BCR-ABL Kinase Domain Research Articles

Targeting Tyrosine Kinase Receptors

Protein tyrosine kinases (PTKs) regulate cell growth and other key functions. Constitutive PTK activation by somatic mutations, overexpression, or abnormal upstream signaling is characteristic of many cancers, including hematologic malignancies, providing a rationale for therapeutically targeting PTKs with small molecules. Imatinib, an ATP-competitive inhibitor of BCR-ABL1, the PTK causal to chronic myeloid leukemia (CML), established a paradigm for tyrosine kinase inhibitors (TKIs) as cancer therapeutics. Although a relatively weak inhibitor, imatinib is effective in most patients with chronic phase CML (CML-CP), while responses are transient in blastic phase (CML-BP). Point mutations in the BCR-ABL1 kinase domain have emerged as a major mechanism of drug resistance. The more potent second-generation TKIs – dasatinib, nilotinib, and bosutinib – induce deeper and faster responses and are active against many imatinib-resistant mutants, with the exception of T315I in the gatekeeper position of the catalytic site. This problem was addressed with ponatinib, a third-generation TKI covering all single BCR-ABL1 mutants, including T315I. Ponatinib has excellent clinical activity in CML-CP patients who failed other TKIs, while responses in CML-BP are short-lived. Some patients fail ponatinib due to BCR-ABL1 compound mutations, suggesting even third-generation TKIs cannot completely prevent mutational escape by the disease-initiating kinase. Another unsolved problem is that TKIs fail to efficiently target CML stem cells, leading to recurrence of active leukemia upon discontinuation. Despite these shortcomings, TKIs have completely changed the face of CML. Unfortunately, repeating this success in other hematologic malignancies has been challenging, likely reflecting differences in disease biology as much as suboptimal design of early compounds. CML-CP represents one extreme of the spectrum, where a single genetic lesion is sufficient to produce the phenotype and the hierarchy of hematopoietic differentiation is maintained. The situation is different in acute myeloid leukemia (AML) with activating FLT3 mutations. Not only these AML cases have mutations in other genes, they typically acquire FLT3 mutations late during disease evolution, implying that the disease-initiating clone will be impervious to FLT3 inhibition. Progress has been made through successive development of more potent TKIs with improved pharmacology, leading to quizartinib. It is clear, however, that FLT3 inhibitors cannot be used as single agents if there is a curative intent and the same may be true for JAK2 inhibitors in myelofibrosis. The first approved JAK2 inhibitor, ruxolitinib, dramatically improves symptoms, but has yet to demonstrate a significant impact on the malignant clone and is certainly not curative. It remains to be seen whether this reflects the fact that JAK2 activation is not the disease–initiating event, lack of inhibitor specificity towards the mutant JAK2 kinase, or other undesirable off-target effects that may be overcome with improved drugs. A completely new chapter was opened with ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK), for the treatment of chronic lymphocytic leukemia (CLL). BTK is essential for signal transduction from the B-cell receptor (BCR). No activating mutations in BTK have been identified in lymphoma or CLL, but constitutive BCR signaling is critical to CLL cell survival in the microenvironment. Early studies show excellent clinical activity in patients with advanced CLL, although many responses are incomplete; much like the imatinib responses in late CML-CP. Ibrutinib may have a similarly profound effect upon CLL as imatinib on CML, but perhaps also similar limitations, such as the inability to eradicate residual leukemia; this of course needs to be tested in frontline studies. TKIs have had a significant albeit uneven impact upon treatment paradigms in hematologic malignancies. Future progress will involve optimizing compounds in terms of potency, selectivity, and pharmacokinetics. Allosteric inhibitors may add to the armamentarium. From the target perspective, it is likely that most activated kinase alleles have been discovered and the focus should shift to identification of disease-critical unmutated kinases. Lastly, identifying synthetically lethal inhibitor combinations will be critical to fully exploit the potential of TKI therapy. Disclosures:Deininger:BMS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; ARIAD: Consultancy, Membership on an entity's Board of Directors or advisory committees; NOVARTIS: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; CELGENE: Research Funding; GENZYME: Research Funding; INCYTE: Consultancy, Membership on an entity's Board of Directors or advisory committees; GILEAD: Research Funding.

High-Throughput Drug Sensitivity and Resistance Testing (DSRT) Platform Reveals Novel Candidate Drugs For Advanced Phase BCR-ABL1-Positive Leukemia

