Specific High-Sensitivity Enzymatic Reporter UnLOCKing-Mediated Detection of Oncogenic BCR::ABL1 and EGFR Rearrangements.

Advances in molecular medicine have placed nucleic acid detection methods at the center of an increasing number of clinical applications. Polymerase chain reaction (PCR)-based diagnostics have been widely adopted for their versatility, specificity, and sensitivity. However, recently reported clustered regularly interspaced short palindromic repeats-based methods have demonstrated equivalent to superior performance, with increased portability and reduced processing time and cost. In this study, we applied Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK) technology to the detection of oncogenic rearrangements. We implemented SHERLOCK for the detection of BCR::ABL1 mRNA, a hallmark of chronic myeloid leukemia (CML), and EGFR DNA oncogenic alleles, frequently detected in glioblastoma and non-small cell lung cancer (NSCLC). SHERLOCK enabled rapid, sensitive, and variant-specific detection of BCR::ABL1 and EGFR alterations. Compared with the gold-standard PCR-based methods currently used in clinic, SHERLOCK achieved equivalent to greater sensitivity, suggesting it could be a new tool in CML and NSCLC, to detect low level of molecular residual disease.

Antimetabolic cooperativity with the clinically approved l-asparaginase and tyrosine kinase inhibitors to eradicate CML stem cells.

OBJECTIVE: Long-term treatment with tyrosine kinase inhibitors (TKI) represents an effective cure for chronic myeloid leukemia (CML) patients and discontinuation of TKI therapy is now proposed to patient with deep molecular responses. However, evidence demonstrating that TKI are unable to fully eradicate dormant leukemic stem cells (LSC) indicate that new therapeutic strategies are needed to control LSC and to prevent relapse. In this study we investigated the metabolic pathways responsible for CML surviving to imatinib exposure and its potential therapeutic utility to improve the efficacy of TKI against stem-like CML cells. METHODS: Using complementary cell-based techniques, metabolism was characterized in a large panel of BCR-ABL+ cell lines as well as primary CD34+ stem-like cells from CML patients exposed to TKI and L-Asparaginases. Colony forming cell (CFC) assay and flow cytometry were used to identify CML progenitor and stem like-cells. Preclinical models of leukemia dormancy were used to test the effect of treatments. RESULTS: Although TKI suppressed glycolysis, compensatory glutamine-dependent mitochondrial oxidation supported ATP synthesis and CML cell survival. Glutamine metabolism was inhibited by L-asparaginases such as Kidrolase or Erwinase without inducing predominant CML cell death. However, clinically relevant concentrations of TKI render CML cells susceptible to Kidrolase. The combination of TKI with Lasparaginase reactivates the intinsic apoptotic pathway leading to efficient CML cell death. CONCLUSION: Targeting glutamine metabolism with the FDA-approved drug, Kidrolase in combination with TKI that suppress glycolysis represents an effective and widely applicable therapeutic strategy for eradicating stem-like CML cells.

Relevance of treatment-free remission recommendations in chronic phase chronic leukemia patients treated with frontline tyrosine kinase inhibitors.

BACKGROUND: Tyrosine kinase inhibitors (TKI) can be safely discontinued in chronic phase chronic myeloid leukemia (CP-CML) patients who had achieved a sustained deep molecular response. Based on the results of discontinuation trials, recommendations regarding patient selection for a treatment-free remission (TFR) attempt had been proposed. The aims of this study were to evaluate the rate of patients eligible for TKI discontinuation and molecular recurrence-free survival (MRFS) after stop according to recommendations. METHODS: Over a 10-year period, newly diagnosed CP-CML patients and treated with first-line TKI in the nine French participating centers were included. Eligibility to treatment discontinuation and MRFS were analyzed and compared according to selection criteria defined by recommendations and first-line treatments. RESULTS: From January 2006 to December 2015, 398 patients were considered. Among them, 73% and 27% of patients received imatinib or either second or third generation tyrosine kinase inhibitors as frontline treatment, respectively. Considering the selection criteria defined by recommendations, up to 55% of the patients were selected as optimal candidates for treatment discontinuation. Overall 95/398 (24%) discontinued treatment. MRFS was 51.8% [95% CI 41.41-62.19] at 2 years and 43.8% [31.45-56.15] at 5 years. Patients receiving frontline second-generation TKI and fulfilling the eligibility criteria suggested by recommendations had the lowest probability of molecular relapse after TKI stop when compare to others. CONCLUSION: One third of CP-CML patients treated with TKI frontline fulfilled the selection criteria suggested by European LeukemiaNet TFR recommendations. Meeting selection criteria and second-generation TKI frontline were associated with the highest MRFS.

