Imatinib Binding Research Articles

Dasatinib in the Treatment of Chronic Myeloid Leukemia

The recognition that the BCR-ABL gene and corresponding protein with deregulated tyrosine kinase (TK) activity is crucial for malignant transformation in chronic myeloid leukemia (CML), led to the synthesis of the smallmolecule drugs designed to interfere with BCR-ABL TK activation. The first tyrosine kinase inhibitor (TKI) was imatinib mesylate, introduced into clinical practice in 1998, which became the first choice drug in chronic phase CML. However, approximately 20-25% of patients initially successfully treated with imatinib will need alternative therapy because of unsatisfactory therapeutic results due to drug resistance. The risk of resistance is even higher in patients in advanced phases of CML. Resistance to imatinib is in about 35%-45% attributed to selection of clones expressing mutant forms of BCRABL which impair imatinib binding but preserve the kinase activity. The availability of second-generation TKIs has provided a new therapeutic option for patients with imatinib resistance. Among them, dasatinib is the first to be approved in the US and European Union for CML patients resistant or intolerant to imatinib. This drug is a dual SRC/ABL kinase inhibitor, 325-fold more potent than imatinib against cells expressing wild-type BCR-ABL and active in most clinically relevant BCR-ABL mutations, except highly resistant T315I. Several clinical trials have demonstrated that dasatinib is effective and generally well tolerated in imatinib resistant or intolerant CML and represents a promising therapeutic option for these patients. Keywords: Chronic myeloid leukemia, tyrosine kinase inhibitors, dual SRC/ABL kinase inhibitor, dasatinib, imatinib, AGP, A-loop, AP, ATP, AUC, BP, CCyR Comp, CFU, CHR, CML, CMoR, CP, CyR, EGFR, HR, IC50, JNK, MAPK, MCyR, MMoR, OS, P-gp, PCyR, PDGFR, PFS, Ph, PI3K, P-loop, SFKs, STAT5, TK, TKI, piperazin-1-yl-2-methylpyrymidin-4-ylamino thiazole-5-carboxamide, family kinase (SFks), ABL kinase domain, BCR-ABL mutants, F317L, Threonine315, M351T, colony-forming-unit (CFU) assay, IC50 0.2 nM), Lck (IC50 1.1 nM), Src (IC50 0.55 nM), Yes (IC500.41 nM), hOCT1 (human organic cation transporter 1), CYP3A4 enzyme

Clinical Outcomes In Patients with Chronic Myelogenous Leukemia (CML) and the BCR-ABL 35 Base Pair (bp) Intron 8 Insertion Mutation.

AbstractAbstract 3408More than 80% of patients (pts) with CML who are treated with imatinib as first line therapy achieve a complete cytogenetic response (CCR), although approximately 20% of these pts may ultimately progress. The most common mechanism for loss of response is acquired resistance to imatinib due to the development of a point mutation in the ABL kinase domain of BCR-ABL that interferes with optimal imatinib binding. An alternatively spliced BCR-ABL mRNA with a 35 bp insertion between exons 8 and 9 was first described in 2008 (Laudadio, 2008: Lee, 2008); this results in a BCR-ABL protein with 10 novel amino acids inserted after amino acid 474 within the C- terminus of the kinase domain followed by a stop codon. However, clinical responses to imatinib, dasatinib, or nilotinib have not been previously described in patients with this mutation. We retrospectively identified 40 pts who had this mutation detected and describe their clinical outcomes. Of the 40 patients, 24 were male, and the median age was 50, (range 16–70). Six patients had received treatment prior to 2001, and all 40 patients began initial therapy with imatinib. Twenty nine pts had mutation testing done because of either disease progression or lack of response to imatinib and in 9 pts, the mutation was detected during retrospective analysis. Two pts had the mutation detected at the time of diagnosis (one is still on imatinib). Six pts had additional mutations detected simultaneously: E255V, E355G, E450G, T315I with F416L, T315I with E255K, and one with another insertion between exons 4 and 5. The median time from start of imatinib to insertion mutation detection was 36 months (range 0–189). Thirty of the 40 pts (75%) either did not tolerate (n=2) or progressed while on therapy with imatinib (n=28). Eleven pts remain on imatinib, although 5 are on doses of 600 mg/day or higher. Eighteen of the 30 pts (60%) who failed imatinib were changed to dasatinib as second line therapy; 10 of these (56%) did not respond and were changed to nilotinib as third line therapy. Ten of the 30 pts (30%) who did not respond to imatinib were changed to nilotinib as second line therapy; 3 pts achieved an MCR. In summary, this report provides the first clinical summary of pts with CML who have the 35 bp intron 8 insertion mutation. Although it is not clear whether this insertion mutation directly leads to imatinib resistance, given that 75% of pts progressed on standard dose imatinib, other alternative therapies should be considered once this mutation is detected. However, given the small numbers of pts treated with dasatinib or nilotinib as second line therapy, it is difficult to determine whether either has a superior effect. Results of biochemical and cellular studies comparing the relative sensitivity of the BCR-ABL 35 bp intron 8 insertion mutation to several ABL kinase inhibitors will be presented.Table 1Response summary for patients with BCR-ABL 35 bp intron 8 insertion mutation.Number of patients (%)Failure or progression on first-line imatinib30/40 (75%)Failure or progression on second-line dasatinib10/18 (56%)Failure or progression on second-line nilotinib7/10 (70%)Kinase domain mutation concurrently detected with 35 bp insertion mutation6/40 (15%)Received non-TKI treatment prior to front-line imatinib6/40 (15%) Disclosures:Berman:Novartis Inc: Research Funding. Kantarjian:Genzyme: Research Funding; Eisai: Research Funding. Druker:Molecular MD: Equity Ownership.

