DCC-2036: A Novel Switch Pocket Inhibitor of ABL Tyrosine Kinase with Therapeutic Efficacy Against BCR-ABL T315I In Vitro and in a CML Mouse Model.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 463-463 ◽  
Author(s):  
Richard A. Van Etten ◽  
Wayne W. Chan ◽  
Virginia M. Zaleskas ◽  
Peter Evangelista ◽  
Katherine Lazarides ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
De Novo ◽  
Acquired Resistance ◽  
Peripheral Blood Leukocyte ◽  
Kinase Domain ◽  
Leukemic Cells ◽  
Prolonged Survival ◽  
Structure Based Drug Design ◽  
Abl Kinase

Abstract Therapy with ATP-competitive ABL kinase inhibitors, including imatinib (Gleevec), dasatinib (Sprycel), and nilotinib (Tasigna), has revolutionized the treatment of chronic myeloid leukemia (CML), but a substantial proportion of patients develop resistance to these agents. A major mechanism of acquired resistance is mutations in the ABL kinase domain that render BCR-ABL insensitive to the drug; in particular, mutation of the ABL kinase “gatekeeper” Thr315 residue to Ile (T315I) confers pan-resistance to all ATP-competitive ABL inhibitors. To address this need, we developed and characterized a novel chemical class of compounds that bind to five structural pockets involved in the endogenous “switch” mechanism used by the ABL kinase to conformationally control its activity state. Using a structure-based drug design approach, diversity in these “switch pockets” between kinases can be exploited to develop inhibitors with high potency and specificity. The resulting ABL inhibitors have several attractive features, as they: act through a non-ATP-competitive mechanism; avoid steric clash with Ile315; inhibit purified ABL that is either unphosphorylated (switch-off) or phosphorylated (switch-on) at Tyr393 with IC50 of 0.8–4.0 nM; co-crystallize with phospho-Y393-ABL WT and T315I; and have very prolonged residency time on the kinase (off-rate ∼400 min vs. 3 min for imatinib). A lead compound, DCC-2036, is highly selective for ABL, FLT3, and SRC family kinases, and has favorable pharmacokinetic and toxicity profiles in animals. In vitro, DCC-2036 inhibited proliferation and induced apoptosis of Ba/F3 cells expressing BCR-ABL WT or several common imatinib-resistant mutants (Y253F, T315I, M351T) with IC50 from 5 to 25 nM, without appreciable inhibition of parental Ba/F3 cells in IL-3 (IC50 >1000 nM). In Balb/c mice injected with Ba/F3-BCR-ABL T315I cells, pharmacodynamic studies indicated >8 hour inhibition of phospho-Stat5 and phospho-ABL in the leukemic cells following a single oral 100 mg/kg dose of DCC-2036, whereas daily dosing of DCC-2036 significantly prolonged survival. In the mouse retroviral bone marrow transduction/transplantation model of CML, DCC-2036 at 100 mg/kg/d also reduced peripheral blood leukocyte counts and significantly prolonged survival vs. vehicle-treated control mice. These results demonstrate that ABL switch pocket inhibitors are a promising new pharmacologic weapon for the treatment of de novo and drug-resistant CML, including BCR-ABL T315I.

FGF2 Mediates Resistance In CML Patients In The Absence Of Kinase Domain Mutations, and Resistance Is Overcome By Ponatinib

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3983-3983
Author(s):  
Elie Traer ◽  
Nathalie Javidi-Sharifi ◽  
Anupriya Agarwal ◽  
Jennifer B Dunlap ◽  
Isabel English ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stromal Cells ◽  
Kinase Inhibitors ◽  
K562 Cells ◽  
Kinase Domain ◽  
Bone Marrow Microenvironment ◽  
Abl Kinase ◽  
Mechanism Of Resistance ◽  
Kinase Domain Mutations

