Добірка наукової літератури з теми "Cytokines; T315I mutation; chronic myeloid leukaemia"

Abstract Introduction Treatment-resistant acute lymphoblastic leukemia (ALL) remains a significant clinical issue. Recently, genomic profiling has identified a new subtype of high-risk ALL termed Philadelphia-chromosome-like (Ph-like) ALL, associated with a poor outcome1. Ph-like ALL has a gene expression profile similar to Ph+ (BCR-ABL1+) ALL, characterized by the presence of fusion genes converging on kinase and cytokine signaling pathways. These pathways have been shown to be targetable both in vitro and in case reports by tyrosine kinase inhibitors (TKIs). Despite well-documented efficacy profiles, it is known from TKI-use in chronic myeloid leukemia (CML) andPh+ ALL that resistance is likely, resulting in relapse. Our study aims to model and understand mechanisms of TKI-resistance inPh-like ALL, informing future therapeutic strategies that may avert or overcome resistance, potentially improving patient outcomes. Methods Three Ph-like ALL lines were generated via retroviral-transduction from plasmids of fusion genes identified in patient cohorts (RANBP2-ABL1, SSBP2-CSF1R and PAX5-JAK2, a kind gift from C. Mullighan)2 into Ba/F3 pro-B cells. Transformation was confirmed via growth of cells in the absence of IL-3. Cells were tested for sensitivity to a panel of TKIs (imatinib, dasatinib, ponatinib, ruxolitinib and BMS-911543) via Annexin-V/7-AAD flow-cytometry and western blotting of downstream effector proteins. Drug resistance was generated through exposure of cells to incrementally increasing concentrations of TKIs over a period of 3-6 months, and cell death LD50 determined byAnnexin-V/7-AAD. Sanger sequencing of the 3-prime partner gene of each fusion was performed to identify the emergence of any kinase-domain mutations. Results Ba/F3 Ph-like cells demonstrated sensitivity to TKIs at clinically relevant doses (RANBP2-ABL1: 1 μM imatinib, 5 nM dasatinib & 5 nM ponatinib; SSBP2-CSF1R: 1 μM imatinib, 6 nM dasatinib; PAX5-JAK2: 1 μM ruxolitinib & 2 μM BMS-911543). This correlated with decreased levels of relevant downstream signaling proteins including p-Stat5, p-Erk and p-CrkL. TKI-resistant Ph-like ALL lines were tolerant to a significantly higher concentration of TKIs compared to control (RANBP2-ABL1: 10 μM imatinib, 200 nM dasatinib & 200 nM ponatinib; SSBP2-CSF1R: 10 μM imatinib, 200 nM dasatinib; PAX5-JAK2: 10μMruxolitinib & 10μM BMS-911543; Table 1). Sequencing analysis revealed that Ba/F3 RANBP2-ABL1 imatinib and dasatinib resistant cells acquired the clinically significant ABL1 T315I (c.944C>T) kinase-domain mutation, which was ultimately targetable using the third-generation TKI ponatinib (LD50: 25 nM). An ABL1 E255K (c.763G>A) and c-terminus deletion was discovered in the ponatinib-resistant line. In Ba/F3 SSBP2-CSF1R cells, a novel CSF1R L785M (c.2566C>A) mutation was identified in imatinib-resistant cells whereas a deletion spanning the SSBP2-CSF1R breakpoint was acquired in the dasatinib-resistant line. A JAK2 Y931C (c.3286A>G) point mutation previously associated with resistance to ATP-competitive inhibitors was acquired in Ba/F3 PAX5-JAK2ruxolitinib and BMS-911543 resistant lines. Conclusion In vitro modeling of Ph-like ALL resistance has identified novel kinase domain mutations and deletions that may arise as a result of targeted TKI therapy. In addition, previously identified mutations (T315I and E255K) were also identified. Detection of these mutations is important because alterations in drug-binding regions are known to result in significantly reduced TKI sensitivity, leading clinically to relapse3. This study describes an in vitro platform that can be utilized to inform future clinical approachesincluding the development of rational therapeutic approaches (and/or combination therapies) to avert resistance inPh-like ALL cases treated with rationally targeted therapies. Abbreviations: ABL1 - Abelson tyrosine protein kinase 1 CSF1R - Colony stimulating factor 1 receptor JAK2 - Janus kinase 2 PAX5 - Paired box 5 RANBP2 - RAN-binding protein 2 SSBP2 - Single-stranded DNA binding protein 2 References: 1 Den Boer et al, Lancet Oncology 2009; 10(2):125-34 2 Roberts et al, Cancer Cell 2012; 22(2):153-66 3 Barouch-Bentov & Sauer, Expert Opinion on Investigational Drugs 2011; 20(2);153-208 Disclosures Hughes: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Australasian Leukaemia and Lymphoma Group (ALLG): Other: Chair of the CML/MPN Disease Group. White:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Ariad: Consultancy, Honoraria, Research Funding.