BackgroundAdvanced BCR-ABL1-positive leukemias (chronic myeloid leukemia in blast crisis and Ph+ALL) remain a therapy challenge despite advances in tyrosine kinase inhibitor (TKI) therapy. Emergence of primary and secondary resistance due to gatekeeper and compound mutations within the BCR-ABL1 kinase domain is common even with the novel 2nd and 3rd generation TKIs (dasatinib, nilotinib, ponatinib). We set out to identify novel candidate drugs for advanced BCR-ABL1-positive leukemias by using an unbiased high-throughput drug testing platform and utilizing both primary patient cells and cell lines. MethodsAs a study material we used 3 CML cell lines representing different types of CML blast phases. In addition to commonly used K562 cells, EM-2 and MOLM-1 cell lines were tested. AML cell lines (AML-193, AP-1060, HL60ATCC, HL60TB, Kasumi-1, KG-1, ME-1, MOLM-13, MONO-MAC-6, MUTZ-2, MV4-11, NOMO-1, SH-2, SHI-1, SIG-M5, SKM-1, THP-1) were used as cell line controls.To verify the results obtained from cell lines, primary bone marrow (BM) cells were derived from 2 TKI-resistant CML BC patients. Patient 1 had developed resistance to imatinib and dasatinib due to a T315I mutation, whereas patient 2 was resistant to nilotinib, dasatinib and ponatinib due to a V299L and a compound mutation. BM cells from 4 healthy individuals were used as controls.The functional profiling of drug responses was performed with a high-throughput drug sensitivity and resistance testing (DSRT) platform comprising of 306 anti-cancer agents (FDA/EMA approved, investigational and experimental compounds). Cells were dispensed to pre-drugged 386-well plates of 5 different concentrations and incubated in a humidified incubator with 5% CO2 at 37 °C for 72 hours. Cell viability was measured by using a luminescent cell viability assay (CellTiter-Glo). From plate reads a Drug Sensitivity Score (DSS) was calculated for each drug as a measure of cytotoxicity.In addition to DSRT, Human Phospho-Kinase Array Kit (R&D systems) was used to analyze the phosphokinase profile in patient samples. ResultsBased on initial comparisons between CML and AML cells lines, nonspecific cytotoxic drugs, which showed high activity in all cell lines, were omitted from further analysis. The DSS scores from different CML cells lines correlated relatively closely (EM-2 vs. K-562, r=0.89; EM-2 vs. MOLM-1, r=0.82; K-562 vs. MOLM-1, r=0.78; p<0.0001 for all correlations).We next ranked the DSRT data according to the DSS values with most sensitive drugs showing the highest DSS scores. The primary cells from CML BC were further normalized against the median values from healthy controls, resulting in leukemia-specific sensitivity scores (sDSS). Ranked results from the DSRT analysis are shown in the Table. [Display omitted] As expected, the cell lines were sensitive to TKIs, with the exception of the MOLM-1, which showed only modest sensitivity. The clinically TKI-resistant patient samples were also TKI-resistant ex vivo, further validating the DSRT assay data.Drugs which showed efficacy in both the cell lines and the TKI-resistant patients included HSP90 inhibitors (NVP-AUY922, BIIB021), a NAMPT inhibitor daporinad and the protein translation inhibitor omacetaxine (homoharringtonine). Phosphokinase antibody array results from the patient samples showed increased expression of the HSP27 protein as a putative biomarker for HSP90 inhibitor response. ConclusionsDSRT is a powerful assay for identifying novel candidate molecules for refractory BCR-ABL1-positive leukemias. Our results indicate that HSP90 and NAMPT inhibitors in particular warrant further clinical evaluation both by analyzing a larger set of primary patient samples and by performing proof-of-concept clinical studies. The results also pave way for designing rational combination therapy strategies. Disclosures:Mustjoki:Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau. Porkka:BMS: Consultancy, Research Funding, Speakers Bureau; Novartis: Consultancy, Research Funding, Speakers Bureau.

Increasing Expression Of The Efflux Transporter ABCB1 May Predispose CML Cells To Overt TKI Resistance

Tyrosine kinase inhibitors (TKIs) result in excellent responses in most Chronic Myeloid Leukemia (CML) patients. However, up to 35% of patients treated with imatinib (IM) exhibit resistance and more recently nilotinib (NIL) and dasatinib (DAS) resistance have also been observed. Mutations in the BCR-ABL kinase domain (KD) are the main cause of secondary TKI resistance. Other mechanisms include overexpression of BCR-ABL, LYN and ABCB1. Predicting patients with susceptibility to mutation development and disease progression is crucial, thus we investigated the kinetics of TKI resistance emergence in vitro and in vivo.ABCB1 is implicated in TKI efflux hence we postulated that overexpression of ABCB1 leads to reduced intracellular TKI concentrations, resulting in inferior inhibition of Bcr-Abl predisposing cells to resistance development. Accordingly, 3 CML blast crisis (BC) cell lines (K562, K562-Dox, KU812) were cultured in increasing concentrations of IM to 2 μM, NIL to 2 μM and DAS to 200 nM until we observed overt resistance defined as a significant increase in survival in cytotoxicity assays and p-Crkl dependent IC50. Mechanisms of resistance were investigated in cell line intermediates: BCR-ABL, ABCB1 and LYN mRNA expression levels were determined by RT-PCR and KD mutation sequencing was performed.In our TKI resistant cell lines (Table 1), an increase in ABCB1 mRNA was the initial change observed prior to the development of additional resistance mechanisms (KD mutations, ABCB1 BCR-ABL and LYN overexpression). Interestingly, in 4/6 cells lines ABCB1 mRNA reduced to basal levels or below following establishment of these additional resistance mechanisms.ABCB1 levels were assessed in 37 de novo CML patients treated with IM who achieved major molecular response (MMR) compared with patients who progressed to BC, lost MMR or developed KD mutations. ABCB1 levels were determined in blood at diagnosis and following therapy (selected patients summarized in Table 2). A sustained >2 fold rise in ABCB1 was observed prior to disease progression in 3/3 patients and in 13/16 patients who did not achieve MMR. Importantly, the same was not observed in patients who achieved MMR (1/6 patients). The fold change of ABCB1 mRNA at day 22 vs diagnosis in patients achieving MMR was significantly different to that in patients not achieving MMR (p=0.004). ABCB1 increased by >2 fold post therapy and decreased following mutation development in 3/12 patients, confirming observations made in vitro, while 6/12 patients demonstrated sustained increase in ABCB1 post mutation similar to results observed in progression patients. ABCB1 mRNA did not change during therapy in 3/12 patients with mutations. While we recognize the majority of cells present in patients who achieve MMR are normal rather than leukemic, it is important to note that in patients who do not achieve MMR, ABCB1 expression increases in the remaining leukemic cells.We conclude ABCB1 overexpression acts as an initial mediator of resistance, providing a favorable environment for development of further resistance. Sustained increased levels of ABCB1 may contribute to disease progression and lack of response to IM. Additionally, ABCB1 may serve as a prognostic indicator (eg: level at day 22) and potentially assist in development of treatment strategies using TKIs in combination with other medications to enhance intracellular TKI concentration. Disclosures:Hughes:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL: Research Funding. White:Novartis: Research Funding; BMS: Research Funding, Speakers Bureau; Ariad: Research Funding; CSL: Research Funding.