Incidences of Deep Molecular Responses and Treatment-Free Remission in de Novo CP-CML Patients.

Background: Tyrosine Kinase Inhibitors (TKIs) discontinuation in patients who had achieved a deep molecular response (DMR) offer now the opportunity of prolonged treatment-free remission (TFR). Patients and Methods: Aims of this study were to evaluate the proportion of de novo chronic-phase chronic myeloid leukemia (CP-CML) patients who achieved a sustained DMR and to identify predictive factors of DMR and molecular recurrence-free survival (MRFS) after TKI discontinuation. Results: Over a period of 10 years, 398 CP-CML patients treated with first-line TKIs were included. Median age at diagnosis was 61 years, 291 (73%) and 107 (27%) patients were treated with frontline imatinib (IMA) or second- or third-generation TKIs (2-3G TKI), respectively. With a median follow-up of seven years (range, 0.6 to 13.8 years), 182 (46%) patients achieved a sustained DMR at least 24 months. Gender, BCR-ABL1 transcript type, and Sokal and ELTS risk scores were significantly associated with a higher probability of sustained DMR while TKI first-line (IMA vs. 2-3G TKI) was not. We estimate that 28% of CML-CP would have been an optimal candidate for TKI discontinuation according to recent recommendations. Finally, 95 (24%) patients have entered in a TFR program. MRFS rates at 12 and 48 months were 55.1% (95% CI, 44.3% to 65.9%) and 46.9% (95% CI, 34.9% to 58.9%), respectively. In multivariate analyses, first-line 2-3G TKIs compared to IMA and TKI duration were the most significant factors of MRFS. Conclusions: Our results suggest that frontline TKIs have a significant impact on TFR in patients who fulfill the selection criteria for TKI discontinuation.

Inhibition of DDR1 enhances in vivo chemosensitivity in KRAS-mutant lung adenocarcinoma.

Platinum-based chemotherapy in combination with immune-checkpoint inhibitors is the current standard of care for patients with advanced lung adenocarcinoma (LUAD). However, tumor progression evolves in most cases. Therefore, predictive biomarkers are needed for better patient stratification and for the identification of new therapeutic strategies, including enhancing the efficacy of chemotoxic agents. Here, we hypothesized that discoidin domain receptor 1 (DDR1) may be both a predictive factor for chemoresistance in patients with LUAD and a potential target positively selected in resistant cells. By using biopsies from patients with LUAD, KRAS-mutant LUAD cell lines, and in vivo genetically engineered KRAS-driven mouse models, we evaluated the role of DDR1 in the context of chemotherapy treatment. We found that DDR1 is upregulated during chemotherapy both in vitro and in vivo. Moreover, analysis of a cohort of patients with LUAD suggested that high DDR1 levels in pretreatment biopsies correlated with poor response to chemotherapy. Additionally, we showed that combining DDR1 inhibition with chemotherapy prompted a synergistic therapeutic effect and enhanced cell death of KRAS-mutant tumors in vivo. Collectively, this study suggests a potential role for DDR1 as both a predictive and prognostic biomarker, potentially improving the chemotherapy response of patients with LUAD.