The development and clinical availability of second-generation tyrosine kinase inhibitors (TKIs) for the treatment of patients who discontinue imatinib therapy has further improved the outlook for patients with chronic phase chronic myeloid leukaemia (CP-CML). There is, however, uncertainty surrounding how best to treat patients after failing second-generation TKIs. A three-section questionnaire was devised by chronic myeloid leukaemia experts to address questions surrounding this issue. Responses were received from 14 out of 34 experts (41.2%). Generally, a reasonable consensus was found among the responses for most issues. There was a complete consensus that ponatinib was suitable for all patients carrying the T315I mutation regardless of the molecular response to prior treatment. There was also complete consensus that allografting is appropriate in any patient who has had blast crises and is back in a second chronic phase. More recommendations for third-line treatment of CP-CML patients are necessary.

Ponatinib is a third generation kinase inhibitor designed to overcome the gatekeeper T315I mutation. In different trials this drug showed inhibitory activity against native BCR-ABL1 kinase and several ABL1 mutations. For this reason, ponatinib is currently indicated for the treatment of chronic myeloid leukaemia (CML) in every phase of disease resistant and/or intolerant to dasatinib and nilotinib and for whom imatinib is not indicated anymore or for patients with T315I mutation. The drug is also indicated for Ph+ acute lymphoblastic leukaemia (ALL). Ponatinib was temporarily suspended in 2013 for the occurrence of cardiovascular thrombotic events. Since then, different investigators analyzed baseline characteristics of patient candidates for ponatinib, especially cardiovascular profile, in order to describe general management recommendations in this setting. In this review, clinical trials data about the use of ponatinib in CML and Ph+ ALL patients will be discussed. It will be focused also about the safety and tolerability profile of the drug and future perspectives of employment.

Abstract The Philadelphia chromosome, is formed as a result of a reciprocal translocation between chromosomes 9 and 22 and results in the formation of the hybrid oncoprotein BCR-ABL which is pathognomic of Chronic Myeloid Leukaemia (CML). Imatinib Mesylate (IM), a tyrosine kinase inhibitor that specifically binds BCR-ABL in its inactive conformation and functions as a competitive inhibitor of ATP binding, has revolutionized therapy for patients with CML. However, resistance develops in a significant proportion of cases and is predominantly mediated by single base-pair substitutions within the BCR-ABL kinase domain leading to changes in the amino acid composition and conformational changes in the kinase domain that inhibit IM binding whilst retaining BCR-ABL phosphorylation capacity. Second generation tyrosine kinase inhibitors such as Dasatinib and Nilotinib retain activity in IM-resistant patients due to less stringent binding requirements and represent viable alternatives for IM-resistant or intolerant CML patients. In this study, we undertook to examine the molecular mechanisms underlying IM resistance. A cohort of 33 patients with either primary or acquired resistance (n=31) to IM or intolerance to IM (n=2) was identified by persistently high or increasing levels of BCR-ABL transcripts determined by standardised real-time quantitative PCR. An initial allele-specific PCR screen was used to sensitively detect the clinically significant T315I mutation, which renders patients insensitive to currently available tyrosine kinase inhibitors: four (11.8%) IM resistant/intolerant patients were T315I positive. To further characterise the molecular mechanisms of mutation induced resistance, the BCR-ABL kinase domain was then screened for the presence of a mutation using a sensitive denaturing high performance liquid chromatography (dHPLC) approach. dHPLC can detect a single base pair substitution within the BCR-ABL kinase domain based on hybridization to a non-mutated wild type control. Mutated samples display reduced hybridization capacity to the dHPLC column and elute at an earlier time-point. Sensitivity of dHPLC (0.1–10%) is