first-in-class cetaxine shows clinical activity against Ph+ CML with a mechanism independent of tyrosine kinase inhibition. Currently available tyrosine kinase inhibitors (TKIs) have no activity against T315I. Methods: Adult Pts with T315I+ CML following TKI failure received OM induction at 1.25 mg/m2 subcutaneous (SC) twice daily (BID) for 14 days every 28 days followed by maintenance at 1.25 mg/m2 SC BID for 7 days every 28 days (maintenance after at least one induction cycle and achievement of hematologic response). Results: 66 pts (39 chronic [CP], 16 accelerated [AP] and 11 blast phase [BP]) have been enrolled. All had failed prior imatinib and 80% failed ≥2 prior TKIs. Median age is 58 yrs. Median disease duration is 58 mos. OM is well tolerated with transient myelosuppression as the primary toxicity. Grade 3/4 non-hematologic events are diarrhea (2%) and fatigue (4%). Efficacy data are available for 44 Pts. In CP Pts, the median number of cycles is 4 (1–22) with 39% having received ≥ 6 cycles of therapy; 64% of Pts have had the T315I clone reduced to below detection limits; the 2-year progression free survival is 70%. Conclusions: Omacetaxine in T315I+ CML Pts results in de-selection of the T315I clone and induces hematologic and cytogenetic responses. [Table: see text] [Table: see text]

scholarly journals Identifying Novel Mechanisms of Resistance to Tyrosine Kinase Inhibitors in Anaplastic Large Cell Lymphoma

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 5060-5060
Author(s):  
Elif Atabay ◽  
Qi Wang ◽  
Ambrogio Chiara ◽  
Taek-Chin Cheong ◽  
Silvia Peola ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Tyrosine Kinase ◽  
Candidate Genes ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Mapk Pathway ◽  
Specific Treatment ◽  
Large Cell ◽  
Anaplastic Lymphoma

INTRODUCTION: Anaplastic large cell lymphomas (ALCL) frequently carry oncogenic fusion proteins as a consequence of chromosomal translocations of the anaplastic lymphoma kinase (ALK) gene. The fusion protein resulting from the translocation between dimerization domain of nucleophosmin (NPM) and intracellular tyrosine kinase domain of ALK activates several signaling pathways, promoting cell growth, transformation, migration, and survival of the cells. Chemotherapy has been used as a standard treatment approach for ALCL patients, yet about 30% of patients relapse. A more specific treatment method is based on targeting ALK tyrosine kinase using tyrosine kinase inhibitors (TKIs). Crizotinib is an ALK TKI that is approved for the treatment of ALK-rearranged lung cancer and has received Breakthrough Therapy designation for lymphoma because of its high activity in chemo refractory ALCL. However, as for lung cancer, also ALCL patient develop crizotinib resistance due to ALK mutations or unknown mechanisms. In this study, we aimed at elucidating unknown by-pass mechanisms of crizotinib resistance in ALCL. METHODS: We used Genome-wide CRISPR-Cas9 Knockout Screening (GeCKO) to identify candidate genes that contribute to resistance to crizotinib. Four different ALCL cell lines were infected with Lenti-GeCKO libraries. After treatment with crizotinib for 14 days, DNA isolation and next generation sequencing was performed on crizotinib resistant cells to identify candidate genes depleted by the GeCKO screening. Top candidates were selected for validation assays and further analyses. RESULTS: We identified two phosphatases, PTPN1 and PTPN2, in different ALCL cell lines as consistent top hits. Functional validation of these candidate genes showed that single loss of either PTPN1 or PTPN2 generate immediate resistance to crizotinib in ALCL cell lines. Analysis of downstream pathways showed that while loss of PTPN1 activates primarily the MAPK pathway, loss of PTPN2 promotes persistent STAT3 and MAPK activation in ALK inhibited cells. Remarkably, in PTPN1 knockout cells we observed hyperactivation of SHP2, an oncogenic phosphatase that positively regulates the RAS-MAPK pathway. On the other hand, over-expression of PTPN1 and PTPN2 partially inhibited SHP2 phosphorylation. A treatment that combined crizotinib and the recently developed SHP2 inhibitor completely blocked the sustained ERK phosphorylation and reverted the crizotinib resistance observed in PTPN1 and PTPN2 deficient lymphoma cells. CONCLUSIONS: GeCKO library screen identified PTPN1 and PTPN2 as specific genes that mediate crizotinib resistance in ALCL cell lines. Loss of PTPN1 and PTPN2 drives resistance by activating MAPK and/or JAK-STAT pathway. Combined inhibition of SHP2 is a potent therapeutic approach to overcome resistance to crizotinib in ALCL cells. Disclosures Gambacorti-Passerini: Bristol-Meyers Squibb: Consultancy; Pfizer: Honoraria, Research Funding.

Activity of the Multi-Targeted Kinase Inhibitor, AT9283 on Imatinib-Resistant CML Models.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1104-1104 ◽  
Author(s):  
Ruriko Tanaka ◽  
Matthew S Squires ◽  
Shinya Kimura ◽  
Asumi Yokota ◽  
Kirsty Mallett ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Cell Lines ◽  
Kinase Inhibitors ◽  
Wild Type ◽  
Once Daily ◽  
Imatinib Resistant ◽  
Mutant Forms ◽  
In Vivo Effects

Abstract CML is caused by a consistent genetic abnormality, termed the Philadelphia chromosome, that results from a reciprocal (9;22) translocation leading to the expression of the BCR-ABL fusion protein. Although treatment has been revolutionized by the introduction of tyrosine kinase inhibitors which target Abl activity, reactivation of Abl signaling via several different point mutations remains problematic. In particular the mutation of Threonine 315 to Isoleucine (T315I) confers resistance to all existing therapies with tyrosine kinase inhibitors in the clinical settings. We describe the in vitro and in vivo effects of AT9283, a potent inhibitor of several protein kinases, including Abl kinase (wild type BCR-ABL and several of the drug resistant mutant variants that have arisen in clinical practice e.g. T315I), JAK2, JAK3 and Aurora kinases A and B, on imatinib-resistant CML cells including those harboring BCR-ABL (T315I). AT9283 has potent anti-proliferative activity in a panel of BaF3 and human cell lines expressing the BCR-ABL or its mutant forms. In BaF3 BCR-ABL wild-type and T315I mutant cells and K562 CML cells we observed inhibition of substrates of both BCR-ABL (STAT5) and Aurora B (Histone H3) at concentrations &gt;300nM and &lt;100nM, respectively, suggesting that AT9283 is capable of inhibiting Aurora and BCR-ABL simultaneously in these cell lines. The in vivo effects of AT9283 were examined in several mouse models engrafted either subcutaneously or intravenously with BaF3, human CML cell lines or primary CML patient samples expressing the BCR-ABL or its mutant forms. Specifically AT9283 prolonged the survival of mice engrafted intravenously with either BaF3 BCR-ABL T315I, or E255K cells when administered intraperitoneally twice daily at doses of either 6.25 or 10mg/kg or once daily at 15mg/kg when administered 5 days in every week repeated twice. Maximal survival advantage was conferred at either 10mg/kg twice daily or 15mg/kg once a day. Similar data were obtained in an intravenous model using primary CML cells taken from a patient harbouring the BCR-ABL E255K mutation. We also present data from ongoing studies showing increased survival rates in these in vivo model systems following multiple cycles of AT9283 administered on the 15mg/kg once daily schedule. These data together support further clinical investigation of AT9283 in patients with treatment resistant CML.

Sign in / Sign up

Export Citation Format

Share Document