Abstract Background Development of resistance to kinase inhibitors remains a challenge in chronic myeloid leukemia (CML). Kinase domain mutations are a common mechanism of resistance, yet the mechanism of resistance in the absence of mutations remains less clear. Recent evidence suggests that the bone marrow microenvironment provides a sanctuary for leukemia cells, and may be involved in mediating resistance to imatinib – particularly in the absence of BCR-ABL kinase domain mutations. We tested selected cytokines, growth factors, and extracellular matrix proteins expressed by cells in the bone marrow microenvironment for their ability to protect CML cells from imatinib. Results We found that fibroblast growth factor 2 (FGF2) was the most protective protein for the K562 CML cell line when exposed to imatinib. FGF2 was not only capable of promoting growth in short-term culture, but uniquely able to promote long-term resistance in vitro (p<0.0001 by 2-way ANOVA analysis). To analyze the mechanism of resistance, we used siRNA to target the FGF receptors 1-4 and found that only siRNA targeting FGFR3 was able to abrogate the protective effect of FGF2. Phospho-chip and Western blot analysis revealed that FGF2 binds FGFR3, which then signals the downstream kinases Ras, c-RAF, MEK1, and ERK1/2 to promote survival in the presence of imatinib. Inhibition of FGFR3 with the specific FGFR inhibitor PD173074 led to dephosphorylation of this signaling cascade, and restored sensitivity to imatinib of FGF2-mediated resistant K562 cells. Resistance could also be overcome with ponatinib, a multi-kinase inhibitor that targets both BCR-ABL and FGFR, whereas imatinib, nilotinib and dasatinib were all ineffective against FGF2-mediated resistant K562 cells. Although ponatinib was rationally designed to circumvent the BCR-ABL T315I gatekeeper mutation, it was also able to achieve major cytogenetic responses in 62% of patients without detectable kinase domain mutations in the recent PACE trial. We theorized that increased FGF2 may drive resistance in the subset of patients without kinase domain mutations who respond to ponatinib, similar to our in vitro findings. To evaluate this possibility, we identified patients without kinase domain mutations who were responsive to ponatinib and quantified bone marrow FGF2 by immunohistochemistry. In comparison to ponatinib-responsive patients with kinase domain mutations, patients without kinase domain mutations had increased FGF2 in their bone marrow (50.5% versus 36.6%, p=0.033). Moreover, FGF2 in the marrow decreased concurrently with response to ponatinib, further suggesting that FGF2-mediated resistance is interrupted by FGFR inhibition (-15.9% versus 0.8%, when compared to the change in FGF2 of patients with kinase domain mutations, p=0.012). Qualitatively, FGF2 was predominantly localized in supportive stromal cells (consistent with previous reports), supporting a paracrine mechanism of resistance. Furthermore, we also evaluated a single patient without kinase domain mutations who was resistant to ponatinib. In this patient’s marrow, there was no elevation in FGF2 or change in FGF2 with ponatinib treatment. Taken together, inhibition of FGFR appears to be critical for the clinical activity of ponatinib in patients without kinase domain mutations. Conclusions In summary, our data supports a model of resistance in which FGF2 production by the marrow stromal cells promotes resistance to multiple ABL kinase inhibitors without the need for mutation of the ABL kinase domain. Resistance occurs via FGF2 ligand-induced activation of the FGFR3/Ras/MAPK pathway, and can be overcome by concomitant inhibition of ABL and FGFR. In combination with recent clinical data with ponatinib, our data suggest that FGF2-mediated resistance is a major mechanism of resistance in CML patients without kinase domain mutations. These results illustrate the clinical importance of ligand-induced resistance to kinase inhibitors and support an approach of developing rational inhibitor combinations to circumvent resistance, particularly in other kinase-driven malignancies that routinely develop resistance to kinase inhibitors. Disclosures: Tyner: InCyte Corporation: Research Funding. Druker:Novartis, Bristol-Myers Squibb, & ARIAD: Novartis, BMS & ARIAD clin trial funding. OHSU holds contracts; no salary/lab research funds. OHSU & Druker have financial interest in MolecularMD; technology used in some studies licensed to MolecularMD. This conflict reviewed and managed by OHSU. Other.