The Expression of Myeloproliferative Neoplasm-Associated Calreticulin Variants Depends on the Functionality of ER-Associated Degradation.

BACKGROUND: Mutations in CALR observed in myeloproliferative neoplasms (MPN) were recently shown to be pathogenic via their interaction with MPL and the subsequent activation of the Janus Kinase - Signal Transducer and Activator of Transcription (JAK-STAT) pathway. However, little is known on the impact of those variant CALR proteins on endoplasmic reticulum (ER) homeostasis. METHODS: The impact of the expression of Wild Type (WT) or mutant CALR on ER homeostasis was assessed by quantifying the expression level of Unfolded Protein Response (UPR) target genes, splicing of X-box Binding Protein 1 (XBP1), and the expression level of endogenous lectins. Pharmacological and molecular (siRNA) screens were used to identify mechanisms involved in CALR mutant proteins degradation. Coimmunoprecipitations were performed to define more precisely actors involved in CALR proteins disposal. RESULTS: We showed that the expression of CALR mutants alters neither ER homeostasis nor the sensitivity of hematopoietic cells towards ER stress-induced apoptosis. In contrast, the expression of CALR variants is generally low because of a combination of secretion and protein degradation mechanisms mostly mediated through the ER-Associated Degradation (ERAD)-proteasome pathway. Moreover, we identified a specific ERAD network involved in the degradation of CALR variants. CONCLUSIONS: We propose that this ERAD network could be considered as a potential therapeutic target for selectively inhibiting CALR mutant-dependent proliferation associated with MPN, and therefore attenuate the associated pathogenic outcomes.

Novel analytical methods to interpret large sequencing data from small sample sizes.

BACKGROUND: Targeted therapies have greatly improved cancer patient prognosis. For instance, chronic myeloid leukemia is now well treated with imatinib, a tyrosine kinase inhibitor. Around 80% of the patients reach complete remission. However, despite its great efficiency, some patients are resistant to the drug. This heterogeneity in the response might be associated with pharmacokinetic parameters, varying between individuals because of genetic variants. To assess this issue, next-generation sequencing of large panels of genes can be performed from patient samples. However, the common problem in pharmacogenetic studies is the availability of samples, often limited. In the end, large sequencing data are obtained from small sample sizes; therefore, classical statistical analyses cannot be applied to identify interesting targets. To overcome this concern, here, we described original and underused statistical methods to analyze large sequencing data from a restricted number of samples. RESULTS: To evaluate the relevance of our method, 48 genes involved in pharmacokinetics were sequenced by next-generation sequencing from 24 chronic myeloid leukemia patients, either sensitive or resistant to imatinib treatment. Using a graphical representation, from 708 identified polymorphisms, a reduced list of 115 candidates was obtained. Then, by analyzing each gene and the distribution of variant alleles, several candidates were highlighted such as UGT1A9, PTPN22, and ERCC5. These genes were already associated with the transport, the metabolism, and even the sensitivity to imatinib in previous studies. CONCLUSIONS: These relevant tests are great alternatives to inferential statistics not applicable to next-generation sequencing experiments performed on small sample sizes. These approaches permit to reduce the number of targets and find good candidates for further treatment sensitivity studies.

Killer immunoglobulin-like receptor genotypes and chronic myeloid leukemia outcomes after imatinib cessation for treatment-free remission.

BACKGROUND: Natural Killer (NK) cells are innate lymphoid cells that can be cytotoxic toward a large panel of solid tumors and hematological malignancies including chronic myeloid leukemia (CML). Such a cytotoxicity depends on various receptors. Killer immunoglobulin-like receptors (KIR) belong to these receptors and are involved in maturation process, then in the activation abilities of NK cells. METHODS: We investigated the prognostic impact of the KIR2DL5B genotype in 240 CML patients included in two clinical trials investigating tyrosine kinase inhibitors (TKI) discontinuation: STIM and STIM2. RESULTS: After adjustment for standard risk factors in CML, we found that the inhibitory receptor KIR2DL5B-positive genotype was independently related to a delayed second deep molecular remission (HR 0.54, 95% CI [0.32-0.91], P = 0.02) after TKI rechallenge but not to time to first deep molecular remission or treatment-free remission rates. CONCLUSION: These results suggest that KIR2DL5B could carry a role in lymphocyte-mediated control of leukemic residual disease control in patient with CML relapse.

