Combination of Bortezomib and Mitotic Inhibitors Down-Modulate Bcr-Abl and Efficiently Eliminates Tyrosine-Kinase Inhibitor Sensitive and Resistant Bcr-Abl-Positive Leukemic Cells

Emergence of resistance to Tyrosine-Kinase Inhibitors (TKIs), such as imatinib, dasatinib and nilotinib, in Chronic Myelogenous Leukemia (CML) demands new therapeutic strategies. We and others have previously established bortezomib, a selective proteasome inhibitor, as an important potential treatment in CML. Here we show that the combined regimens of bortezomib with mitotic inhibitors, such as the microtubule-stabilizing agent Paclitaxel and the PLK1 inhibitor BI2536, efficiently kill TKIs-resistant and -sensitive Bcr-Abl-positive leukemic cells. Combined treatment activates caspases 8, 9 and 3, which correlate with caspase-induced PARP cleavage. These effects are associated with a marked increase in activation of the stress-related MAP kinases p38MAPK and JNK. Interestingly, combined treatment induces a marked decrease in the total and phosphorylated Bcr-Abl protein levels, and inhibits signaling pathways downstream of Bcr-Abl: downregulation of STAT3 and STAT5 phosphorylation and/or total levels and a decrease in phosphorylation of the Bcr-Abl-associated proteins CrkL and Lyn. Moreover, we found that other mitotic inhibitors (Vincristine and Docetaxel), in combination with bortezomib, also suppress the Bcr-Abl-induced pro-survival signals and result in caspase 3 activation. These results open novel possibilities for the treatment of Bcr-Abl-positive leukemias, especially in the imatinib, dasatinib and nilotinib-resistant CML cases.     

Phosphorylation of c-Cbl and p85 PI3K driven by all-trans retinoic acid and CD38 depends on Lyn kinase activity

The leukocyte antigen CD38 is expressed after all-trans retinoic acid (ATRA) treatment in HL-60 myelogenous leukemia cells and promotes induced myeloid differentiation when overexpressed. We found that Vav1 and SLP-76 associate with CD38 in two cell lines, and that these proteins complex with Lyn, a Src family kinase (SFK) upregulated by ATRA. SFK inhibitors PP2 and dasatinib, which enhance ATRA-induced differentiation, were used to evaluate the involvement of Lyn kinase activity in CD38-driven signaling. Cells treated with ATRA for 48 h followed by one hour of PP2 incubation show SFK/Lyn kinase inhibition. We observed that Lyn inhibition blocked c-Cbl and p85/p55 PI3K phosphorylation driven by the anti-CD38 agonistic mAb IB4 in ATRA-treated HL-60 cells and untreated CD38 + transfectants. In contrast, cells cultured for 48 h following concurrent ATRA and PP2 treatment did not show Lyn inhibition, suggesting ATRA regulates the effects on Lyn. 48 h of co-treatment preserved CD38-stimulated c-Cbl and p85/p55 PI3K phosphorylation indicating Lyn kinase activity is necessary for these events. In contrast another SFK inhibitor (dasatinib) which blocks Lyn activity with ATRA co-treatment prevented ATRA-induced c-Cbl phosphorylation and crippled p85 PI3K phosphorylation, indicating Lyn kinase activity is important for ATRA-propelled events potentially regulated by CD38. We found that loss of Lyn activity coincided with a decrease in Vav1/Lyn/CD38 and SLP-76/Lyn/CD38 interaction, suggesting these molecules form a complex that regulates CD38 signaling. Lyn inhibition also reduced Lyn and CD38 binding to p85 PI3K, indicating CD38 facilitates a complex responsible for PI3K phosphorylation. Therefore, Lyn kinase activity is important for CD38-associated signaling that may drive ATRA-induced differentiation.     