Comparison of Imatinib, AMN107 and Dasatinib in an Accelerated Cell-Based Mutagenesis Screen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 691-691 ◽  
Author(s):  
Michael W.N. Deininger ◽  
Heather Bradeen ◽  
Taiping Jia ◽  
Thomas O’Hare ◽  
Stephanie G. Willis ◽  
...  
Keyword(s):  
Structural Changes ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Drug Concentrations ◽  
A Cell ◽  
Vitro Model ◽  
Proliferation Assays ◽  
Abl Kinase Inhibitors

Abstract Background. Mutations in the Bcr-Abl kinase domain (KD) are the leading cause of acquired imatinib (IM) resistance. Dasatinib (BMS354825) and AMN107 are potent alternate Abl inhibitors with activity at nanomolar levels against wild type Bcr-Abl and most KD mutants, with the exception of T315I. In a cell-line based mutagenesis assay we compared incidence and type of Bcr-Abl mutants emerging in the presence of IM, AMN107 and dasatinib. Methods. BaF3-p210Bcr-Abl cells were mutagenized by 24 hours exposure to 0.42 μM N-ethyl-N-nitrosourea (ENU), a dose with minimal cytotoxicity. After ENU washout cells were seeded at 5 x 105/well in 96-well plates and observed for growth for up to 4 weeks. Cells from wells with growth were expanded and subjected to BCR-ABL KD sequencing. Results: The frequency of wells with growth decreased with higher doses of all 3 inhibitors (table 1) and growth tended to occur later. Only isolated wells had growth without ENU exposure. At ≥2 μM IM (2-fold the IC90 in cell proliferation assays) 18 different mutations were seen, with highly resistant mutants prevailing at higher concentrations (table 2). At 50 nM AMN107 (2-fold the IC90) Y253H, G250E, F359C, E255K, L384M, L387F, E292V and T315I were detected, at 500 nM Y253H, E255V and T315I were recovered and only T315I at 2000 nM. At 5 nM dasatinib (2-fold the IC90), E255K, L284V, F317V were detected in addition to T315I, at 10 nM T315I, F317V/I and V299L were found and at 25 nM only T315I. All resistant clones growing out at ≥4 μM IM, 500 nM AMN107 or 10 nM dasatinib were KD mutant, suggesting that KD mutations were the sole cause of the observed resistance. Conclusions: (i) At drug concentrations corresponding to 2-fold IC9018 different mutations were recovered with IM, 9 with AMN107 and 6 with dasatinib, suggesting that the conformational requirements for dasatinib binding to Abl may be least stringent. If free plasma trough levels ≥25 nM dasatinib or ≥2000 nM AMN107 are achievable, the only mutant predicted to emerge clinically is T315I. (ii) No additional mutations were observed with AMN107 compared to IM, suggesting the structural changes in AMN107 compared to IM did not generate novel vulnerable sites. (iii) At least in this in vitro model, resistance to Abl kinase inhibitors is entirely dependent on Bcr-Abl, despite the fact that ENU treatment is expected to induce multiple additional mutations. Thus a T315I inhibitor combined with AMN107 or dasatinib may be effective at preventing the emergence of resistance to Abl kinase inhibitors. Table 1 Recovery of resistant clones (representative experiment) Imatinib (microM) Wells with mutations/wells sequenced/wells with growth Dasatinib (nM) Wells with mutations/wells sequenced/wells with growth AMN107 (nM) Wells with mutations/wells sequenced/wells with growth 2 62/62/82 5 8/24/96 10 0/24/96 4 74/74/74 10 38/38/56 50 20/24/96 8 27/27/42 25 22/22/24 500 45/45/46 16 12/12/12 100 20/20/21 2000 23/23/24 Table 2 Percentage of resistant clones with T315I mutations Imatinib (μM) number of different mutations/% T315I Dasatinib (nM) number of different mutations/% T315I AMN107 (nM) number of different mutations/% T315I 2 16/27.8 5 4/16.7 10 0/0.0 4 7/43.2 10 3/63.2 50 8/20.8 8 4/37.4 25 1/100 500 3/62.0 16 4/50.0 100 1/100.0 2000 1/100.0

Dynamics of BCR-ABL kinase domain mutations in chronic myeloid leukemia after sequential treatment with multiple tyrosine kinase inhibitors

Blood ◽  
2007 ◽  
Vol 110 (12) ◽  
pp. 4005-4011 ◽  
Author(s):  
Jorge Cortes ◽  
Elias Jabbour ◽  
Hagop Kantarjian ◽  
C. Cameron Yin ◽  
Jianqin Shan ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Differential Sensitivity ◽  
Mutation Site ◽  
Abl Kinase