Minor Subclones Harboring Small Insertions and Deletions Probably Due To Aberrant Splicing Can Frequently Be Detected By Deep Sequencing of The BCR-ABL Kinase Domain

Background and Aimalthough BCR-ABL kinase domain (KD) mutations can frequently be identified in patients who develop resistance to tyrosine kinase inhibitors (TKIs) in Philadelphia-positive (Ph+) leukemias, other mechanisms may play a role. Small insertions and deletions within the BCR-ABL KD have occasionally been reported in chronic myeloid leukemia (CML) patients who failed TKI therapy and have been hypothesized to have a causative role in drug resistance. Some were in-frame insertions and deletions, others were predicted to result in truncated BCR-ABL proteins. However, the detection of these sequence variations is hampered by the fact that they are almost always confined to subclones co-exisiting with full length BCR-ABL. This has most likely resulted in an underestimation of their frequency and complexity, since i) they can be confused with background noise/reduced quality readings in direct sequencing chromatograms and ii) cloning would be needed to better resolve overlapping sequences in these samples. The recent development of Deep Sequencing (DS) technologies has opened the way to a more accurate characterization of molecular aberrations. DS enables greater sensitivity, quantitation of sequence variant abundance and clonal analysis of a given DNA region. We thus took advantage of DS to better characterize the spectrum of insertions and deletions in CML and Ph+ acute lymphoblastic leukemia (ALL) patients with response or resistance to TKI therapy. Methodsa total of 110 samples of 41 CML and 16 Ph+ ALL patients who received one or multiple lines of TKI therapy were analyzed. DS was performed on a Roche GS Junior instrument, according to an amplicon sequencing design and protocol set up and validated in the framework of the IRON-II international study. Runs were designed to achieve high sequencing depth; this allowed to reliably identify and characterize deletions or insertions with a lower detection limit of 0.1%. In order to reconstruct the dynamics of evolution of these sequence variations in relation to the TKI administered and to the level of response achieved, we evaluated their presence in serial follow-up samples collected during TKI therapy in 15 patients. ResultsDS revealed a 35-base pair (bp) insertion in 35/41 (85%) CML and 14/16 (87%) Ph+ ALL patients. This sequence variation, already reported in the literature as ‘35INS', consists in the retention of 35 nucleotides (nt) from intron 8 at the exon 8 to exon 9 border. It leads to a truncated BCR-ABL variant having 10 a.a. encoded by intron 8 sequences but lacking 653 C-terminal a.a., including 22 a.a. of the KD, along with the entire C-terminal region. 35INS was detected with variable abundance (range 0.1%-96% of all BCR-ABL transcripts), but in only three samples abundance was higher than 15-20% - thus detectable also by conventional sequencing. Re-sequencing a set of samples in the same and independent runs confirmed the presence of the 35INS and demonstrated that this variant was not a PCR or sequencing artifact. Longitudinal analysis showed that the expression of 35INS fluctuated over time with no apparent correlation with response levels. In addition, DS detected one in-frame deletion in 20/41 (48%) CML patients and 7/16 (44%) Ph+ ALL patients, with an abundance ranging from 0.2% to 19%. This previously unreported variant consisted of a 72bp deletion (nt.1233-1304) at the junction of exon 6 to exon 7, that causes the loss of 24 residues (a.a. 359-383) of the KD. ConclusionsOur results further underline that DS technologies allow more accurate sequence characterization in comparison to conventional methods. Minor clones harboring insertions or deletions (always involving intron/exon junctions - which implicates alternative or aberrant splicing mechanisms) were found to be very frequent both in CML and in Ph+ ALL patients but, apparently, did not correlate with response or resistance to TKI therapy. In line with our findings, a very recent functional study has demonstrated that the truncated BCR-ABL protein resulting from the 35INS is kinase-inactive and should not play any role in TKI-resistance - in contrast to what had initially been hypothesized. However, further analysis of a larger number of samples would be needed to better understand the biological and clinical meaning of these minor clones surviving TKI therapy.Supported by PRIN 2009 (prot.2009JSMKY), Fondazione CARISBO, AIL, AIRC, FP7 ‘NGS-PTL', IGA NT 11555/13899. Disclosures:Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Gugliotta:Novartis: Consultancy, Honoraria; Bristol-Myers Squibb: Consultancy, Honoraria. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Cavo:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Onyx: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Martinelli:NOVARTIS : Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