Real-Time Analysis of Imatinib- and Dasatinib-Induced Effects on Chronic Myelogenous Leukemia Cell Interaction with Fibronectin

Attachment of stem leukemic cells to the bone marrow extracellular matrix increases their resistance to chemotherapy and contributes to the disease persistence. In chronic myelogenous leukemia (CML), the activity of the fusion BCR-ABL kinase affects adhesion signaling. Using real-time monitoring of microimpedance, we studied in detail the kinetics of interaction of human CML cells (JURL-MK1, MOLM-7) and of control BCR-ABL-negative leukemia cells (HEL, JURKAT) with fibronectin-coated surface. The effect of two clinically used kinase inhibitors, imatinib (a relatively specific c-ABL inhibitor) and dasatinib (dual ABL/SRC family kinase inhibitor), on cell binding to fibronectin is described. Both imatinib and low-dose (several nM) dasatinib reinforced CML cell interaction with fibronectin while no significant change was induced in BCR-ABL-negative cells. On the other hand, clinically relevant doses of dasatinib (100 nM) had almost no effect in CML cells. The efficiency of the inhibitors in blocking the activity of BCR-ABL and SRC-family kinases was assessed from the extent of phosphorylation at autophosphorylation sites. In both CML cell lines, SRC kinases were found to be transactivated by BCR-ABL. In the intracellular context, EC50 for BCR-ABL inhibition was in subnanomolar range for dasatinib and in submicromolar one for imatinib. EC50 for direct inhibition of LYN kinase was found to be about 20 nM for dasatinib and more than 10 µM for imatinib. Cells pretreated with 100 nM dasatinib were still able to bind to fibronectin and SRC kinases are thus not necessary for the formation of cell-matrix contacts. However, a minimal activity of SRC kinases might be required to mediate the increase in cell adhesivity induced by BCR-ABL inhibition. Indeed, active (autophosphorylated) LYN was found to localize in cell adhesive structures which were visualized using interference reflection microscopy.     

Src Drives Growth of Antiestrogen Resistant Breast Cancer Cell Lines and Is a Marker for Reduced Benefit of Tamoxifen Treatment

The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.     

Proliferation and Survival Signaling from Both Jak2-V617F and Lyn Involving GSK3 and mTOR/p70S6K/4EBP1 in PVTL-1 Cell Line Newly Established from Acute Myeloid Leukemia Transformed from Polycythemia Vera

The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),−16,+21,−22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.     

Adhesion structures in leukemia cells and their regulation by Src family kinases

Interaction of leukemia blasts with the bone marrow extracellular matrix often results in protection of leukemia cells from chemotherapy and in persistence of the residual disease which is on the basis of subsequent relapses. The adhesion signaling pathways have been extensively studied in adherent cells as well as in mature haematopoietic cells, but the adhesion structures and signaling in haematopoietic stem and progenitor cells, either normal or malignant, are much less explored. We analyzed the interaction of leukemia cells with fibronectin (FN) using interference reflection microscopy, immunofluorescence, measurement of adherent cell fraction, real-time microimpedance measurement and live cell imaging. We found that leukemia cells form very dynamic adhesion structures similar to early stages of focal adhesions. In contrast to adherent cells, where Src family kinases (SFK) belong to important regulators of focal adhesion dynamics, we observed only minor effects of SFK inhibitor dasatinib on leukemia cell binding to FN. The relatively weak involvement of SFK in adhesion structure regulation might be associated with the lack of cytoskeletal mechanical tension in leukemia cells. On the other hand, active Lyn kinase was found to specifically localize to leukemia cell adhesion structures and a less firm cell attachment to FN was often associated with higher Lyn activity (this unexpectedly occurred also after cell treatment with the inhibitor SKI-1). Lyn thus may be important for signaling from integrin-associated complexes to other processes in leukemia cells.     

Application of Multiplexed Kinase Inhibitor Beads to Study Kinome Adaptations in Drug-Resistant Leukemia

Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCβ, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.     