The Novel BCR-ABL1 V304D Mutation Induces Pan-Tyrosine Kinase Inhibitor Resistance by a Unique Kinase Lateral Escape Mechanism and Is Associated with Very Poor Prognosis In Patients (PTS) with Chronic Myeloid Leukemia (CML).

AbstractAbstract 3402We discovered a novel BCR-ABL1 mutation (V304D) in pts with CML failing imatinib by DNA expansion of specific clones followed by DNA sequencing of ≥10 clones. BCR-ABL1V304D was detected in a median of 37% (range, 20% to 80%) resistant clones from 13 (18%) of 70 imatinib-resistant pts with CML in chronic phase (CP). Pts received imatinib for a median of 35 months (range 2 to 66) at doses ≥600 mg/d. No pt achieved a cytogenetic response. Four received nilotinib: 3 had hematologic resistance and 1 progressed to blast phase (BP). All pts died with negligible response to second-line TKIs: 8/12 pts on dasatinib had disease progression and 4 responded (2 hematologic and 2 transient minor cytogenetic responses). BCR-ABL1V304D failed to induce cytokine-independence or activate Stat5 in Ba/F3 cells. Phosphorylation of CrkL and specific BCR-ABL1 substrates were detectable but diminished compared to unmutated BCR-ABL1-transduced cells. BCR-ABL1V304D failed to catalyze autophosphorylation and the catalytic domain of ABL1V304D demonstrated deficient kinase activity. Enforced expression of BCR-ABL1V304Din CML cells induced quiescence and protection from imatinib-induced apoptosis. In vitro analyses of cells from a pt in CP expressing BCR-ABL1V304D in 50% of clones failed to detect CrKL phosphorylation in the presence of normal BCR-ABL1 protein levels, suggesting that BCR-ABL1V304D encodes a kinase-deficient protein and is associated with remarkable TKI resistance and extremely poor prognosis.To determine the mechanism of resistance imposed by BCR-ABL1V304D, we modeled this mutation in water and counterions and compared it to unmutated and mutant BCR-ABL1 isoforms. We first correlated the free energy of binding (DGbind) to the corresponding IC50 (DGbind = -RT lnIC50) and calculated the difference in free energy of binding between wild-type and mutant kinases (DDGbind = DGbind(WT) – DGbind(MUT)). DGbind <0 indicates a tighter binding to a TKI of the unmutated kinase relative to the mutant kinase. A negative increase of 1.4 kcal/mol in DGbind corresponds to a decrease by a factor of 10 in the IC50 value. The DGbind (IC50) values of imatinib for wild-type, Y253H, and T315I kinases were -10.47kcal/mol (21nM), -7.45 kcal/mol (3.4mM), and -6.38 kcal/mol (21mM), similar to published experimental data (25nM, 1.8–3.9mM, and >10mM, respectively), thus validating our modeling. DGbind and IC50 values for imatinib and dasatinib against V304D are -9.86kcal/mol (59nM) and -12.27 kcal/mol (1.02nM), respectively. 3D images generated from an equilibrated frame of 10 ns molecular dynamics (MD) simulations demonstrated that the 304 position is not in direct contact with imatinib, nor does it directly alter imatinib binding. Rather, V304D disturbs the position of the regulatory αC helix (Figure1). Longer standard molecular dynamics simulations coupled with steered MD recipes indicate that V304D induces a rearrangement of the ATP/drug binding pocket and water-mediated disruption of some fundamental hydrogen bonds regulating the transition of the activation loop to a “semi-open” conformation and the apt overall conformation of the SH3-binding segment of the TK (residues K294-F311). Furthermore, a decrease in the number of total interactions causes unidirectional drug translation toward the binding site exit. Iterative simulations revealed significant ATP/inhibitor diversion with subsequent complete imatinib expulsion. Thus, the V304D-induced semi-opened conformation of the activation loop favors 1) the lateral escape of imatinib, thus increasing the rate of TKI dissociating from the kinase and 2) does not allow the passage of ATP to reach deep into the binding pocket, thus hampering tyrosine phosphorylation. A similar phenomenon is observed in the activation loop in the active conformation of the V304D kinase bound to dasatinib, which results in greater exposure to water solvent of a part of the binding site and almost complete loss of hydrophobic contacts in the opposite end of the binding site. [Display omitted] In summary, BCR-ABL1V304D results in kinase inactivation, pan-TKI resistance mediated by a novel mechanism of lateral escape at the kinase domain, less control of protein autoinhibition via perturbation of the SH3 binding domain and very poor prognosis. Complete modeling data against a panel of novel TKIs and potential modes of overcoming this novel mechanism of resistance will be presented. Disclosures:Kantarjian:Bristol Myers Squibb: Research Funding; ARIAD: Research Funding; Nerviano: Research Funding. Cortes:Bristol Myers Squibb: Research Funding; ARIAD: Research Funding; Nerviano: Research Funding.