significantly greater than that of sequencing (15–25%). Following dHPLC analysis, samples showing evidence of mutation were examined by direct sequencing to identify the mutation(s) present. Kinase domain mutations have been identified in 18 of the 33 (55%) patients examined to date and these include p-loop mutations (M244V, G250E, Q252H), IM-binding domain mutations (T315I & F317L), catalytic domain mutations (M351T & E355G), and an activation-loop mutation (L387M). Three previously unreported mutations that may be associated with IM resistance (T267A, L273M, K291Q) were identified. The L273M positive patient also has an M244V mutation and has shown primary resistance to IM & is currently being treated with Nilotinib; the T267A patient has rising BCR-ABL transcripts, while the K291Q patient has had a 3 log reduction of BCR-ABL transcripts following IM dose escalation. In addition to the above mutations, 2 SNPs were identified at E275E and at L248L, which may not be clinically relevant. The identification of clinically significant mutations facilitates selection of alternative approaches to therapy such as IM dose escalation, second generation tyrosine kinase inhibitors or allogeneic stem cell transplantation, if eligible, allowing patient specific approaches to therapy.

CML (chronic myeloid leukaemia) is a myeloproliferative disease that originates in an HSC (haemopoietic stem cell) as a result of the t(9;22) translocation, giving rise to the Ph (Philadelphia chromosome) and bcr-abl oncoprotein. The disease starts in CP (chronic phase), but as a result of genomic instability, it progresses over time to accelerated phase and then to BC (blast crisis), becoming increasingly resistant to therapy. bcr-abl is a constitutively active tyrosine kinase that has been targeted by TKIs (tyrosine kinase inhibitors), including IM (imatinib mesylate), nilotinib and dasatinib. We have developed various flow cytometry techniques to enable us to isolate candidate CML stem cells from CP patients at diagnosis that efflux Hoechst dye, express CD34, lack CD38 and are cytokine-non-responsive in culture over periods of up to 12 days in growth factors. These stem cells have been shown to regenerate bcr-abl-positive haemopoiesis in immunocompromised mice upon transplantation. We previously demonstrated that IM was antiproliferative for CML stem cells but did not induce apoptosis. Clinical experience now confirms that IM may not target CML stem cells in vivo with few patients achieving complete molecular remission and relapse occurring rapidly upon drug withdrawal. Our recent efforts have focused on understanding why CML stem cells are resistant to IM and on trying to find novel ways to induce apoptosis of this population. We have shown that CML stem cells express very high levels of functional wild-type bcr-abl; no kinase domain mutations have been detected in the stem cell population. Dasatinib, a more potent multitargeted TKI than IM, inhibits bcr-abl activity more efficiently than IM but still does not induce apoptosis of the stem cell population. Most recently, we have tested a number of novel drug combinations and found that FTIs (farnesyl transferase inhibitors) have activity against CML. BMS-214662 is the most effective of these and induces apoptosis of phenotypically and functionally defined CML stem cells in vitro, as a single agent and in combination with IM or dasatinib. The effect against CML stem cells is selective with little effect on normal stem cells. The drug is also effective against BC CML stem cells and equally effective against wild-type and mutant bcr-abl, including the most resistant mutant T315I. In association with apoptosis, there is activation of caspase 8 and caspase 3, inhibition of the MAPK pathway, IAP-1 (inhibitor of apoptosis protein-1), NF-κB (nuclear factor κB) and iNOS (inducible nitric oxide synthase). Furthermore, BMS-214662 synergizes with MEK1/2 [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase 1/2] inhibitors, suggesting a second mechanism other that RAS inhibition for induction of apoptosis. Our intentions are now to explore the activity of BMS-214662 in other cancer stem cell disorders and to move this preclinical work to a clinical trial combining dasatinib with BMS-214662 in CML.