AbstractDasatinib and nilotinib are potent tyrosine kinase inhibitors (TKIs) with activity against many imatinib-resistant chronic myeloid leukemia (CML) clones with BCR-ABL kinase domain (KD) mutations, except T315I. We assessed for changes in the BCR-ABL KD mutation status in 112 patients with persistent CML who received a second-generation TKI after imatinib failure. Sixty-seven different KD mutations were detected before the start of therapy with a second TKI, with T315I seen in 15%. Equal numbers of patients received nilotinib or dasatinib following imatinib, and 18 received 3 TKIs. Response rates were similar for patients with and without mutations, regardless of mutation site except for T315I. Overall, 29 patients (26%) developed new KD mutations after therapy with a second (n = 24) or third (n = 5) TKI, but only 4 (4%) developed T315I. In 73% of cases, the KD mutations that persisted or developed following switch to new TKI were at sites also found in prior in vitro TKI mutagenesis assays. Although there is only a mild increase in mutation frequency with sequential TKI treatment, novel mutations do occur and mutation regression/acquisition/persistence generally reflects the in vitro differential sensitivity predicted for each TKI. In this study, there was no marked increase in development of T315I.

Kinase domain mutations of BCR-ABL frequently precede imatinib-based therapy and give rise to relapse in patients with de novo Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL)

Blood ◽  
2007 ◽  
Vol 110 (2) ◽  
pp. 727-734 ◽  
Author(s):  
Heike Pfeifer ◽  
Barbara Wassmann ◽  
Anna Pavlova ◽  
Lydia Wunderle ◽  
Johannes Oldenburg ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Kinase Inhibitors ◽  
De Novo ◽  
Cell Clone ◽  
Remission Rate ◽  
Lymphoblastic Leukemia ◽  
Kinase Domain ◽  
Leukemic Cells ◽  
Newly Diagnosed ◽  
Imatinib Resistance

Abstract Acquired imatinib resistance in advanced Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) has been associated with mutations in the kinase domain (KD) of BCR-ABL. We examined the prevalence of KD mutations in newly diagnosed and imatinib-naive Ph+ ALL patients and assessed their clinical relevance in the setting of uniform frontline therapy with imatinib in combination with chemotherapy. Patients enrolled in the German Multicenter Study Group for Adult Acute Lymphoblastic Leukemia (GMALL) trial ADE10 for newly diagnosed elderly Ph+ ALL were retrospectively examined for the presence of BCR-ABL KD mutations by denaturing high-performance liquid chromatography (D-HPLC), cDNA sequencing, and allele-specific polymerase chain reaction (PCR). A KD mutation was detected in a minor subpopulation of leukemic cells in 40% of newly diagnosed and imatinib-naive patients. At relapse, the dominant cell clone harbored an identical mutation in 90% of cases, the overall prevalence of mutations at relapse was 80%. P-loop mutations predominated and were not associated with an inferior hematologic or molecular remission rate or shorter remission duration compared with unmutated BCR-ABL. BCR-ABL mutations conferring high-level imatinib resistance are present in a substantial proportion of patients with de novo Ph+ ALL and eventually give rise to relapse. This provides a rationale for the frontline use of kinase inhibitors active against these BCR-ABL mutants.

Dynamics of BCR-ABL Kinase Domain Mutations in Patients with Chronic Myeloid Leukemia (CML) after Treatment with One, Two or Three Tyrosine Kinase Inhibitors (TKI).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 750-750 ◽  
Author(s):  
Elias Jabbour ◽  
Dan Jones ◽  
Hagop Kantarjian ◽  
Susan O’Brien ◽  
Guillermo Garcia-Manero ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Imatinib Resistance ◽  
Mutation Status ◽  
Abl Kinase ◽  
Kinase Domain Mutations ◽  
New Mutations