Abstract 3687: Expression of a Src-family kinase in CML cells induces resistance to imatinib in a kinase-dependent manner

Abstract Chronic Myelogenous Leukemia (CML) is characterized by the presence of Philadelphia Chromosome, which results in the expression of the 210 kDa Bcr-Abl tyrosine kinase. Bcr-Abl constitutively activates signaling pathways important for the proliferation, survival and cytokine independence of myeloid progenitors, including the Src family kinases (SFKs). Imatinib, an inhibitor of Bcr-Abl tyrosine kinase activity, is the frontline therapy for CML. Although imatinib is remarkably effective in the chronic phase of the disease, patients with accelerated or blast crisis CML often develop resistance. Several recent studies performed on clinical specimens from imatinib-resistant patients with wild-type Bcr-Abl found that the Src family kinase members Hck and Lyn are overexpressed or highly active, suggesting that Src kinases may play a role in imatinib resistance. While the role of Lyn has been addressed in some detail, the contribution of Hck to this type of imatinib resistance is less well understood. To test whether Hck overexpression in CML cells induces resistance to imatinib in a kinase-dependent manner, we employed a Hck mutant (Hck-T338A) that is uniquely sensitive to the pyrazolo-pyrimidine inhibitor, NaPP1. In vitro, Hck-T338A was 48 times more sensitive to NaPP1 than the wild-type kinase. Importantly, this “gatekeeper” mutation was functionally silent and did not confer a loss or gain of function on Hck in a control fibroblast transformation assay. Expression of wild-type Hck or the T338A mutant in K562 CML cells resulted in resistance to imatinib-induced apoptosis and inhibition of soft-agar colony formation. Treatment with NaPP1 restored sensitivity to imatinib in a concentration-dependent manner only in cells expressing the Hck-T338A mutant. In contrast, cells expressing wild-type Hck were not affected by NaPP1, demonstrating the selectivity of NaPP1 for the Hck-T338A mutant. This result shows that Hck-induced imatinib resistance requires Hck kinase activity. In addition, NaPP1 also reduced Hck-mediated phosphorylation of Bcr-Abl at sites that may affect imatinib binding exclusively in cells expressing Hck-T338A. Together, these data establish a direct cause and effect relationship between Hck overexpression and imatinib resistance in CML cells. Furthermore, our results show that imatinib resistance requires Hck kinase function and may occur via direct Hck-induced trans-phosphorylation of Bcr-Abl. Selective drug targeting of Hck may be of therapeutic benefit in imatinib-resistant CML patients with increased Hck expression or activity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3687.

Abstract 632: Cross-resistance of myeloid cells transformed with Bcr-Abl imatinib-resistant mutant T315I to Src-family kinase inhibitors

Abstract Bcr-Abl, the constitutively active protein-tyrosine kinase arising from the Philadelphia chromosome translocation, plays a key role in the etiology of chronic myelogenous leukemia (CML). Treatment of CML with the Abl kinase inhibitor imatinib has been a clinical success in the chronic phase of the disease. However, treatment failure in the blast crisis phase is attributable to imatinib resistance caused by point mutations in the kinase domain of Bcr-Abl. One of the most clinically challenging resistance mutations involves substitution of the gatekeeper threonine with isoleucine (T315I). This residue is located within the ATP-binding site and is essential for imatinib binding. Src family kinases (SFKs) are important mediators of Bcr-Abl signal transduction and oncogenesis in CML. Bcr-Abl binds to and activates multiple SFKs in CML cells, including Hck and Lyn. In turn, active SFKs directly phosphorylate regulatory tyrosine residues in the SH3 domain, the SH2-kinase linker, and the Bcr-derived portion of Bcr-Abl. Previous work from our group showed that global inhibition of SFK activity with the ATP-competitive pyrrolo-pyrimidine A-419259 leads to growth arrest and apoptosis in CML cell lines and in primary CD34+ CML cells. In addition, expression of an Hck mutant with engineered resistance to A-419259 protects CML cells against the antiproliferative and apoptotic effects of this compound. Other studies also correlate CML progression and drug resistance with overexpression and activation of Hck and Lyn. Given the important role of SFKs in Bcr-Abl-mediated signaling and drug resistance, we studied the effect of A-419259 on human TF-1 myeloid cells transformed with clinically relevant imatinib-resistant forms of Bcr-Abl (E255V, Y253H and T315I). Proliferation of TF-1 cells transformed with Bcr-Abl E255V and Y253H was markedly inhibited by A-419249. The anti-proliferative effects of the inhibitor correlated with suppression of overall SFK activity. However, TF-1 cells transformed with the gatekeeper mutant T315I were completely cross-resistant to the SFK inhibitor A-419259. Surprisingly, SFK activity in TF-1 cells expressing Bcr-Abl T315I was not inhibited by this compound. Immunoprecipitation experiments show that endogenous Lyn remains active in A-419259-treated TF-1 cells transformed with Bcr-Abl T315I. In addition, over-expressed Hck remains active in TF-1/T315I cells upon A-419259 treatment, but is completely inhibited in TF-1 cells transformed with wild-type Bcr-Abl. These results suggest that in general, SFKs are important targets to overcome imatinib resistance in CML. However, our data also suggest a new mechanism of Bcr-Abl T315I-induced resistance to drugs that target SFKs in CML. We speculate that this effect may be due to an acquired ability of Bcr-Abl T315I to phosphorylate SFKs at novel sites that interfere with drug action. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 632.