Incidence and outcome of BCR-ABL mutated chronic myeloid leukemia patients who failed to tyrosine kinase inhibitors.

PURPOSE: To assess the incidence of BCR-ABL kinase domain (KD) mutation detection and its prognostic significance in chronic phase chronic myeloid leukemia (CP-CML) patients treated with tyrosine kinase inhibitors (TKIs). PATIENTS AND METHODS: We analyzed characteristics and outcome of 253 CP-CML patients who had at least one mutation analysis performed using direct sequencing. Of them, 187 patients were early CP (ECP) and 66 were late CP late chronic phase (LCP) and 88% were treated with Imatinib as first-line TKI. RESULTS: Overall, 80 (32%) patients harbored BCR-ABL KD mutations. A BCR-ABL KD mutation was identified in 57% of patients, who progressed to accelerated or blastic phases (AP-BP), and 47%, 29%, 35%, 16% and 26% in patients in CP-CML at the time of mutation analysis who lost a complete hematologic response, failed to achieve or loss of a prior complete cytogenetic and major molecular response, respectively. Overall survival and cumulative incidence of CML-related death were significantly correlated with the disease phase whatever the absence or presence of a mutation was and for the latter the mutation subgroup (T315I vs P-loop vs non-T315I non-P-loop) (P<.001). Considering patients who were in CP at the time of mutation analysis, LCP mutated patients had a significantly worse outcome than ECP-mutated patients despite a lower incidence of T315I and P-loop mutations (P<.001). With a median follow-up from mutation analysis to last follow-up of 5 years, T315I and P-loop mutations were not associated with a worse outcome in ECP patients (P = .817). CONCLUSION: Our results suggest that early mutation detection together with accessibility to 2nd and 3rd generation TKIs have reversed the worst outcome associated with BCR-ABL KD mutations whatever the mutation subgroup in CP-CML patients.

CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations.

CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.

Impact of second decline rate of BCR-ABL1 transcript on clinical outcome of chronic phase chronic myeloid leukemia patients on imatinib first-line.

Early molecular response has been associated with clinical outcome in chronic myeloid leukemia (CML) patients treated with tyrosine kinase inhibitors. The BCR-ABL1 transcript rate decline from baseline to 3 months has been demonstrated to be more predictive than a single BCR-ABL1 level at 3 months (M3). However, it cannot be used routinely because ABL1, as an internal gene control, is not reliable for BCR-ABL1 quantification above 10%. This study aimed to compare clinical outcome and molecular response of chronic phase CML patients, depending on the percentage of BCR-ABL1 transcript decrease from month 3 to month 6 using ABL1 as an internal control gene. Two hundred sixteen chronic phase CML patients treated with imatinib 400 mg for whom M3 and month 6 molecular data were available were included in the study. Associations with event-free (EFS), failure-free (FFS), progression-free (PFS), and overall survivals (OS) molecular response 4 log and 4.5 log were assessed. The percentage of BCR-ABL1 decline from month 3 to month 6 was significantly linked to the EFS and the FFS (p < 0.001). A common cut-off of 67% of decline predicted the better risk of event. Patients with a decrease below 67% have worse EFS and FFS as compared to those having a higher decrease (p < 0.001). The impact was confirmed by multivariate analysis. Since the slope between diagnosis and 3 months cannot be reliable using ABL1 as an internal gene control, the second decline rate of BCR-ABL1 transcript between month 3 and month 6 could efficiently identify patients at higher risk of event.