Short interfering RNA (siRNA) targeting the Lyn kinase induces apoptosis in primary, and drug-resistant, BCR-ABL1(+) leukemia cells

We studied the effects of Lyn ablation on the survival of drug-resistant chronic myelogenous leukemia (CML) blast crisis cells using siRNA. Lyn siRNA reduced Lyn protein in both normal hematopoietic cells and BCR-ABL1-expressing (BCR-ABL1(+)) blasts by 80-95%. Within 48 h, siRNA-treated BCR-ABL1(+) blasts underwent apoptosis, whereas normal cells remained viable. This increased dependence on Lyn signaling for BCR-ABL1(+) blast survival provides the basis for rational treatment of drug-resistant CML blast crisis, particularly when lymphoid in nature.     

Drug repurposing for chronic myeloid leukemia: in silico and in vitro investigation of DrugBank database for allosteric Bcr-Abl inhibitors

Chronic myeloid leukemia (CML) is caused by chromosomal rearrangement resulting in the expression of Bcr-Abl fusion protein with deregulated Abl tyrosine kinase activity. Approved drugs – imatinib, dasatinib, nilotinib, and ponatinib – target the ATP-binding site of Abl kinase. Even though these drugs are initially effective, long-term usefulness is limited by the development of resistance. To overcome this problem, targeting the allosteric site of Abl kinase, which is remote from the ATP-binding site is found to be a useful strategy. In this study, structure-based and ligand-based virtual screening methods were applied to narrow down possible drugs (from DrugBank database) that could target the allosteric site of Abl kinase. Detailed investigations of the selected drugs in the allosteric site of Abl kinase, using molecular dynamics and steered molecular dynamics simulation shows that gefitinib, an EGFR inhibitor approved for the treatment of lung cancer, could bind effectively to the allosteric site of Bcr-Abl. More interestingly, gefitinib was found to enhance the ability of imatinib to bind at the ATP-binding site of Bcr-Abl kinase. Based on the in silico findings, gefitinib was tested in combination with imatinib in K562 CML cell line using MTT cell proliferation assay and found to have a synergistic antiproliferative activity. Further detailed mechanistic study could help to unravel the full potential of imatinib – gefitinib combination for the treatment of CML.     

Inhibition of PI3K/mTOR Overcomes Nilotinib Resistance in BCR-ABL1 Positive Leukemia Cells through Translational Down-Regulation of MDM2

Chronic myeloid leukemia (CML) is a cytogenetic disorder resulting from formation of the Philadelphia chromosome (Ph), that is, the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein. Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds with which to treat CML. t(9;22) is not restricted to CML, 20-30% of acute lymphoblastic leukemia (ALL) cases also carry the Ph. However, TKIs are not as effective in the treatment of Ph+ ALL as in CML. In this study, the Ph+ cell lines JURL-MK2 and SUP-B15 were used to investigate TKI resistance mechanisms and the sensitization of Ph+ tumor cells to TKI treatment. The annexin V/PI (propidium iodide) assay revealed that nilotinib induced apoptosis in JURL-MK2 cells, but not in SUP-B15 cells. Since there was no mutation in the tyrosine kinase domain of BCR-ABL1 in cell line SUP-B15, the cells were not generally unresponsive to TKI, as evidenced by dephosphorylation of the BCR-ABL1 downstream targets, Crk-like protein (CrkL) and Grb-associated binder-2 (GAB2). Resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib unresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 cells with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, while combining nilotinib and BEZ235 led to a synergistic effect. The main role of PI3K/mTOR inhibitor BEZ235 and the reason for apoptosis in the nilotinib-resistant cells was the block of the translational machinery, leading to the rapid downregulation of the anti-apoptotic protein MDM2 (human homolog of the murine double minute-2). These findings highlight MDM2 as a potential therapeutic target to increase TKI-mediated apoptosis and imply that the combination of PI3K/mTOR inhibitor and TKI might form a novel strategy to combat TKI-resistant BCR-ABL1 positive leukemia.     