Molecular Dynamics Simulations Show That Conformational Selection Governs the Binding Preferences of Imatinib for Several Tyrosine Kinases

Tyrosine kinases transmit cellular signals through a complex mechanism, involving their phosphorylation and switching between inactive and active conformations. The cancer drug imatinib binds tightly to several homologous kinases, including Abl, but weakly to others, including Src. Imatinib specifically targets the inactive, so-called "DFG-out" conformation of Abl, which differs from the preferred, "DFG-in" conformation of Src in the orientation of a conserved Asp-Phe-Gly (DFG) activation loop. However, recent x-ray structures showed that Src can also adopt the DFG-out conformation and uses it to bind imatinib. The Src/Abl-binding free energy difference can thus be decomposed into two contributions. Contribution i measures the different protein-imatinib interactions when either kinase is in its DFG-out conformation. Contribution ii depends on the ability of imatinib to select or induce this conformation, i.e. on the relative stabilities of the DFG-out and DFG-in conformations of each kinase. Neither contribution has been measured experimentally. We use molecular dynamics simulations to show that contribution i is very small, 0.2 +/- 0.6 kcal/mol; imatinib interactions are very similar in the two kinases, including long range electrostatic interactions with the imatinib positive charge. Contribution ii, deduced using the experimental binding free energy difference, is much larger, 4.4 +/- 0.9 kcal/mol. Thus, conformational selection, easy in Abl, difficult in Src, underpins imatinib specificity. Contribution ii has a simple interpretation; it closely approximates the stability difference between the DFG-out and DFG-in conformations of apo-Src. Additional calculations show that conformational selection also governs the relative binding of imatinib to the kinases c-Kit and Lck. These results should help clarify the current framework for engineering kinase signaling.

Outcome of Patients with Philadelphia Positive Chronic Myeloid Leukemia Post Imatinib Failure: Prognostic Significance of ABL Mutation and Additional Clonal Abnormality.

Abstract 4673Point mutation within the ABL kinase domain of the BCR-ABL gene has been associated with clinical resistance to imatinib in chronic myeloid leukemia (CML). However, several other mechanisms have been proposed to underlie the development of imatinib resistance such overexpression and amplification of the BCR-ABL gene locus; development of additional chromosomal abnormalities (ACA), activation of BCR-ABL independent mechanisms; increased drug efflux through multidrug resistance gene and binding of imatinib to serum alfa-1 acid glycoprotein. In this regard, we report the outcome of 54 patients Ph+ CML who were resistant to imatinibe and the association with point mutation, ACA and overexpression of BCR-ABL through in situ hybridization (FISH). Patients and MethodsOf 54 CML patients, 16 were in early chronic phase (ECP), 24 in late chronic phase (LCP) and 14 in accelerated phase (AP) before imatinib treatment. They were considered imatinib resistant for the following reasons: hematologic resistant or recurrence (7 patients), cytogenetic resistance or recurrence (30 patients) or progression to more advanced phase (17 patients). Mutational analysis was carrying out by direct sequencing and FISH analysis using commercial BCR/ABL t(9;22), dual color, dual fusion probe (Kreatech, Poseidon, The Netherlands). Subsequent therapy was also analyzed, as to whether they underwent to allogeneic SCT, chemotherapy, or received, dasatinib or nilotinib. ResultsA total of 24 BCR-ABL kinase domain mutations were detected in 54 patients. Two patients had more than 1 mutation. The most frequent mutations were F359V (6/24), T315I (5/24) and F317L (3/24). P-loop mutations accounted for 60% of the remaining amino acid substitutions. Twelve patients were treated with nilotinib 400 mg bid, 50% presented mutation at resistance (3 F359V, 1 Q252H, 1 E355G, and 1 T315I), and all alive but one with T315I mutation died after 1 month. Dasatinib, 100-140 mg daily, was dispensed for 37 patients, mutations were identified in 40%, where 10 patients are still alive (2 T315I, 2 F317L, and 6 others). Allogeneic SCT was performed in 2 patients (1 G250E), and both died. Two patients died (1 F359V) after systemic chemotherapy and 1 patient who received 800 mg of IM is in CCyR after 40 months. There was no difference in overall survival when mutation was present or not at the moment of resistance (log rank, p=0.9). We also compared the karyotype tacked before imatinib treatment with the karyotype performed at the time of resistance; 17 patients developed ACA and were associated with worse overall survival (log rank, p=0,046) when compared with those patients without ACA. FISH analysis did not identify overexpression of BCR-ABL, but 4 cases presented 3 signals for BCR-ABL indeed had an extra Ph chromosome. ConclusionsSecond-generation TK inhibitors improve overall survival of those patients who presented mutations at resistance. The presence of ACA during imatinib treatment was related with a higher mortality despite the treatment applied and the presence or not of mutations. We also conclude that overexpression of BCR-ABL was not responsible for resistance to imatinib in our study population. Disclosures:No relevant conflicts of interest to declare.

Nilotinib for the treatment of chronic myeloid leukemia: An evidence-based review

Chronic myelogenous leukemia (CML) is a progressive and often fatal hematopoietic neoplasm. The Bcr-Abl tyrosine kinase inhibitor imatinib mesylate represented a major therapeutic advance over conventional CML therapy, with more than 90% of patients obtaining complete hematologic response, and 70%-80% of patients achieving a complete cytogenetic response. Despite the high efficacy of imatinib, a minority of patients in chronic phase CML and more patients in advanced phases are resistant to imatinib, or develop resistance during treatment. This is attributed, in 40% to 50% of cases, to the development of mutations in the Bcr-Abl tyrosine kinase domain that impair imatinib binding. Attempts to circumvent resistance led to the discovery of nilotinib (Tasigna), a novel, potent and selective oral Bcr-Abl kinase inhibitor. To review the evidence for the use of nilotinib in the management of CML. Preclinical and clinical investigations demonstrate that nilotinib effectively overcomes imatinib resistance, and has further improved the treatment of CML. Nilotinib is currently indicated for patients with CML in chronic and accelerated phases following imatinib failure. Randomized studies are ongoing to assess the efficacy of nilotinib in patients with newly diagnosed CML.