The Impact Of Novel Splicing Abnormalities Of BCR-ABL On The Pathogenesis Of CML

BackgroundChronic myeloid leukemia (CML) is effectively treated with tyrosine kinase inhibitors (TKIs), but reactivation of BCR-ABL frequently occurs through acquisition of kinase domain point mutations. The mechanism of resistance in patients without BCR-ABL kinase domain point mutation is still elusive. Previous studies have revealed the abnormal splicing of BCR-ABL kinase domain, including Exon8/9 junction 35bp insertion and exon-skipping of Exon 7 (T O'Hare et al, Blood 2011. Gaillard JB, et al. Mol Cancer Ther 2010). The insertion of 35 intronic nucleotides at the exon 8/9 splice junction introduces a stop codon after 10 intron-encoded residues and inactive tyrosine kinase activity. The effect of these splicing abnormalities on susceptibility of cells against TKIs is still controversial. Furthermore, the conventional direct sequence techniques could not evaluate splicing abnormalities in major molecular response (MMR)- complete molecular response (CMR) CML patients, who achieved clinically leukemia-free state with a small number residual CML stem cells. AimsThe aim of this study is to evaluate the frequency and the patterns of splicing abnormalities of BCR-ABL in CML patients, especially who achieved MMR-CMR by TKI treatment. MethodsWe analyzed peripheral blood samples from healthy individuals and CML chronic phase patients. We extracted total RNA from these samples and synthesized cDNA, and then performed PCR-amplification of BCR-ABL kinase domain in CML patients and ABL kinase domain in healthy individuals, respectively. PCR products were subjected to the amplicon sequence: We deeply sequenced BCR-ABL fusion gene transcripts, and evaluated splicing forms of BCR-ABL by using HiSeq 2000 (illumina). ResultsWe successfully established a novel analysis method, which can detect the pattern of splicing abnormalities even in MMR-CMR patients. Using the amplicon sequence technique, we detected abnormal splicing patterns of BCR-ABL in 5 out of 15 CML patients. We also found that the splicing abnormalities were not restricted to 35bp insertion at the exon8/9 junction, thus intronic retention of intron 8 and intron 9 could be frequently detected with or without the 35bp insertion in CML patients (Table 1). Of note, these abnormal splicing patterns always co-existed with wild type BCR-ABL transcripts in all 5 cases analyzed. In addition to the novel splicing abnormalities in CML, we unexpectedly found in healthy individuals that splicing abnormalities such as 35bp insertion at the exon8/9 junction and intronic retention could be detected in ABL1 transcripts (Table 1). This result suggests that this sort of splicing abnormalities could occur at a certain frequency in steady state human hematpoiesis, and is not specific to BCR-ABL. Summary / ConclusionWe have newly established an analysis system to efficiently detect splicing abnormalities of BCR-ABL even in MMR-CMR CML patients. Using this highly efficient amplicon sequence technique, we identified novel splicing abnormalities both in healthy individuals and CML patients, and found that the wild type BCR-ABL transcripts always co-exist with abnormally spliced BCR-ABL transcripts. These results collectively suggest that splicing abnormalities within the ABL1 kinase domain are not specific to CML patients treated with TKIs, and that the detection of such kinase domain splicing abnormalities do not reflect insusceptibility of the remaining cells during TKI treatment. Disclosures:No relevant conflicts of interest to declare.

Activity Of Omacetaxine Mepesuccinate Against Ponatinib Resistant Philadelphia Chromosome Positive Leukemia Cells

Chronic myeloid leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric tyrosine kinase BCR-ABL. ABL tyrosine kinase inhibitor (TKI) therapy (e.g. imatinib, nilotinib and dasatinib) has improved the survival of Ph-positive leukemia patients. However, despite the impressive efficacy of these agents, disease relapse has been observed in clinically. Mutations in the BCR-ABL kinase domain can cause of ABL TKI resistance. In particular, one of the BCR-ABL kinase domain mutations (e.g. T315I) is associated with a high level of resistance to all available ABL TKIs. Ponatinib (formally, AP24534) is a multi-target TKI. Recently, in the PACE (Ponatinib Ph+ acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML) Evaluation) trial, ponatinib showed significant efficacy against Ph-positive leukemia in patients with multi-resistant T315I mutations. However, in some patients, especially those with Ph-positive ALL, ponatinib resistant clones were identified. Omacetaxine mepesuccinate, formally known as homoharringtonine is a natural alkaloid obtained from various Cephalotaxus species. Omacetaxine is a first-in-class cephalotaxine in clinical development as anti-leukemic therapy. Omacetaxine acts by binding to the A-site cleft of ribosomes and thereby transiently inhibits protein synthesis. Omacetaxine was approved for the treatment adult patients with chronic or the accelerated phase of CML resistant to other therapies. We investigated the efficacy of omacetaxine against ponatinib resistant Ph-positive cells. Ba/F3 ponatinib resistant cells (Ba/F3 ponatinib-R) have three BCR-ABL point mutations (Y253H, E255K and T315I: data not shown). With 72 h omacetaxine treatment, the cell growth of Ba/F3 ponatinib-R and Ph-positive ALL cell line was significantly reduced even at a low concentration and it is also effective to the other hematological malignancies such as acute myeloid leukemia. In contrast, Ba/F3 ponatinib-R was resistant to ponatinib. With 48 h treatment, omacetaxine dependent apoptosis was increased. Although anti-apoptotic proteins were not increased in this cell line compared to parental cells, as compound mutations such as E255V/T315I confer high-level resistance to ponatinib, these three point mutant was associated with vitro resistance to ponatinib. We also examined intracellular signaling. The phosphorylations of BCR-ABL and a down-stream molecule, Crk-L, were decreased. Protein expressions of BCR-ABL and Crk-L were also decreased. However, caspase-3 and cleaved Poly (ADP-ribose) polymerase (PARP) levels were significantly increased in low concentration. In a previous study, omacetaxine was shown to induce apoptosis in leukemic cells due to a selective decrease in short-lived proteins. We found that omacetaxine reduced the expression of BCR-ABL and heat shock protein 90(HSP90) which is stabilize BCR-ABL protein. We also found that omacetaxine reduced the expression of anti-apoptotic protein, Bcl-2. The protein expression of c-myc was also reduced. We next examined a ponatinib resistant primary Ph+ ALL and chronic phase CML samples. The ponatinib resistant primary cells have several BCR-ABL point mutations (e.g. Q252H, E255K/V, and T315I). We found the growth of primary cells was resistant to ponatinib but to be reduced after omacetaxine treatment and similar signaling events were occurred in OM-treated primary ALL cells. Omacetaxine is an inhibitor of protein synthesis. Because omacetaxine inhibits the BCR-ABL, Bcl-2 and HSP90 pathways in BCR-ABL positive leukemia cells through reduced the levels of these proteins, Omacetaxine has anti-tumor activity and promotes apoptosis. Our findings suggest that omacetaxine may benefit patients with leukemic BCR-ABL mutant cells, possibly allowing ponatinib resistant clones to be overcome. Disclosures:Ohyashiki:Novartis: Honoraria, Research Funding.