Dasatinib-Loaded Erythrocytes Trigger Apoptosis in Untreated Chronic Myelogenous Leukemic Cells: A Cellular Reservoir Participating in Dasatinib Efficiency.

Dasatinib is an ABL1 tyrosine kinase inhibitor (TKI) with a short in vivo plasmatic half-life but with good efficiency, which is not fully understood. We investigated the possibility that circulating erythrocytes store and then provide dasatinib to target cells. In vitro coincubation of dasatinib-treated cells with naïve leukemic cells followed by analysis of kinase inhibition, apoptosis induction, fluorescent molecule exchanges, and dasatinib dosage were performed. Cells incubated with clinically relevant concentrations of dasatinib for a short time retained, after a washout procedure, an intracellular pool of dasatinib which was transferable to naïve BCR-ABL1 expressing cells and induced their apoptosis. This was verified in total blood where the huge cellular volume of erythrocytes constituted a large reservoir of dasatinib able to induce apoptosis in naïve BCR-ABL1 cell lines and primitive chronic myeloid leukemia (CML) CD34+ cells. This dasatinib transfer necessitated a contact between donor and acceptor cells. A component exchange occurred during this contact, carrying dasatinib and other TKIs such as nilotinib or the fluorescent sunitinib. An active pool of dasatinib could be buried inside the circulating erythrocytes, out of reach of detoxifying mechanisms, but still available for target cells and thus extending the acute effect of the plasmatic pool of the drug.

A new mechanism of resistance to ABL1 tyrosine kinase inhibitors in a BCR-ABL1-positive cell line.

Tyrosine kinase inhibitors (TKI) constitute the frontline treatment for chronic myeloid leukemia patients. Dasatinib, a second-generation TKI, was developed to overcome TKI resistances. However, dasatinib resistances are also described but remain less characterized. To mimic in vivo acquired dasatinib resistance, the BCR-ABL1-positive cell line K562 was transiently treated with a pharmacological concentration of dasatinib, for a short time in the presence of stem cell factor. A dasatinib resistant counterpart (K562 RES) was developed. Investigation of resistance mechanisms using kinase substrate arrays revealed that FYN was overactivated in K562 RES. The FYN inhibitor KX2-391 cooperated with dasatinib to block K562 RES proliferation. Cell tracking experiments showed that activated FYN support cell proliferation independently of BCR-ABL1 in K562 RES cells. Moreover, the MEK-ERK pathway was found hyper-phosphorylated in K562 RES cells even in the presence of dasatinib. Actually, ERK1/2 activity supported viability in K562 RES only in the absence of BCR-ABL1 activity. Finally, BCR-ABL1 and MEK inhibitor combination was sufficient to induce cell death even in non-proliferating resistant cells. Considering the conditions used to generate this dasatinib resistant cell line, such a resistance mechanism could be found in dasatinib treated patients. Consequently, it is valuable to know that inhibition of the MEK-ERK1/2 axis can overcome this resistance.

Loss of mutL homolog-1 (MLH1) expression promotes acquisition of oncogenic and inhibitor-resistant point mutations in tyrosine kinases.

Genomic instability drives cancer progression by promoting genetic abnormalities that allow for the multi-step clonal selection of cells with growth advantages. We previously reported that the IL-9-dependent TS1 cell line sequentially acquired activating substitutions in JAK1 and JAK3 upon successive selections for growth factor independent and JAK inhibitor-resistant cells, suggestive of a defect in mutation avoidance mechanisms. In the first part of this paper, we discovered that the gene encoding mutL homolog-1 (MLH1), a key component of the DNA mismatch repair system, is silenced by promoter methylation in TS1 cells. By means of stable ectopic expression and RNA interference methods, we showed that the high frequencies of growth factor-independent and inhibitor-resistant cells with activating JAK mutations can be attributed to the absence of MLH1 expression. In the second part of this paper, we confirm the clinical relevance of our findings by showing that chronic myeloid leukemia relapses upon ABL-targeted therapy correlated with a lower expression of MLH1 messenger RNA. Interestingly, the mutational profile observed in our TS1 model, characterized by a strong predominance of T:A>C:G transitions, was identical to the one described in the literature for primitive cells derived from chronic myeloid leukemia patients. Taken together, our observations demonstrate for the first time a causal relationship between MLH1-deficiency and incidence of oncogenic point mutations in tyrosine kinases driving cell transformation and acquired resistance to kinase-targeted cancer therapies.