Abstract Dasatinib (D) and nilotinib (N) are potent tyrosine kinase inhibitors (TKIs) with activity against many imatinib (IM) resistant BCR-ABL kinase domain mutants, except T315I. In vitro mutant models have selected specific mutations occurring after incubation with IM, D and N. Therapy with these new TKI may select for patients with T315I or other mutations relatively insensitive to them. We assessed the change in mutation status of the bcr-abl kinase domain (codons 220 to 500) in 113 patients (pts) with CML who received therapy with D and/or N after imatinib failure. Median age was 60 years (range, 21 to 82 years). Seventy-one (63%) pts received prior interferon (IFN). Median time on imatinib was 28 months (range, 2 to 78 months). At the time of imatinib failure, mutations were detected in 46 of 85 (54%) pts who had DNA sequencing. The evolution of mutations after a second TKI was as follows (Table 1). Twenty pts received a third TKI after failing IM and a second TKI. The evolution of mutations in this cohort was as follows (Table 2). Overall, 19 of 101 evaluable pts (19%), cases had new mutations emerge following TKI switch 17 after a 2nd TKI (12 nilotinib, 5 dasatinib), and 2 after a 3rd TKI (2 dasatinib). We analyzed whether these N- and D-associated new mutations were at sites that have been detected following D and N treatment in vitro (Burgess et al, PNAS 2005; Bradeen et al, Blood 2006; Von Bubnoff et al, Blood 2006). Only 14/46 (30%) kinase domain mutations that developed after D (7) or N (7) corresponded with an in vitro-identified site. Only 5 of 134 (4%) mutations identified were T315I (3 after dasatinib, 2 after nilotinib), but the mutation status of these patients was unknown after IM. We conclude that the spectrum of mutations that develops in vivo after TKI switch is broader and includes common imatinib-resistance sites as well. There appears to no marked increase in the incidence of T315I mutation after TKI switch. Table 1. Dynamics of mutations after 2nd TKI Post IM mutation No. Post-2nd TKI Mutation (New + Same + Lost) *1 pt acquired new mutation with persistence of pre-existing mutation, 1 lost 3 mutations and acquired 1, and 1 pt lost 2 mutations. Nilotinib Dasatinib Absent 39 8+NA+NA/21 3+NA+NA/18 Present 46 3+20+3/26 2+16+2*/20 Unknown 28 8/9 13/19 Table 2. Dynamics of mutations after 3rd TKI Post IM mutation No. Post-3nd TKI Mutation (compared to status after 2nd TKI) (New + Same + Lost) Nilotinib Dasatinib Absent 5 0/1 1+NA+NA/4 Present 12 0+1+0/1 2+6+3/11 Unknown 3 1/3 NA

De Novo ABL Kinase Domain Mutations in Murine Bone Marrow Cells Retrovirally Transduced with Wild-Type p210 BCR-ABL and Amplified in Culture with Imatinib Mesylate.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1990-1990
Author(s):  
Stephane Flamant ◽  
Martine Guillier ◽  
Marie-Laure Bonnet ◽  
Jean-Pierre Lecouedic ◽  
William Vainchenker ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Imatinib Mesylate ◽  
Cell Lines ◽  
De Novo ◽  
Bone Marrow Cells ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Kinase Domain Mutations ◽  
Marrow Cells

Abstract Imatinib mesylate (IM) is a tyrosine kinase inhibitor which is highly efficient in chronic myelogenous leukemia (CML), especially in the firts chronic phase of the disease. Recent data showed, however, that resistance to IM can develop in patients in more aggressive phases of their disease, which mainly occurs through mutations within the ABL kinase domain that interfere with IM binding, leading to IM-resistant relapses. The mechanisms of the occurrence of ABL kinase domain mutations in patients on IM therapy are not well understood, and in some of them, a mutation pre-existing to the introduction of IM was described, suggesting the possibility of a clonal selection under IM therapy. To determine if ABL kinase domain mutations could be induced de novo in primary marrow cells, we used an ecotropic BCR-ABL retrovirus (MIGR-p210 vector, 5.105 viral particles / ml) and infected 5-FU-treated bone marrow cells from C57BL/6 mice. Retrovirally transduced cells (30% GFP+) were transplanted in lethally irradiated animals in which they induced lethal leukemia in 3 weeks. Both BCR-ABL-transduced and control C57BL/6 bone marrow cells were seeded in liquid cultures (104 cells/well) in the presence of 0.25 μM IM with weekly half medium changes during which the concentration of IM was increased gradually from 0.25 to 2 μM over 2 months. In these conditions, no growth could be obtained from normal bone marrow cells (0/192 wells) whereas in 10/192 wells containing BCR-ABL-transduced cells, we observed significant growth on IM. These cells were then amplified in the presence of murine stromal MS-5 cells and 2 μM IM for over 6 months and 2 clones (C3 and C10) exhibiting persistent growth were further characterized. At cytological analysis both cell lines had a typical mast cell morphology. Flow cytomery analyses demonsrated the presence of CD41 marker on both cell lines, with absence of myeloid (Gr1), erythroid (Ter119) and B-cell (B220) markers. Cells were not polyploid and interestingly, they exhibited higher growth rates in the presence of IM, with reduced growth upon IM deprivation. Both cell lines had evidence of BCR-ABL vector integration by PCR analysis and were highly GFP+. To explore the mechanisms of IM-resistance in these cells, we extracted high molecular weight genomic DNA and amplified a BCR-ABL fragment of 1236 bp encompassing the ABL kinase domain of the integrated construct. We then sequenced the ABL kinase domain using internal primers in both 5′-3′ directions. Plasmid DNA from the original MIGR-p210 BCR-ABL vector served as control. In both clones (C3 and C10) ABL kinase point mutations were readily detectable which were not found in the BCR-ABL retroviral vector. C3 carried two mutations interesting the C helix (E300K) and the SH2 contact region (E371K) of ABL kinase domain, whereas C10 carried a single mutation in the C helix (D295N). These mutations were previously detected in a random in vitro mutagenesis assay of BCR-ABL in bacterial systems. Thus, our model is the first demonstration of the occurrence of ABL kinase domain mutations and the concomittant generation of an IM-resistant phenotype in primary marrow stem cells transduced with BCR-ABL vector DNA and selected in the presence of IM. The mechanisms of the occurrence of these mutations in vitro are currently under study but these results suggest that de novo ABL kinase mutations could also occur in vivo in CML patients treated with IM.