Dasatinib in the treatment of imatinib refractory chronic myeloid leukemia

The development of imatinib for the treatment of chronic myeloid leukemia (CML) has proven to be an example of medical success in the era of targeted therapy. However, imatinib resistance or intolerance occurs in a substantial number of patients. Additionally, patients who have progressed beyond the chronic phase of CML do relatively poorly with imatinib therapy. Mechanisms of imatinib resistance include BCR-ABL point mutations resulting in decreased imatinib binding, as well as mutation-independent causes of resistance such as SRC family kinase dysregulation, BCR-ABL gene amplification, drug influx/efflux mechanisms and other poorly understood processes. The options for therapy in these patients include stem cell transplantation, imatinib dose escalation as well as the use of second-generation tyrosine kinase inhibitors. Dasatinib is a second-generation multi-kinase inhibitor with several theoretical and mechanistic advantages over imatinib. Moreover, several studies have evaluated dasatinib in patients who have progressed on imatinib therapy with encouraging results. Other novel agents such as mTOR inhibitors, bosutinib and INNO 406 have also shown promise in this setting. Although treatment options have increased, the choice of second-line therapy in patients with CML is influenced by concerns surrounding the duration of response as well as toxicity. Consequently, there is no agreed upon optimal second-line agent. This paper reviews the current data and attempts to address these issues.

Dasatinib in the treatment of imatinib refractory chronic myeloid leukemia

Dasatinib in the treatment of imatinib refractory chronic myeloid leukemia Radhakrishnan Ramchandren, Charles A SchifferDivision of Hematology/Oncology, Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, MI, USAAbstract: The development of imatinib for the treatment of chronic myeloid leukemia (CML) has proven to be an example of medical success in the era of targeted therapy. However, imatinib resistance or intolerance occurs in a substantial number of patients. Additionally, patients who have progressed beyond the chronic phase of CML do relatively poorly with imatinib therapy. Mechanisms of imatinib resistance include BCR-ABL point mutations resulting in decreased imatinib binding, as well as mutation-independent causes of resistance such as SRC family kinase dysregulation, BCR-ABL gene amplification, drug influx/efflux mechanisms and other poorly understood processes. The options for therapy in these patients include stem cell transplantation, imatinib dose escalation as well as the use of second-generation tyrosine kinase inhibitors. Dasatinib is a second-generation multi-kinase inhibitor with several theoretical and mechanistic advantages over imatinib. Moreover, several studies have evaluated dasatinib in patients who have progressed on imatinib therapy with encouraging results. Other novel agents such as mTOR inhibitors, bosutinib and INNO 406 have also shown promise in this setting. Although treatment options have increased, the choice of second-line therapy in patients with CML is influenced by concerns surrounding the duration of response as well as toxicity. Consequently, there is no agreed upon optimal second-line agent. This paper reviews the current data and attempts to address these issues. Keywords: chronic myeloid leukemia (CML), dasatinib, imatinib, resistance (imatinib resistance), nilotinib, tyrosine kinase inhibitor

Understanding the Role of Mutations in Therapeutic Decision Making for Chronic Myeloid Leukemia

In patients with chronic myeloid leukemia (CML) resistant to imatinib, resistance is commonly associated with mutations in the BCR-ABL protein. Approximately 85% to 90% of resistance-associated mutations occur within the ABL kinase domain, and confer resistance either directly, by blocking imatinib binding, or indirectly, by altering the conformation of BCR-ABL. The degree of resistance depends on the mutation, with some remaining sensitive to imatinib. Imatinib dose escalation may overcome resistance in some of these patients or therapy can be switched to the second-generation tyrosine kinase inhibitors (TKIs) nilotinib or dasatinib. The long-term efficacy of second-generation TKIs may also be related to specific BCR-ABL mutations, with the T315I mutant remaining resistant to all currently available TKIs. Other treatments, including investigational agents, may be options for patients with this mutation. The choice of therapy should be guided by multiple factors, including mutational analysis, disease phase, patient characteristics, and the safety profile of the agents.

A conserved protonation-dependent switch controls drug binding in the Abl kinase

In many protein kinases, a characteristic conformational change (the "DFG flip") connects catalytically active and inactive conformations. Many kinase inhibitors--including the cancer drug imatinib--selectively target a specific DFG conformation, but the function and mechanism of the flip remain unclear. Using long molecular dynamics simulations of the Abl kinase, we visualized the DFG flip in atomic-level detail and formulated an energetic model predicting that protonation of the DFG aspartate controls the flip. Consistent with our model's predictions, we demonstrated experimentally that the kinetics of imatinib binding to Abl kinase have a pH dependence that disappears when the DFG aspartate is mutated. Our model suggests a possible explanation for the high degree of conservation of the DFG motif: that the flip, modulated by electrostatic changes inherent to the catalytic cycle, allows the kinase to access flexible conformations facilitating nucleotide binding and release.

A Review of the Management of Chronic Myeloid Leukemia with Dasatinib

Dasatinib is a multitargeted tyrosine kinase inhibitor (TKI), recommended for chronic myeloid leukemia (CML) in chronic, accelerated and blastic phases that are resistant or intolerant to imatinib. The recognition of BCR-ABL gene and corresponding protein with deregulated tyrosine kinase (TK) activity as crucial for malignant transformation in CML, led to synthesis of the small-molecule drugs designed to interfere with Bcr-Abl TK activation. The first TKI introduced into clinical practice in 1998, was imatinib mesylate, which became the first choice drug in chronic phase CML. However, approximately 20%-25% of patients initially successfully treated with imatinib, develop resistance to this drug, mainly because of selection of clones expressing mutant forms of BCR-ABL with impairing imatinib binding. Dasatinib is 325-fold more potent than imatinib against cell expressing wild-type BCR-ABL and showing activity against most imatinib-resistant Bcr-Abl mutants. Several clinical trials demonstrated that dasatinib is effective and generally well tolerated in imatinib resistant or intolerant CML and represents a promising therapeutic option for these patients.