A single nucleotide polymorphism in cBIM is associated with a slower achievement of major molecular response in chronic myeloid leukaemia treated with imatinib.

PURPOSE: BIM is essential for the response to tyrosine-kinase inhibitors (TKI) in chronic myeloid leukaemia (CML) patients. Recently, a deletion polymorphism in intron 2 of the BIM gene was demonstrated to confer an intrinsic TKI resistance in Asian patients. The present study aimed at identifying mutations in the BIM sequence that could lead to imatinib resistance independently of BCR-ABL mutations. EXPERIMENTAL DESIGN: BIM coding sequence analysis was performed in 72 imatinib-treated CML patients from a French population of our centre and in 29 healthy controls (reference population) as a case-control study. Real-time quantitative PCR (RT qPCR) was performed to assess Bim expression in our reference population. RESULTS: No mutation with amino-acid change was found in the BIM coding sequence. However, we observed a silent single nucleotide polymorphism (SNP) c465C>T (rs724710). A strong statistical link was found between the presence of the T allele and the high Sokal risk group (p = 0.0065). T allele frequency was higher in non responsive patients than in the reference population (p = 0.0049). Similarly, this T allele was associated with the mutation frequency on the tyrosine kinase domain of BCR-ABL (p<0.001) and the presence of the T allele significantly lengthened the time to achieve a major molecular response (MMR). Finally, the presence of the T allele was related to a decreased basal expression of the Bim mRNA in the circulating mononuclear cells of healthy controls. CONCLUSION: These results suggest that the analysis of the c465C>T SNP of BIM could be useful for predicting the outcome of imatinib-treated CML patients.

ABT-737 increases tyrosine kinase inhibitor-induced apoptosis in chronic myeloid leukemia cells through XIAP downregulation and sensitizes CD34(+) CD38(-) population to imatinib.

Chronic myeloid leukemia (CML) tumorigenicity is driven by the oncogenic BCR-ABL tyrosine kinase. Specific tyrosine kinase inhibitors (TKI) have been designed and are now used for the treatment of CML. These TKI induce apoptosis in leukemic cells in a BIM-dependent mechanism. We hypothesized that an increase in BIM activity could sensitize CML cells to TKI. We blocked the anti-apoptotic proteins of the Bcl-2 family by using ABT-737, a Bcl-2 and Bcl-XL inhibitor. ABT-737 modified Bcl-2 protein interactions toward a pro-apoptotic phenotype. Its combination with TKI resulted in a strong synergism in CML cell lines. The association also induced a large decrease in X-linked inhibitor of apoptosis (XIAP), followed by caspase-3 activation. This XIAP decrease was due to post-translational events. The mitochondrial serine protease HtrA2/Omi was identified as being responsible for this off-target effect. Then, ABT-737 and TKI cooperate at several levels to induce apoptosis of CML cells lines, and the benefit of this association was also observed in CML hematopoietic progenitors. Interestingly, a lethal effect was also observed in the more immature CD34(+)CD38(-) TKI-insensitive population. Combination therapy might by an interesting strategy for the treatment of CML patients.

α-defensin 1-3 and α-defensin 4 as predictive markers of imatinib resistance and relapse in CML patients.