Next-Generation Sequencing Of The BCR-ABL1 Kinase Domain May Be Beneficial In Decision Making Among Chronic Myeloid Leukemia Patients With Tyrosine Kinase Inhibitor Resistance

Background and AimBCR-ABL1 mutation testing is recommended for chronic myeloid leukemia (CML) patients who have suboptimal response and/or treatment failure with tyrosine kinase inhibitor (TKI) therapy. BCR-ABL1 mutations in the kinase domain (KD) of ABL1 account for at least 40-50% of all TKI resistant cases. Thus, detection of low-level mutations after development of resistance may offer critical information to guide subsequent therapy selection. The current gold standard for BCR-ABL1 mutation detection is Sanger sequencing (SS), which has an analytical sensitivity of approximately 10-20%. In this study, our aim was to detect low level BCR-ABL1 variants in follow up samples of CML patients with TKI resistance using next-generation sequencing (NGS) approach. MethodsEight patients with CML who were resistant to imatinib had been routinely sequenced with SS for BCR-ABL1 KD mutations between December 2009 and December 2012. We then retrospectively analyzed these samples with NGS. RT and long range PCR was performed to amplify BCR-ABL1 fusion transcripts and the PCR products sequenced bidirectional after library preparation. We performed a fusion transcript based BCR-ABL1 mutation assay using Roche 454 amplicon deep-sequencing technology that is suited for detecting low level variants in pooled amplicon samples. Sequencing data was analyzed with GS Amplicon Variant Analyzer (AVA) software, and the variant frequency cut-off was adjusted to 1%. ResultsClinical features, sequencing results, and the outcomes of the patients were summarized in Table 1. Four patients were male, and the median age was 37 years (range, 20-60 years). The patients were all in chronic phase at the time of the diagnosis. After imatinib resistance, 4 patients had received dasatinib (DAS), and 2 were given nilotinib (NIL) as second line TKI treatment. The remaining two patients had both received DAS and NIL (Table 1). In a set of 20 clinical samples, at different time points, NGS not only identified all the mutations detected by SS, but additionally identified low level variants present between 1 – 28.12 %. T315I and E255K/V were the most common mutations, which were detected in four patients, both by SS and NGS at the same time points (Table 1). Two patients (patient #1 and #4) had T315I, and they both progressed to blastic phase and died. E255K was detected in patients #2 and #3, and patient #2 had achieved and maintained complete cytogenetic and major molecular responses with 100 mg daily DAS, whereas patient #3 had received both NIL and DAS, but she was deceased due to myeloid blastic crisis. Among 4 patients (patients #5, #6, #7, and #8), mutation analysis was performed at eleven different time points, and these patients were wild-type with SS. We also did not detect any clinically significant mutations in these patients by NGS. Most probably mechanisms other than KD mutations were responsible for the TKI resistance among these four patients. ConclusionsPolyclonal mutations in BCR-ABL1 KD are commonly identified in TKI resistant patients. Thus, detection of low-level mutations after development of resistance offers critical information to guide subsequent therapy selection. An inappropriate kinase inhibitor selection could highly increase the risk of treatment failure with clonal expansion of the resistant mutant. In our imatinib resistant cohort, we detected low level variants accompany to known mutations which may constitute background genetic variations. Although we had expected to detect mutations earlier by NGS (i.e. before these mutations can be detected by SS), we did not observe such finding in our patients. The patients' samples may not show a stable mutation spectrum between time points. Hence, it is not always possible to spot a mutation before patients show resistance to therapy. Regular NGS analysis might detect these mutations in earlier phases, which might help clinicians to choose the most suitable individual treatment modality for the patients. AcknowledgmentThe authors would like to thank the Interlaboratory Robustness of Next-generation sequencing (IRON) Phase II study group members, especially to Simona Soverini and Alexander Kohlmann who designed BCR-ABL primers and plates. We also would like to thank the Research Fund of the Istanbul University (Project no. 24244) and Turkish Society of Hematology for supporting the study.▪ Disclosures:Sayitoglu:Roche Diagnostics: Research Support Other.

Inhibition Of PI3K/mTOR Overcomes Nilotinib Resistance In BCR-ABL1 Positive Leukemia Cells

Expression of the Philadelphia chromosome (Ph), i.e., the t(9;22) chromosomal translocation and the formation of the BCR-ABL1 fusion protein, is the hallmark of chronic myeloid leukemia (CML). Tyrosine kinase inhibitors (TKI), such as imatinib and nilotinib, have emerged as leading compounds to treat CML. Translocation t(9;22) does not only occur in CML, 20-30% of acute lymphoblastic leukemia (ALL) are also found to 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 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. There was no mutation in the tyrosine kinase domain of BCR-ABL1 in both cell lines. SUP-B15 cells were not generally irresponsive to TKI, evidenced by dephosphorylation of the BCR-ABL1 downstream target GAB2 (Grb-associated binder-2). The resistance to apoptosis after nilotinib treatment was accompanied by the constitutive and nilotinib irresponsive activation of the phosphoinositide 3-kinase (PI3K) pathway. Treatment of SUP-B15 with the dual PI3K/mammalian target of rapamycin (mTOR) inhibitor BEZ235 alone induced apoptosis in a low percentage of cells, combination of nilotinib and BEZ235 led to a synergistic effect. Caspase 3 and PARP (poly ADP ribose polymerase) cleavage confirmed that apoptosis was induced after combined treatment. 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 down-regulation of anti-apoptotic protein MDM2 (human homolog of the murine double minute 2, hMDM2). The current findings suggest that MDM2 may be a therapeutic target to increase TKI-mediated apoptosis and that the combination of PI3K/mTOR dual inhibitor and TKI inhibitor might turn out to be a novel strategy for TKI-resistant BCR-ABL1 positive leukemia. Disclosures:No relevant conflicts of interest to declare.