BCR-ABL alternative splicing as a common mechanism for imatinib resistance: evidence from molecular dynamics simulations.

Rare cases of chronic myelogenous leukemia (CML) express high levels of alternatively spliced BCR-ABL mRNA with a 35-bp insertion (35INS) between ABL kinase domain exons 8 and 9. This insertion results in a frameshift leading to the addition of 10 residues and truncation of 653 residues due to early termination. Sensitive PCR-based testing showed that 32 of 52 (62%) imatinib-resistant CML patients in chronic phase and 8 of 38 (21%) in accelerated or blast crisis expressed varying levels of the alternatively spliced BCR-ABL mRNA. A three-dimensional structural model of the 35INS ABL kinase domain complexed with imatinib was built using homology modeling, followed by molecular dynamics simulations. Simulation results showed that the new residues cause a significant global conformational change, altering imatinib binding in a way similar to that of the T315I mutation and, therefore, providing resistance to imatinib that depends on the level of expression.

Outcome of Patients with Chronic Myeloid Leukemia with T315I BCR-ABL Mutation

The most frequent causes of imatinib resistance are BCR-ABL mutations, usually selected during treatment. Most of them destabilize the inactive conformation necessary for imatinib binding. The T315I mutation, located at the ATP binding pocket is resistant to imatinib and other available kinase inhibitors and is associated with a poor outcome. The aim of this study was to evaluate the frequency of T315I mutation in patients with CML resistant to imatinib and to describe their outcome. Mutation analysis was performed in two institutions, through direct automated sequencing. Total RNA was extracted from peripheral blood or bone marrow and amplification of the kinase domain of ABL from BCR/ABL was performed, using a semi-nested RT-PCR, to cover amino acids 244–486. PCR product was submitted to direct automated sequencing and compared with normal sequences of BCR-ABL gene (M14752, GenBank). A total of 173 resistant imatinib resistant patients were analyzed. BCR-ABL mutations were detected in 47 out of 173 (27%) patients. Among these patients, 14 (30%) presented T315I mutation. The mutation was found in ten patients during imatinib treatment, in one before start of imatinib, in three after dasatinib (2) and nilotinib (1) failure. Three patients have more than one mutation analysis, performed during different time-points of treatment: imatinib resistance, dasatinib resistance (3) and bone marrow transplantation relapse (1). Overall survival from diagnosis was 39,5% and 17,5% from mutation detection, in a median time of 69 and 13 months respectively. After imatinib resistance, eight patients were treated with dasatinib, one with nilotinib and had no hematological response; tree were treated with Hydrea and two were submitted to allogeneic bone marrow transplantation. Five patients died due to disease progression in median time of 19 months after mutation detection. One patient died after bone marrow transplantation, due to GVHD and infection. Eight patients are alive, three in CP, two in BC, two in hematological response and one in complete cytogenetic response after bone marrow transplantation. In conclusion, in this study we confirmed the high frequency of T315I mutation in imatinib resistant patients and their poor prognosis. Most of them started imatinib in a late chronic or advanced phase, with exception of two patients which started imatinib 800mg in early chronic phase, suggesting that there was inhibition of Ph positive sensitive cells and selection of resistant clones.

Determining the Rise in BCR-ABL RNA That Optimally Predicts a Kinase Domain Mutation in Patients with CML on Imatinib.