OBJECTIVE: Imatinib mesylate is a tyrosine kinase inhibitor used as first line treatment in chronic myeloid leukaemia. Despite a remarkable effectiveness, treatment failure cases have been reported in 20 percent of CML patients. The identification of biomarkers which can predict the response to imatinib is our point of interest. METHODS: Gene expression profiling microarray was carried out on secondary imatinib resistant patients. Longitudinal studies were performed on imatinib treated responder/resistant patients. Then, Q-RT/PCR studies were realized on patients prior imatinib initiation. RESULTS: For imatinib responder patients, we observed a strong and lasting decrease of α-defensin 1-3 and α-defensin 4 expression. For relapse patients, we observed a dramatic increase of α-defensin 1-3 and α-defensin 4 expression before BCR-ABL transcript increase. Moreover, before imatinib initiation, α-defensin 1-3 and α-defensin 4 expression was significantly lower in the resistant group than in the responder group. CONCLUSION: The variation of expression of α-defensin 1-3 and α-defensin 4 in peripheral blood is associated with imatinib resistance and may reflect an adequate immune control of the disease. Monitoring of α-defensin 1-3 and α-defensin 4 could be helpful to predict the patients who are not going to respond to the treatment.

Evidence that resistance to nilotinib may be due to BCR-ABL, Pgp, or Src kinase overexpression.

Targeting the tyrosine kinase activity of Bcr-Abl is an attractive therapeutic strategy in chronic myeloid leukemia (CML) and in Bcr-Abl-positive acute lymphoblastic leukemia. Whereas imatinib, a selective inhibitor of Bcr-Abl tyrosine kinase, is now used in frontline therapy for CML, second-generation inhibitors of Bcr-Abl tyrosine kinase such as nilotinib or dasatinib have been developed for the treatment of imatinib-resistant or imatinib-intolerant disease. In the current study, we generated nilotinib-resistant cell lines and investigated their mechanism of resistance. Overexpression of BCR-ABL and multidrug resistance gene (MDR-1) were found among the investigated mechanisms. We showed that nilotinib is a substrate of the multidrug resistance gene product, P-glycoprotein, using verapamil or PSC833 to block binding. Up-regulated expression of p53/56 Lyn kinase, both at the mRNA and protein level, was found in one of the resistant cell lines and Lyn silencing by small interfering RNA restored sensitivity to nilotinib. Moreover, failure of nilotinib treatment was accompanied by an increase of Lyn mRNA expression in patients with resistant CML. Two Src kinase inhibitors (PP1 and PP2) partially removed resistance but did not significantly inhibit Bcr-Abl tyrosine kinase activity. In contrast, dasatinib, a dual Bcr-Abl and Src kinase inhibitor, inhibited the phosphorylation of both BCR-ABL and Lyn, and induced apoptosis of the Bcr-Abl cell line overexpressing p53/56 Lyn. Such mechanisms of resistance are close to those observed in imatinib-resistant cell lines and emphasize the critical role of Lyn in nilotinib resistance.

Multidrug resistance gene (MDR1) polymorphisms are associated with major molecular responses to standard-dose imatinib in chronic myeloid leukemia.

Despite the excellent efficacy of imatinib in chronic myeloid leukemia (CML), the response in patients is heterogeneous, which may in part be caused by pharmacogenetic variability. Imatinib has been reported to be a substrate of the P-glycoprotein pump. In the current study, we focused on the ABCB1 (MDR1) genotype. We analyzed the 3 most relevant single nucleotide polymorphisms of MDR1 in 90 CML patients treated with imatinib. Among the patients homozygous for allele 1236T, 85% achieved a major molecular response versus 47.7% for the other genotypes (P = .003). For the 2677G>T/A polymorphism, the presence of G allele was associated with worse response (77.8%, TT/TA; vs 47.1%, GG/GA/GT; P = .018). Patients with 1236TT genotype had higher imatinib concentrations. One of the haplotypes (1236C-2677G-3435C) was statistically linked to less frequent major molecular response (70% vs 44.6%; P = .021). Hence, we demonstrated the usefulness of these single nucleotide polymorphisms in the identification of CML who may or may not respond optimally to imatinib.