BCR-ABL P-Loop Deletion Detected in Imatinib-Resistant CML Patients Corresponds to Splice Variant Associated with L248V Point Mutation, Retains BCR-ABL Kinase Activity, and Responds to Dasatinib Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2168-2168
Author(s):  
Nikolas von Bubnoff ◽  
Philipp Erben ◽  
Martin Müller ◽  
Tanja Lahaye ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Splice Variant ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Point Mutations ◽  
Kinase Domain ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Exon 4

Abstract Clonal selection of cells harboring point mutations of the BCR-ABL kinase domain are considered a major cause of resistance to imatinib. More than 40 different point mutations have been described that cause a variable degree of imatinib resistance, and display a differential response to alternative kinase inhibitors, like dasatinib or nilotinib. Here, we describe three cases (2 m, 1 f) with imatinib resistant chronic myelogenous leukemia (CML) associated with a specific deletion of 81 bp of ABL exon 4. Patients were diagnosed with chronic phase (CP) CML at the age of 52, 54, and 68 years. After initial interferon alpha based therapies for 32, 60, and 71 mo, imatinib therapy was initiated at dosages between 400–800 mg per day. After 18, 24, and 29 mo patients lost hematologic response in CP CML (n=2) or progressed to lymphoid blast crisis (BC, n=1). Molecular analysis of the ABL kinase domain revealed a deletion of a 81 bp fragment associated with a loss of amino acids 248–274 in all cases. In one patient, an additional M351T mutation was found. In the two cases with CP CML, dasatinib was commenced for imatinib resistance, resulting in a partial hematologic and minor cytogenetic response (60 and 70% Ph+ metaphases, respectively) after 14 mo of therapy. The patient with lymphoid BC was treated with vincristine and prednisone and died 24 mo after appearance of imatinib resistance. In two cases, sequencing of genomic DNA revealed an underlying CTG/GTG mutation associated with a L248V amino acid switch. The point mutation activated a cryptic splice site within ABL exon 4 leading to an in-frame splice variant characterized by the loss of a 81 bp 3′ portion of exon 4. We sought to evaluate the BCR-ABL kinase activity of the splice variant and the response to tyrosine kinase inhibitors in vitro. The 81 bp deletion of p210 BCR-ABL was cloned using cDNA from one of the patients. Using this construct, retrovirally transduced Ba/F3 cells were transformed upon growth factor withdrawal. These cells expressed BCR-ABL at the transcript and protein levels. Presence of the 81 bp deletion was confirmed by sequencing. Despite the presence of the corresponding 27 amino acid P-loop deletion (Δ248–274), Western blot indicated strong autophosphorylation of BCR-ABL, which decreased in the presence of imatinib to non-detectable levels at concentrations of 1.25μM and above. In the presence of imatinib/nilotinib/dasatinib, the growth of BCR-ABL expressing Ba/F3 cells was shifted from an IC50 of 125/30/0.5nM for wild-type BCR-ABL to 470/185/1.9nM for Δ248–274 cells. Thus, in vitro data demonstrate that deletion of almost the entire P-loop does not abrogate BCR-ABL kinase activity and results in only marginal resistance towards ABL kinase inhibitors. We conclude that deletions of BCR-ABL may be the result of alternative splicing generated by point mutations associated with resistance to imatinib. The Δ248–274 splice variant retains BCR-ABL kinase activity and sensitivity to imatinib, nilotinib, and dasatinib.