Patients With Philadelphia-Positive Leukemia With BCR-ABL Kinase Mutations Prior To Allogeneic Transplantation Predominantly Relapse With The Same Mutation

IntroductionTyrosine kinase inhibitors (TKIs) remain the front-line therapy for chronic myeloid leukemia (CML). TKIs also improve remission rates when incorporated into induction and maintenance regimens for Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL). Unfortunately, resistance to TKIs can occur, and is commonly associated with a point mutation in the ABL kinase domain of BCR-ABL. Allogeneic hematopoietic stem cell transplant (HSCT) is indicated for patients with advanced phase CML or for those who fail TKI therapy, and is also often used in Ph+ ALL patients with suitably matched donors. Sadly, relapse after HSCT is fairly common in both advanced phase CML and Ph+ ALL. Studies have explored the benefit of TKIs given post-HSCT to prevent relapse, but there is limited data as to guide their selection and administration. An important consideration is that the “second generation” TKIs may be associated with more toxicity, particularly cytopenias, in the post-HSCT setting. The question arises whether pre-HSCT mutation status should guide prophylactic TKI selection. Given that resistance can occur with a mix of wild type and mutant clones, which clones “win” in the post-transplant setting? Thus, the purpose of this study is to investigate if specific ABL kinase domain mutations persist or recur in CML and Ph+ ALL patients after HSCT. MethodsIn this retrospective analysis, subjects were at least 18 years of age, had undergone allogeneic HSCT at our center between 2000 and 2010 for CML or Ph+ ALL, and had a positive p210 or p190 BCR-ABL transcript by polymerase chain reaction (PCR) in both the pre- and post-HSCT settings. Patients without available records for chart review and those without available pre- and post-HSCT RNA were excluded. Bone marrow and peripheral blood samples obtained prior to HSCT were analyzed for mutations in BCR-ABL. Total RNA was extracted using a TRIzol reagent (Invitrogen, CA, USA) method. A nested PCR, including an initial RT-PCR step for p210 and/or p190 transcripts, was used to amplify a 928-bp product spanning exons 4-9 of the ABL kinase domain. Sanger sequencing of the PCR product was performed using an Applied Biosystems (ABI, CA, USA) 3730xl Analyzer. In patients with a mutation identified, post-HSCT samples were then sequenced. ResultsA total of 95 CML patients and 20 Ph+ ALL patients who underwent HSCT were included in the study. History was notable for pre-HSCT TKI therapy in 64.2% of CML and 90.0% of Ph+ ALL patients, correlating with a mean duration of pre-HSCT TKI exposure of 12.3 and 10.6 months, respectively. At a mean of 2.1 years of follow-up, 30.5% of CML and 70.0% of Ph+ ALL patients had received a TKI post-HSCT either for prophylaxis or relapsed/refractory disease.Sequencing revealed pre-HSCT ABL kinase mutations in 10 (10.5%) of CML and in 4 (20.0%) of Ph+ ALL patients. All 14 harbored at least one mutation known to be associated with resistance to one or more TKIs. Mutations occurred across the kinase domain, including the TKI-binding site, P-loop and A-loop: L248V, G250E, Q252H, Y253H, T315I, F317L, M351T, F359V, R362G, E450K, E459K and F486S. In 9 of the 14 (64.2%), the mutation conferring TKI resistance was also detectable post-HSCT at an average of day +191 (range +25 to +559). Seven of the 14 patients had refractory or relapsed disease by last follow-up and, of these, 5 (71.4%) received a TKI known to be resistant to the pre-HSCT mutation and failed to respond. DiscussionWe have shown that resistance mutations often continue to be detectable in patients with molecular relapse or persistently positive BCR-ABL transcripts after HSCT, attributable to persistence or relapse of a TKI-resistant leukemic clone. This finding suggests that in these patients at greatest risk for disease relapse, resistance patterns may be greatly influenced by any drug-resistant clone present before transplant. Thus, in choosing a TKI for use in the prophylactic setting, if no other clinical features (such as co-morbidities) force the exclusion of a particular TKI, the selection of the TKI with predicted activity against the mutant clone might “hedge the bet.” Notably, our overall rate of ABL mutations in CML is somewhat lower than expected, though this may be attributable to the number of patients without TKI exposure. To our knowledge, this is the largest analysis of ABL kinase domain mutations in a transplant population. Further analysis of additional samples is also underway. Disclosures:No relevant conflicts of interest to declare.

Combination Chemotherapy With Tyrosine Kinase Inhibitors Can Overcome Bcr-Abl Mutations In Acute Lymphoblastic Leukemia Or Blast Crisis CML

Background Point mutations in the kinase domain of bcr-abl confer resistance to tyrosine kinase inhibitors (TKIs) in patients with blast phase chronic myeloid leukemia (CML-BC) and Philadelphia positive acute lymphoblastic leukemia (Ph+ ALL). In particular the presence of T315I mutation is highly resistant to most current available TKIs.

Kinetics Of Low-Level Mutant Clones Detected By Subcloning and Sequencing In Tyrosine Kinase Inhibitor Resistant CML