The most common mechanism of imatinib resistance in patients with CML is point mutations in the BCR-ABL kinase domain (KD) that impair imatinib binding to its target. As the second-line BCR-ABL inhibitors nilotinib and dasatinib show activity in most patients with KD mutations, early identification of mutations may prevent relapse. Current consensus recommendations are to perform mutation analysis if patients fail to achieve certain milestones of response, or experience loss of response. Since most patients are routinely monitored by BCR-ABL quantitative polymerase chain reaction (RQ-PCR), it is necessary to determine the optimal increase in BCR-ABL RNA that should trigger mutation testing, while minimizing both false-positive and false-negative test results. Recognizing the lack of a consensus, expert panels and the NCCN guidelines have provisionally recommended mutation screening in cases with a 10-fold or greater increase of BCR-ABL RNA. To address, in an unbiased fashion, the optimal transcript level rise associated with a mutation, and thus advise when mutation screening should be performed, 150 CML patients were serially monitored (median 46 months post-imatinib) by RQ-PCR and KD DNA sequencing. Thirty-five different mutations were identified in 53 patients, at a median of 28 months after imatinib initiation. During 22.5 months of follow-up after mutation screening, relapses occurred in 85% of the patients with a mutation (median 10. 4 months later) versus 46% of those with no mutation (median 32.4 months later) (hazard ratio = 3.2; P<0.0001). An unbiased receiver operating characteristic analysis identified a 2.6-fold increase in BCR-ABL RNA as the optimal cutoff for predicting a concomitant KD mutation, with a sensitivity of 77% for samples drawn at the same time as mutation discovery, and a sensitivity of 94% if including subsequent samples. At this 2.6-fold threshold, the negative predictive value was 97%, implying a small 3% risk of failing to detect a deleterious point mutation when the BCR-ABL RNA increase does not reach this optimized cutoff level. The 2.6-fold threshold approximated the analytical precision limit of our PCR assay. The table shows the diagnostic sensitivities, specificities, negative predictive values, and odds ratios for predicting a concomitant KD mutation at various rising RQ-PCR cutoff thresholds, including the optimal cutoff value of a 2.6-fold increase in BCR-ABL RNA. All cutoff values between a 2- and 4.5-fold increase were significantly predictive of a concomitant mutation, with 2.6-fold being the optimal cutoff level by several criteria (Youden index, negative predictive value, and odds ratio). In contrast, cutoff rises of 5- or 10-fold had poor diagnostic sensitivity (missing those 53–74% of mutation cases with lower levels of transcript rises), and were not significantly predictive of mutations. We conclude that a transcript level rise of >2.6-fold is the optimal cutoff to trigger reflex mutation screening in imatinib-treated CML. Furthermore, the currently recommended 10-fold threshold to trigger mutation screening may be overly stringent and is not universally applicable.RQ-PCR Increase (fold change)Sensitivity (95% CI) (%)Specificity (%)Negative Predictive Value (95% CI) (%)Odds Ratio (95% CI)P2.077 (62–88)4488 (80–94)2.6 (1.2–5.4)0.012.577 (62–88)4689 (81–94)2.8 (1.4–5.9)0.0052.677 (62–88)4789 (81–94)2.9 (1.4–6.0)0.0052.6* (include subsequent sample)94 (82–99)4797 (91–99)13 (3.9–43)<0.00013.074 (60–86)4888 (80–94)2.7 (1.3–5.5)0.0083.564 (49–77)5486 (78–91)2.1 (1.1–4.1)0.034.060 (44–74)6085 (78–91)2.2 (1.1–4.2)0.024.555 (40–70)6485 (78–90)2.2 (1.2–4.2)0.025.047 (32–62)6884 (77–89)1.9 (1.0–3.6)0.061026 (14–40)8382 (75–87)1.7 (0.8–3.7)0.2*Except for the one indicated row, the table includes only those patients with successful RQ-PCR and sequencing performed on the same sample. If samples after the initial genotype determination are included, at the optimal 2.6-fold cutoff, the sensitivity, NPV, and OR each increase.

Detection of Twelve Nucleotides Instertion in the BCR-ABL Kinase Domain in an Imatinib-Resistant but Dasatinib-Sensitive Patient with Bi-Phenotypic Acute Leukemia.

Although targeted inhibition of BCR-ABL with imatinib (IM) is an effective therapy for patients with Philadelphia chromosome-positive leukemia, a minority of patients (most of them in advanced phase) acquire mutations in the BCR-ABL kinase domain (KD) and relapse. The spectrum of the mutations described to date consists in-almost exclusively-single nucleotide substitutions affecting IM binding or impairing the conformational changes of the BCR-ABL KD. Recently, rare cases of intron/truncation mutations of 35 nucleotides, presumably inserted by alternative splicing events but inducing no conservation of the open reading frame (ORF), have been described in CML patients undergoing IM therapy (Laudadio et al. J Mol Diagn. 2008; 10:177–180). Here, we describe a novel mutation involving the BCR-ABL KD, acquired at the moment of the IM resistance, consisting in an insertion of 12 nucleotides leading to the conservation of the ORF. The 57-year old female patient was diagnosed in July 2006 with bi-phenotypic acute leukemia based on peripheral blood and bone marrow findings (hyperleukocytosis at 48 G/l with 47% of blasts exhibiting myeloid and lymphoid features (CD13+, CD33+, CD19+, CD10+ and CD22+), on karyotypic analysis found a t(9;22)(q34;q11) in all mitosis and on molecular analysis detected M BCR-ABL transcript. There was no sibling donor identified. Induction chemotherapy (Hyper C-VAD) and IM at 800mg/day were given. She achieved a complete remission (CR), a complete cytogenetic response and the BCR-ABL/ABL ratio was 2.3% (RT-RQ PCR performed according to the ELN recommendations with ICF 0.51). Consolidation therapy with alternating high dose methotrexate plus cytarabine and Hyper C-VAD plus IM was given and the BCR-ABL/ABL ratio dropped to 0.06%. After a successful PBSC collection with G-CSF alone, she was autografted in April 2007. IM was re-introduced at 600mg/day leading to a sustained drop in transcript level to 0.035% 6 months later. Then the transcript ratio rose rapidly within 3 months (0.7%;2.4%;8.1%) and a mutation screen performed in April 2008 revealed the insertion of 12 nucleotides in 100% of the BCR-ABL transcripts inducing the insertion of 4 amino acid residues (A, F, G and P) between A293 and K294 in the BCR-ABL KD. This insertion into exon 5 of ABL in the BCR-ABL transcript sequences did not seem related to alternative splicing events or to a constitutional polymorphism because this insertion was present at the genomic level in DNA extracted from leukemic cells but was missing in genomic DNA extracted from the patient's epithelial mouth cells. Moreover, retrospective analysis with a fluorescent probe and allele specific oligonucleotide performed by RQ-PCR on the cDNA from patient at diagnosis did not show the insertion at diagnosis, suggesting that the mutational mechanism was induced or selected by IM therapy. The increase of IM up to 800mg/day during six weeks led to the loss of hematologic response. Dasatinib was introduced at 70mg twice daily in may 2008 and molecular monitoring performed monthly showed a strong reduction of the BCRABL/ABL ratio to 0.7%; 0.2% and 0.07% respectively. The mutant ratios performed on the latest follow-up did not show any change in the proportion (100%) of the mutated BCR-ABL transcripts among total BCR-ABL transcripts suggesting no resurgence of the wild-type BCR-ABL clone. Therefore dasatinib seems to efficiently target the mutated clone as well as the wild-type leukemic clone and appears to be highly effective on this kind of mutation.