scholarly journals Leukemogenesis induced by wild-type and STI571-resistant BCR/ABL is potently suppressed by C/EBPα

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1353-1362 ◽  
Author(s):  
Giovanna Ferrari-Amorotti ◽  
Karen Keeshan ◽  
Michela Zattoni ◽  
Clara Guerzoni ◽  
Giorgio Iotti ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Drug Binding ◽  
Myelogenous Leukemia ◽  
Abl Kinase ◽  
Kinase Domain Mutation

Abstract Chronic phase–to–blast crisis transition in chronic myelogenous leukemia (CML) is associated with differentiation arrest and down-regulation of C/EBPα, a transcription factor essential for granulocyte differentiation. Patients with CML in blast crisis (CML-BC) became rapidly resistant to therapy with the breakpoint cluster region–Abelson murine leukemia (BCR/ABL) kinase inhibitor imatinib (STI571) because of mutations in the kinase domain that interfere with drug binding. We show here that the restoration of C/EBPα activity in STI571-sensitive or -resistant 32D-BCR/ABL cells induced granulocyte differentiation, inhibited proliferation in vitro and in mice, and suppressed leukemogenesis. Moreover, activation of C/EBPα eradicated leukemia in 4 of 10 and in 6 of 7 mice injected with STI571-sensitive or -resistant 32D-BCR/ABL cells, respectively. Differentiation induction and proliferation inhibition were required for optimal suppression of leukemogenesis, as indicated by the effects of p42 C/EBPα, which were more potent than those of K298E C/EBPα, a mutant defective in DNA binding and transcription activation that failed to induce granulocyte differentiation. Activation of C/EBPα in blast cells from 4 patients with CML-BC, including one resistant to STI571 and BMS-354825 and carrying the T315I Abl kinase domain mutation, also induced granulocyte differentiation. Thus, these data indicate that C/EBPα has potent antileukemia effects even in cells resistant to ATP-binding competitive tyrosine kinase inhibitors, and they portend the development of anti-leukemia therapies that rely on C/EBPα activation.

scholarly journals Long-term outcome of patients with chronic myeloid leukemia treated with second-generation tyrosine kinase inhibitors after imatinib failure is predicted by the in vitro sensitivity of BCR-ABL kinase domain mutations

Blood ◽  
2009 ◽  
Vol 114 (10) ◽  
pp. 2037-2043 ◽  
Author(s):  
Elias Jabbour ◽  
Daniel Jones ◽  
Hagop M. Kantarjian ◽  
Susan O'Brien ◽  
Constantine Tam ◽  
...  
Keyword(s):  
Overall Survival ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Inhibitory Concentration ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Abl Kinase

AbstractSecondary imatinib resistance in chronic myeloid leukemia (CML) is associated in approximately 50% of cases with mutations in the BCR-ABL kinase domain, necessitating switch to one of several new tyrosine kinase inhibitors (TKIs) that act differentially on mutated BCR-ABL. We assess here whether scoring mutation based on in vitro inhibitory concentration of each TKI-mutation pair can predict long-term clinical outcome. Among 169 patients with CML after imatinib failure, mutations were detected before TKI switch in 41 (48%) treated with dasatinib and 45 (52%) treated with nilotinib. Inhibitory concentration values for each TKI-mutation pair were stratified into high (n = 42), intermediate (n = 25), low (T315I, n = 9), or unknown sensitivity (n = 10). Hematologic and cytogenetic response rates were similar for patients with or without mutations. For patients in chronic phase, hematologic and cytogenetic responses correlated with mutation score; tumors with low and intermediate scores had lower response rates than those with highly sensitive mutations, and worse event-free and overall survival. These correlations with overall survival were not seen for advanced phases. Mutation scoring can predict outcome in CML-chronic phase with imatinib failure treated with second-generation TKIs and can help in therapy selection. More complex prognostic models will be required for advanced stages of disease.

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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