BackgroundBCR-ABL1 kinase domain (KD) point mutation causes resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML) patients through impaired binding of TKI to the target site. Recent studies have reported that multiple mutations detected in 2-9% of patients with imatinib (IM)-resistant CML were associated with poor response rate and survival outcomes. However, biological characteristics and dynamics of multiple low-level mutations are still not assessed with a quantitative serial follow-up data in the same populations. AimsThe aim of this study was to investigate biological characteristics and dynamics of low-level mutations in the serial samples from the patients carrying multiple mutations using subcloning and sequencing. MethodsSince 2002, 414 CML patients were screened for mutation analysis due to sign of resistance to TKIs including imatinib (IM), nilotinib (NIL), dasatinib (DAS), bosutinib (BOS), radotinib (RAD) or ponatinib (PON) at Seoul St Mary's Hospital using direct sequencing and allele specific oligonucleotide-polymerase chain reaction (ASO-PCR). Among them, 31 patients carried ≥ 2 BCR-ABL1 kinase domain mutations. We analyzed 137 samples from these 31 patients using subcloning and sequencing (in total, 2737 colonies were sequenced). By cloning and sequencing, two or more missense mutations present in the same clone were defined as compound mutation and co-existence of single missense mutations in the separated clones was defined as polyclonal mutation. Co-existence of single missense mutation and compound mutation harboring two or more missense mutations in the same clone was defined as mixed mutation. Missense mutations detected by direct sequencing are defined as predominant mutations. Missense mutations detected by cloning and sequencing but not by direct sequencing are defined as low-level mutations. ResultsIn a total of 2737 colonies from 137 samples, 1596 (58%) colonies harbored ≥ 2 missense mutations with a median 2 (range, 2 – 7) mutations, and 905 (33%) colonies with a single mutation and 236 (9%) colonies with wild type were observed.In 2737 colonies, 692 different low-level mutations were detected by cloning and sequencing but not by direct sequencing. Among them, M244V, G250E, Y253H, E279K, T315I, F317L, M351T, E355A, F359I, and F359V were detected by direct sequencing in the followed-up samples, and the others remain undetectable by direct sequencing. To address whether these low-level mutations were distinct in the patients with TKI resistance, we applied the cloning and sequencing to samples from 3 healthy controls and 3 patients with optimal response to IM. 38 different mutations were detected healthy controls. 52 different mutations were detected in optimal responders. S349P, N374S, E450G, and S485P were detected in both healthy controls and optimal responders. All mutations detected in optimal responders except 3 missense mutations (V335A, F382V, and A395S) were detected as low-level mutations in the 31 patients' cohort with TKI resistance. Of 38 different mutations detected in healthy control, 31 mutations except 7 missense mutations (Y264H, M343R, A350V, E373G, G298R, E462K, and T495K) were also detected as low-level mutations in the 31 patients' cohort. ConclusionsWe showed basic characteristics and dynamics of low-level mutations by subcloning and sequencing. Some of the low-level mutant clones harboring previously known (clinically common) TKI resistant mutations changes to the predominant in the followed-up samples. Except them, most low-level mutant clones were not detected repeatly and did not increase gradually in the serial samples, implying that they may not have clinical significance. Disclosures:No relevant conflicts of interest to declare.

Rapid Identification Of Compound Mutations In Patients With Ph-Positive Leukemias By Long-Range Next Generation Sequencing

Mutations in the BCR-ABL1 tyrosine kinase domain (TKD) are regarded as the most important mechanism of resistance to tyrosine kinase inhibitors (TKIs) in patients with Ph-positive leukemias. The occurrence of two or more mutations on the same DNA molecule, the so-called compound mutations, can be associated with particularly high resistance to multiple TKIs. Recent reports indicate that the frequency of compound mutations is rather high, thus rendering their reliable detection an important diagnostic challenge 1,2. Analysis of PCR amplicons of the BCR-ABL1 TKD by next generation sequencing (NGS) has become the method of choice for sensitive detection of compound mutations. This approach is, however, hampered by the requirement of 3-4 overlapping amplicons to cover the entire TKD due to the limited read length offered by most current NGS technologies. This prevents the assignment of nucleotide substitutions located on different amplicons to the same TKD/DNA molecule, and therefore requires additional laborious steps to facilitate unequivocal identification of such constellations. To overcome this limitation, we have established a long-range NGS approach on the FLX instrument (Roche) permitting the coverage of the entire TKD length of ∼0.9 kb in a single read. By testing a series of individual and consecutive specimens derived from five patients with chronic myeloid leukemia, we demonstrate that long-range NGS analysis readily permits the identification of mutations and their assignment to the same or to separate subclones at a limit of sensitivity comparable to NGS-based sequencing of shorter amplicons. In addition to the detection of individual and compound mutations, this approach also facilitates an interpretable documentation of insertions and deletions in the TKD. To address the possibility of artifacts inherent in the technique that could lead to incorrect identification of single and compound mutations, the NGS findings were reevaluated by independent technical approaches. Point mutations were confirmed by Sanger sequencing, LD-PCR 3 and pyrosequencing 4. In select cases, PCR amplicons of the BCR-ABL1 TKD derived from individual specimens were subcloned into pGEM®T easy plasmids, and >100 clones were subjected to analysis by Sanger sequencing. The observations made by NGS analysis including various single mutations (e.g. G250E, Y253H, T315A, F317I, Q252H, T315I), compound mutations (e.g. G250E/Y253H, G250E/T315A, G250E/F317I), and combinations of point mutations with small insertions or deletions (e.g. E459K/C475fs, Q252H/R362fs, T315I/R362fs) as well as large deletions involving multiple exons, could be confirmed in individual clones by Sanger sequencing, thereby documenting the reliability of the long-range NGS technology. The technical advancement presented therefore provides an economic approach to the identification of compound mutations and other genetic alterations in the entire BCR-ABL1 TKD, thus extending the diagnostic armamentarium for rapid assessment of impending resistant disease.1. Khorashad JS, Kelley TW, Szankasi P, et al. BCR-ABL1 compound mutations in tyrosine kinase inhibitor-resistant CML: frequency and clonal relationships. Blood. 2013;121(3):489-498.2. Soverini S, De Benedittis C, Machova Polakova K, et al. Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain. Blood. 2013.3. Preuner S, Denk D, Frommlet F, Nesslboeck M, Lion T. Quantitative monitoring of cell clones carrying point mutations in the BCR-ABL tyrosine kinase domain by ligation-dependent polymerase chain reaction (LD-PCR). Leukemia. 2008;22(10):1956-1961.4. Alikian M, Gerrard G, Subramanian PG, et al. BCR-ABL1 kinase domain mutations: methodology and clinical evaluation. Am J Hematol. 2012;87(3):298-304. [Display omitted] Disclosures:Valent:Novartis: Honoraria, Research Funding. Lion:Novartis, Bristol-Myers- Squibb, Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Sensitivity, Reproducibility and Clinical Utility Of Next-Generation Sequencing (NGS) for BCR-ABL1 Kinase Domain Mutation Screening: Results From The CML Work Package Of The Iron-II (Interlaboratory RObustness Of Next-Generation Sequencing) International Study