Abstract Abstract 4280 Introduction Imatinib (Imatinib) offers outstanding results in the treatment of chronic phase of chronic myeloid leukaemia (CML). However, there are still some patients who do not achieve an optimal response. There are several possible mechanisms both for primary refractoriness and for acquired resistance, but very often the blame falls in the appearance of ABL-kinase-domain (KD) mutations. Different mutations are related to variable degrees of resistance, ranging from the extreme refractoriness of the mutation T315I down to some which appear to have no clinical significance. Aims To define the clinical characteristics and outcome of patients treated in our centre, in which ABL-kinase-domain mutations through direct DNA sequencing have been analysed. Results In 24 of a total 86 patients, KD mutations were looked for. Screening for mutations was undertaken for one of three reasons: at diagnosis in 6 patients, suboptimal response (Leukemia-Net criteria) in 10 cases and Treatment-Failure (LN criteria) in 8 cases. Median time from diagnosis to ABL mutation detection was 38 months. No risk factors at diagnosis for the appearance of KD mutations have been found. Of the 7 mutations, 5 cases corresponded to treatment failures and 2 to patients in so-called suboptimal responses. One patient, after achieving a complete response to Imatinib, developed a T315I KD mutation and died due to blastic crisis despite having received second generation TKI. Another patient in failure (mutation G250E) achieved a molecular response with dasatinib. No mutations were found in 6 patients checked before treatment, but one of these developed a mutation G250E after 18 months, along with the criteria of a suboptimal response and achieved a mayor molecular response with dasatinib. Conclusions KD mutations are found in a proportion of patients in the situation of suboptimal response or failure to IM, more frequently in the latter in our experience, but the clinical significance of some of them is still unclear. On the contrary, DNA sequencing for screening at the moment of diagnosis offers little relevance in chronic phase CML. Disclosures: No relevant conflicts of interest to declare.

The treatment outcome in patients with chronic myeloid leukaemia (CML) in blast crisis (BC) is unsatisfactory despite the use of allogeneic stem cell transplantation (ASCT). Moreover, in some patients ASCT is contraindicated, with limited treatment options. We report the case series of two patients with lymphoid BC CML in whom ASCT was not approachable. The first patient developed BC two months after diagnosis in association with dic(7;9)(p11.2;p11.2) and T315I mutation. Blast crisis with central nervous system leukemic involvement and K611N mutation of the SETD2 gene developed abruptly in the second patient five years after ceasing treatment with nilotinib in major molecular response (MMR) at the patient’s request. Both underwent one course of chemotherapy in combination with rituximab and imatinib, followed by dasatinib and interferon α (INFα) treatment in the first and dasatinib alone in the second case. Deep molecular response (DMR; MR 4.0) was achieved within a short time in both cases. It is probable that DMR was caused by a specific immune response to CML cells, described in both agents. The challenging medical condition that prompted these case series, and the subsequent results, suggest a re-visit to the use of a combination of well-known drugs as an area for further investigation.