Monocytic myeloid‐derived suppressor cells as prognostic factor in chronic myeloid leukaemia patients treated with dasatinib

Myeloid suppressor cells are a heterogeneous group of myeloid cells that are increased in patients with chronic myeloid leukaemia (CML) inducing T cell tolerance. In this study, we found that therapy with tyrosine kinase inhibitors (TKI) decreased the percentage of granulocytic MDSC, but only patients treated with dasatinib showed a significant reduction in the monocytic subset (M‐MDSC). Moreover, a positive correlation was observed between number of persistent M‐MDSC and the value of major molecular response in dasatinib‐treated patients. Serum and exosomes from patients with CML induced conversion of monocytes from healthy volunteers into immunosuppressive M‐MDSC, suggesting a bidirectional crosstalk between CML cells and MDSC. Overall, we identified M‐MDSC as prognostic factors in patients treated with dasatinib. It might be of interest to understand whether MDSC may be a candidate predictive markers of relapse risk following TKI discontinuation, suggesting their potential significance as practice of precision medicine.     

Lyn regulates creatine uptake in an imatinib-resistant CML cell line

BackgroundImatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells.MethodsStable isotope metabolic labeling, media creatine depletion, and Na⁺/K⁺-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na⁺/K⁺-ATPase and creatine uptake.ResultsInhibition of the Na⁺/K⁺-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na⁺/creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na⁺/K⁺-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na⁺/K⁺-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na⁺/K⁺-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells.ConclusionsThese data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na⁺/K⁺-ATPase pump activity.General significanceThese studies identify kinase regulation of the Na⁺/K⁺-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.     

ATRA-Induced Cellular Differentiation and CD38 Expression Inhibits Acquisition of BCR-ABL Mutations for CML Acquired Resistance

Acquired resistance through genetic mutations is a major obstacle in targeted cancer therapy, but the underlying mechanisms are poorly understood. Here we studied mechanisms of acquired resistance of chronic myeloid leukemia (CML) to tyrosine kinase inhibitors (TKIs) by examining genome-wide gene expression changes in KCL-22 CML cells versus their resistant KCL-22M cells that acquire T315I BCR-ABL mutation following TKI exposure. Although T315I BCR-ABL is sufficient to confer resistance to TKIs in CML cells, surprisingly we found that multiple drug resistance pathways were activated in KCL-22M cells along with reduced expression of a set of myeloid differentiation genes. Forced myeloid differentiation by all-trans-retinoic acid (ATRA) effectively blocked acquisition of BCR-ABL mutations and resistance to the TKIs imatinib, nilotinib or dasatinib in our previously described in vitro models of acquired TKI resistance. ATRA induced robust expression of CD38, a cell surface marker and cellular NADase. High levels of CD38 reduced intracellular nicotinamide adenine dinucleotide (NAD⁺) levels and blocked acquired resistance by inhibiting the activity of the NAD⁺-dependent SIRT1 deacetylase that we have previously shown to promote resistance in CML cells by facilitating error-prone DNA damage repair. Consequently, ATRA treatment decreased DNA damage repair and suppressed acquisition of BCR-ABL mutations. This study sheds novel insight into mechanisms underlying acquired resistance in CML, and suggests potential benefit of combining ATRA with TKIs in treating CML, particularly in advanced phases.     

Design and synthesis of novel dasatinib derivatives as inhibitors of leukemia stem cells

We used the concept of bioisosteres to design and synthesize a novel series of dasatinib derivatives for the treatment of leukemia. Unfortunately, most of the dasatinib derivatives did not show appreciable inhibition against leukemia cell lines K562 and HL60. However, acrylamide compound 2c had comparable inhibitory activity with dasatinib against K562 cells (IC₅₀?=?0.039?nM vs. 0.069?nM). And amide compound 2a and acrylamide compound 2c also had comparable inhibitory activity with dasatinib against the leukemia cell line HL60 (IC₅₀?=?0.25?nM and 0.26?nM vs. 0.11?nM). Against the leukemia progenitor cell line KG1a, triazole compounds 15a and 15d–15f and oxadiazole compounds 24a–24d were more potent than dasatinib. In particular, the hydroxyl compounds 15a and 24a were about 64 and 180 fold more potent than dasatinib against KG1a cells (IC₅₀?=?0.14?μM and 0.05?μM vs. 8.98?μM). Compounds 15a and 24a also inhibited colony formation in MCF-7 cells and inhibited cell migration in the cell wound scratch assay in B16BL6 cells. Moreover, hydroxyl compounds 15a and 24a had low toxicity in vivo.     