Determination of the Activity Profile of Bosutinib, Dasatinib and Nilotinib against 18 Imatinib Resistant Bcr/Abl Mutants

The treatment of Chronic Myeloid Leukemia (CML) has been radically modified by the discovery of imatinib (IM), a selective inhibitor of the fusion protein Bcr-Abl, the cause of the disease. A variable portion of CML patients experience resistance to IM therapy. Resistance can arise from different mechanisms but in the vast majority of cases is due to point mutations into the protein sequence that alter directly or indirectly the drug-protein binding. Mutation sites can be schematically clustered in four region: the P loop, the IM binding site, the catalytic domain and the activation loop (A loop). At present more than 70 mutations conferring different levels of resistance have been found in CML patients. Recently, several new inhibitors have been developed in order to obtain an increased potency and a broad range of activity against IM resistant mutants. Nilotinib (NIL) is an IM derivative about 30-fold more potent than IM. Dasatinib (DAS) is a dual-specific Src/Abl inhibitor, structurally unrelated to IM and characterized by an activity 100 to 300-fold higher than IM. Bosutinib (BOS) is a dual Src/Abl inhibitor that shows an activity 10 to 30-fold higher than IM. It is known that resistance to second generation TKIs can also arise and the analysis of mutation profiles reveals substantial differences among different TKIs. Presently the choice of a TKI to treat a patient resistant to IM is mostly based on an empirical basis, e.g. the fact that a certain patient has not been previously exposed to that particular TKI. The possibility to directly compare the different activities of TKIs against a given mutation is of remarkable importance in clinical practice. Such a tool could be used similarly to an antibiogram for bacterial diseases, guiding the choice of the most appropriate inhibitor for each patient.In our study, we investigated the activity of BOS, DAS, IM and NIL against a panel of 18 mutated forms of BCR/ABL chosen to cover the most common mutations found in patients. Stable Ba/F3 transfectant cell lines were generated and the TKIs antiproliferative activity was determined by tritiated thymidine incorporation assay. The relative IC50 increase over wild type BCR/ABL (Relative Resistance RR) was calculated. We classified the RR values in three categories: sensitive (RR≤2), resistant (between 2.01 and 10) or highly resistant (>10) as presented in the table.IC50-fold increase (WT=1)ImatinibBosutinibDasatinibNilotinibParental10.7838.31>5038.45WT1111P-LOOPL248V3.542.975.112.80G250E6.864.314.454.56Q252H1.390.813.052.64Y253F3.580.961.583.23E255K6.029.475.616.69E255V16.995.533.4410.31D276G2.180.601.442.00C-HelixE279K3.550.951.642.05V299L1.5426.108.651.34Active siteT315117.5045.4275.0339.41F317L2.602.424.462.22SH2-contactM351T1.760.700.880.44Active siteF359V2.860.931.495.16A-LOOPL384M1.280.472.212.33H396P2.430.431.072.41H396R3.910.811.633.10G398R0.351.160.690.49C terminal lobeF486S8.102.313.041.85Sensitive≤2Resistant2.01–10Highly resistant>10(Updated table available online at http://www.dimep.medicina.unimib.it/en/staff_174.php?docente_id=32)Our study points out at the differences in the activity spectrum of the 4 TKIs against the 18 Bcr/Abl mutations considered. The activity pattern presented in this work will help to reach a rational and tailored therapy offering to physicians a tool to use the new TKIs in the most efficient way for their patients.

Abl tyrosine kinase inhibitors for overriding Bcr–Abl/T315I: from the second to third generation

Treatment of chronic myeloid leukemia (CML) has changed drastically with the emergence of the Abl tyrosine kinase inhibitor (TKI), imatinib mesylate. However, primary and secondary resistance have frequently been reported, particularly in patients with advanced-stage disease. Point mutations within the Abl kinase domain that interfere with imatinib binding are the most critical cause of imatinib resistance. In order to override this resistance, several second generation ATP-competitive Abl TKIs including dasatinib, nilotinib, bosutinib and INNO-406 have been developed. Despite promising clinical results from these novel Abl TKIs for most mutations, the frequently observed mutant T315I is not effectively targeted by any of these agents. Thus, identification of novel agents and the development of new strategies for the effective treatment of CML patients with the T315I mutation are important and challenging tasks. In this review, the current status of novel agents for CML treatment is overviewed as follows: pathogenesis and features of CML; imatinib and second-generation Abl TKIs; why Abl TKIs are not effective against T315I; and novel agents that may override the T315I mutation.

Nilotinib: a novel Bcr-Abl tyrosine kinase inhibitor for the treatment of leukemias

The successful introduction of the tyrosine kinase inhibitors has initiated a new era in the management of chronic myeloid leukemia (CML). Imatinib mesilate therapy has significantly improved the prognosis of CML. A minority of patients in chronic-phase CML – and more patients in advanced phases – are resistant to imatinib, or develop resistance during treatment. This is attributed, in 40 – 50% of cases, to the development of mutations in the Bcr-Abl tyrosine kinase domain that impair imatinib binding. Nilotinib (Tasigna®) is a novel potent selective oral kinase inhibitor. Preclinical and clinical investigations demonstrate that nilotinib effectively overcomes imatinib resistance, and has induced high rates of hematologic and cytogenetic responses in CML post imatinib failure.