Abstract Mutations of the ABL kinase domain (KD) are the most frequent cause of acquired resistance to imatinib in patients with chronic myeloid leukaemia (CML), most likely due to selection of mutated clones on imatinib treatment. As new Bcr-Abl inhibitors become available, precise quantification of low level mutation will be required to monitor response. Here we report our results evaluating patients with advanced phase or imatinib resistant CML for KD mutations using a newly developed, sensitive and quantitative Ligation PCR (L-PCR) assay in comparison to direct sequencing. Twenty eight CML patients on imatinib (17 male, 11 female) with a median age of 62 (range 20 to 75) years in blast crisis (n=4), accelerated phase (n=12) or imatinib failure (n= 12) were analysed using both approaches. Sequencing of the ABL KD was performed using forward and reverse primers to ABL exons 4 and 7, while the L-PCR analysis focussed initially on the E255K and T315I mutations. Briefly, pairs of probes specific for either wild type (WT) or mutant BCR-ABL were added to the RT-PCR amplified ABL KD, then ligated under conditions optimized for specificity. Ligated probe pairs were than amplified in a quantitative PCR using universal primers. Quantification was performed using internal cell-in-cell dilutions of BaF3 cell lines expressing wt and mutant BCR/ABL and values were expressed as % BCR-ABLmut/ BCR-ABLWT. In our hands, this assay can detect 0.05 – 0.1% T315I and 0.01–0.05% E255K in a BCR-ABL WT background. The inter-assay variation at the lowest detection level was only 6.7 and 4,7% for the mutations T315I and E255K respectively. Results were scored positive only if two independent runs showed amplification exceeding the lowest controls. All patients were treated with a median imatinib dose of 600 (range 500–800) mg for a median of 10.5 (range 1 to 74) months. Dose reductions due to toxicity were necessary in 8 (29%) patients. Direct sequencing revealed E255K or T315I mutations in three patients, each with more than 20% mutated allele. L-PCR revealed these three patients plus three more with lower levels of mutation (T315I, 0.46% and E255K, 0.16, 0.17%). The patient with 0.46% T315I also showed G250E by sequencing. This patient was subsequently treated with Dasatinib but failed to respond. In twenty two patients negative by L-PCR, sequencing of exons 4 and 7 showed 15 (53%) to be WT, 6 (21%) to have KD mutations F317L, F359V (n=2), H396R, M315T with F359V, Y253F with the K247R polymorphism and one to have an 80 base pair deletion 3′ of the p-loop. In conclusion the L-PCR assay was able to detect T315I and E255K mutations in twice as many patients as did direct sequencing. These low-level mutations would most probably also have been missed by the D-HPLC-based screening. The fact that only L-PCR detected this mutation in a patient who failed to respond to dasatinib implies that the assays generate clinically useful information. mutations. The study is currently being expanded to include further mutations and longitudinal monitoring of a larger cohort of patients.

Abstract CML is a clonal myeloproliferative disease in which Bcr-Abl deregulated tyrosine kinase (TK) activity impairs blood cells homeostasis leading to altered growth, homing and apoptosis. Imatinib mesylate (Glivec, Novartis), an ATP competitor molecule, is the gold standard therapy for Bcr-Abl positive diseases, but resistance is increasingly encountered, mainly due to point mutations in the abl kinase portion of the molecule. Second generation TK inhibitors (Dasatinib, Brystol-Myers Squibb and Nilotinib, Novartis) can bind mutated forms of the protein, but T315I mutation appear unresponsive to TK treatments. Apoptosis is a cell suicide mechanism activated to remove redundant, damaged, or infected cells. An essential component of the apoptotic machinery, the caspase family, consists of a group of intracellular cysteine proteases that can be divided into initiator/upstream caspases (2, 8, 9, and 10 caspases), which activate the downstream/effector caspases (3, 6 and 7 caspases). Cleavage of a selected group of substrates by effector caspases is responsible for dismantling of essential cell components, which results in morphological and biochemical changes that characterise apoptotic cell death: cytoskeletal rearrangement, cell membrane blebbing, nuclear condensation and DNA fragmentation. Based on cell line experiments we focused our attention on some members of the pyrrolo[1,2-b][1,2,5]benzothiadiazepines (PBTDs) family to test their potential apoptotic activity in CML. Important apoptotic activity was demonstrated on K562, K562 R-IM, HL -60 ,U937 and Jurkatt cell lines, as evidenced with the concentration and the percentage of the cell death quantified by measuring PI-uptake by flow cytometry, DNA fragmentation, and analyzed by agarose gel electrophoresis generating a characteristic ladder pattern of discontinuous DNA fragments. Three PBTDs coumpounds (RS 678, RS779, RS 2630 RS) were then incubated with peripheral blood and/or bone marrow cells isolated from 30 CML untreated, 25 imatinib-resistant, 10 Dasatinib resistant, and 1 Nilotinib resistant patients. Cells were incubated at the concentration 10μM for,16,24, and 48 hours. Apoptotic DNA fragmentation was tested by agarose gels electrophoresis and was present in all but one patients in a percentage varying from 60 to 85%, compared to 0 to 5% of internal controls. Among the 25 imatinib-resistant patient 23 were tested for abl mutation using D-HPLC. Five patients showed T315I, one F317L, one each M351V, D276G, Y253H, and 2 patients showed 2 mutations (F317l and M315T; and G250G, and F359V). Only one patient Dasatinib reistant carrying M351V mutation showed very low apoptosis (5–10%), while the 5 patients with T315I mutation underwent high apoptotic DNA fragmentation. Apoptosis seem to be triggered by PBTDs through activation of initiator caspases 8 and 9 and effector caspases 3 as eveidenced by western blotting. Although more experiments are needed, these compounds appear promising for a preclinical approach in CML.

Since the introduction of imatinib, the outlook for patients with chronic myeloid leukaemia (CML) has dramatically improved. However, approximately 30-40% of patients develop intolerance or resistance to the drug and cease therapy. Among those who develop resistance, the most common cause is the development of point mutations in the kinase domain (KD) of BCR-ABL1 which impair drug binding and hence result in the loss of kinase inhibition. The T315I mutation (gatekeeper mutation) is of particular significance as it is one of the most common mutations and it is resistant to all 3 tyrosine kinase inhibitors (TKI) currently approved for therapeutic use in Australia. There is evidence to suggest that KD mutations in BCR-ABL1 may alter the biological activity of Bcr-Abl. Both in vitro and clinical studies have suggested that the T315I mutation results in greater oncogenic potential. Patients harbouring this mutation have poorer prognoses and a significantly higher rate of progression compared to other mutations. There is also ample evidence to suggest that secretion of cytokines and growth factors play a role in rendering BCR-ABL1 positive cells resistant to TKI. This thesis focuses on the role of cytokines in the resistance mechanism of cells with the T315I mutation and how this mechanism is achieved. K562-T315I cells were developed in our laboratory by exposing them to increasing levels of dasatinib over several months. HL60 cell lines were virally transduced with the BCR-ABL1p210 [p210 in superscript] and BCR-ABL1T315I [T315I in superscript] constructs. Investigations have identified that several soluble factors are preferentially secreted by cells with the T315I mutation namely, FGF-2, IL-8, MCP-1 and G-CSF. Additionally, the supernatant of K562-T315I cells also contains higher concentrations of GM-CSF and IL-6. These studies also identified that FGF-2 was able to protect K562 naïve cells from TKI-induced cell death and suggests that this occurs via activation of the MAPK and STAT5 pathways. Thus, in addition to acquiring point mutations that result in the inability of TKIs to bind Bcr-Abl, the T315I mutation also results in overexpression of FGF-2 which can confer resistance to non-mutated cells. Furthermore, in the presence of imatinib, dasatinib and nilotinib, K562-T315I cells proliferate and survive better than in the absence of a TKI. This is due to hyperactivation of the MAPK pathway whereas signalling of other pathways, JAK/STAT5 and PI3K/Akt are not increased. This phenomenon was demonstrated especially with nilotinib but studies did not indicate a cytokine mediated effect through an autocrine hypersecretion by the K562-T315I cells. Nevertheless, this finding may be one of the reasons why the T315I mutation confers a worse outcome in patients with CML if they remain on imatinib, nilotinib and dasatinib treatment.
Thesis (M.Phil.) -- University of Adelaide, School of Medicine, 2014