Allosteric enhancement of the BCR-Abl1 kinase inhibition activity of nilotinib by cobinding of asciminib

Inhibitors that bind competitively to the ATP binding pocket in the kinase domain of the oncogenic fusion protein BCR–Abl1 are used successfully in targeted therapy of chronic myeloid leukemia (CML). Such inhibitors provided the first proof of concept that kinase inhibition can succeed in a clinical setting. However, emergence of drug resistance and dose-dependent toxicities limit the effectiveness of these drugs. Therefore, treatment with a combination of drugs without overlapping resistance mechanisms appears to be an appropriate strategy. In the present work, we explore the effectiveness of combination therapies of the recently developed allosteric inhibitor asciminib with the ATP-competitive inhibitors nilotinib and dasatinib in inhibiting the BCR–Abl1 kinase activity in CML cell lines. Through these experiments, we demonstrate that asciminib significantly enhances the inhibition activity of nilotinib, but not of dasatinib. Exploring molecular mechanisms for such allosteric enhancement via systematic computational investigation incorporating molecular dynamics, metadynamics simulations, and density functional theory calculations, we found two distinct contributions. First, binding of asciminib triggers conformational changes in the inactive state of the protein, thereby making the activation process less favorable by ∼4 kcal/mol. Second, the binding of asciminib decreases the binding free energies of nilotinib by ∼3 and ∼7 kcal/mol for the wildtype and T315I-mutated protein, respectively, suggesting the possibility of reducing nilotinib dosage and lowering risk of developing resistance in the treatment of CML.     

Dasatinib treatment can overcome imatinib and nilotinib resistance in CML patient carrying F359I mutation of BCR-ABL oncogene

Point mutations of bcr-abl tyrosine kinase are the most frequent causes of imatinib resistance in chronic myeloid leukaemia (CML) patients. In most CML cases with BCR-ABL mutations leading to imatinib resistance the second generation of tyrosine kinase inhibitors (TKI- e.g. nilotinib or dasatinib) may be effective. Here, we report a case of a CML patient who during imatinib treatment did not obtain clinical and cytogenetic response within 12 months of therapy. The sequencing of BCR-ABL kinase domains was performed and revealed the presence of a F359I point mutation (TTC-to-ATC nucleotide change leading to Phe-to-Ile amino acid substitution). After 1 month of nilotinib therapy a rapid progression of clinical symptoms was observed. In the presence of the F359I point mutation only dasatinib treatment overcame imatinib and nilotinib resistance.     

Stochastic dynamics of leukemic cells under an intermittent targeted therapy

The evolutionary dynamics of cancerous cell populations in a model of Chronic Myeloid Leukemia (CML) is investigated in the presence of an intermittent targeted therapy. Cancer development and progression is modeled by simulating the stochastic evolution of initially healthy cells which can experience genetic mutations and modify their reproductive behavior, becoming leukemic clones. Front line therapy for the treatment of patients affected by CML is based on the administration of tyrosine kinase inhibitors, namely imatinib (Gleevec) or, more recently, dasatinib or nilotinib. Despite the fact that they represent the first example of a successful molecular targeted therapy, the development of resistance to these drugs is observed in a proportion of patients, especially those in advanced stages. In this study, we simulate an imatinib-like treatment of CML by modifying the fitness and the death rate of cancerous cells and describe the several scenarios in the evolutionary dynamics of white blood cells as a consequence of the efficacy of the different modeled therapies. The patient response to the therapy is investigated by simulating a drug administration following a continuous or pulsed time scheduling. A permanent disappearance of leukemic clones is achieved with a continuous therapy. This theoretical behavior is in a good agreement with that observed in previous clinical investigations. However, these findings demonstrate that an intermittent therapy could represent a valid alternative in patients with high risk of toxicity. A suitable tuned pulsed therapy can also reduce the probability of developing resistance.