Background and AimsIn chronic myeloid leukemia (CML) and Philadelphia chromosome-positive (Ph+) acute lymphoblastic leukemia (ALL) patients resistant to tyrosine kinase inhibitors (TKIs), BCR-ABL1 mutation status is an essential component of the therapeutic decision algorithm. Capillary Sanger sequencing (SS) is currently the gold standard for mutation screening of the BCR-ABL1 kinase domain (KD), despite key technical limitations including limited sensitivity and no discrimination between compound and polyclonal mutations. Benchtop next-generation sequencers have recently been introduced as potential diagnostic platforms and there is growing interest in their clinical application. In the framework of the IRON-II (Interlaboratory RObustness of Next-generation sequencing) international consortium, 10 laboratories from 7 countries (Italy, Germany, United Kingdom, Spain, Austria, Turkey, Czech Republic) have engaged in the set-up, standardization and validation of a laboratory-developed screening assay for BCR-ABL1KD mutations based on the Roche 454 amplicon deep-sequencing technology. MethodsFusion primers were designed to generate four partially overlapping amplicons by nested reverse transcription (RT)-polymerase chain reaction (PCR), the first amplification step needed to select for the translocated ABL1 allele. Fusion primers were barcoded with multiplex identifiers (MIDs) consisting of 10-base pair tags allowing multiplexing of twelve clinical samples (forty-eight amplicons) in a single NGS run. The assay was designed in a ready-to-use 96-well plate format containing lyophilized oligonucleotide primers. ResultsDifferent primer designs and primer-MID combinations were evaluated for their performances. Sequencing runs generated an average of 97,432 reads (range, 59,459-151,335). For the primer design selected for further evaluation, the coverage per amplicon ranged between 1,449 and 5,997 sequencing reads. To explore the sensitivity and accuracy of the assay, serial dilutions of BaF3 cell lines harboring four different known mutations (Y253F, E255K, T315I, M351T) into an unmutated BaF3 cell line (50%:50%; 25%:75%; 10%:90%; 5%:95%; 2%:98%; 1%:99%) were sequenced in parallel in two distinct laboratories (Bologna and Jena). In both centers, results showed a high linearity of mutation calling and accuracy of mutation detection and quantitation over the entire range of dilutions, down to 1% mutation abundance. Intra-run reproducibility and inter-run reproducibility were confirmed by a series of experiments in which a set of samples was resequenced in the same and in independent runs, respectively, with and without repetition of the RT and PCR steps. Importantly, we demonstrated that reproducibility could be maintained over a wide dynamic range of amplicon coverage (from 100 to 5,000 independent sequencing reads). A total of 554 clinical samples (2,216 amplicons) were analyzed by the 10 laboratories - including 517 clinical samples analyzed in parallel by NGS and SS and 30 clinical samples analyzed in parallel by NGS, SS and conventional pyrosequencing. Three hundred and ninety-four of 398 (99%) variants detected by SS were also detected by NGS. In addition, comparison between NGS, SS and conventional pyrosequencing results showed very good concordance with respect to the estimation of variant abundance. NGS allowed to detect additional, low level mutations (>1% but<10-15%, i.e. undetectable by SS) in 294/554 (53%) samples. In a subset of twenty randomly selected samples, low level mutations were confirmed by independent methods (restriction fragment length polymorphism or allele-specific oligonucleotide-PCR). Compound mutations as against polyclonality could be resolved in all the clinical samples harboring multiple mutations mapping 450 bp apart or closer. Longitudinal retrospective analysis of CML and Ph+ ALL clinical samples showed that NGS could have identified TKI-resistant mutations earlier than SS, thus allowing more timely therapeutic intervention. ConclusionsOur results indicate the technical feasibility, accuracy and robustness of NGS for BCR-ABL1 KD mutation screening and represent an important step forward towards its routine application in a clinical setting. An international ring trial to test inter-laboratory reproducibility of BCR-ABL1 mutation detection by NGS is now about to start. Disclosures:Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Machova Polakova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Lion:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Hochhaus:ROCHE: Research Funding. Martinelli:NOVARTIS: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; PFIZER: Consultancy; ARIAD: Consultancy.

An Unbiased shRNA Library Screen Identifies Nucleocytoplasmic Transport As a Potential Target For Treatment Of Chronic Myeloid Leukemia

IntroductionBCR-ABL1 kinase domain mutations are detected in 30-60% of patients who develop resistance to tyrosine kinase inhibitors (TKIs) such as imatinib. However, the underlying mechanism(s) of resistance in the remaining patients are not known. To identify BCR-ABL1-independent mechanisms of resistance to TKIs, we used K562 cells that were adapted for long-term growth in 1 µM imatinib (K562-R). These cells lack BCR-ABL1 kinase domain mutations and survive despite continued suppression of BCR-ABL1 kinase activity. To screen for novel genes associated with BCR-ABL1-independent resistance, parental K562 and K562-R cells were lentivirally infected with a pooled shRNA library containing 27,000 shRNAs targeting ∼5,000 genes with known roles in cell signaling (Cellecta, Human Module 1). Using an unbiased, customized algorithm, we identified 18 genes with multiple shRNA hits that were depleted more than three-fold in K562-R cells compared to parental controls. Among the top five genes were RAN and XPO1 (CRM1), both of which encode components of a nucleocytoplasmic transport complex. Parental K562 cells also showed depletion for RAN and XPO1 shRNAs, suggesting a potential role in pathogenesis of CML and magnified activity in TKI resistance. Recently, enhanced expression of XPO1 was shown in Philadelphia chromosome positive leukemia and investigated as a potential therapeutic target in blast crisis CML (Walker et al. ASH 2012). MethodsTo validate the data from our shRNA library screen, we cloned a RAN shRNA into a tet-inducible lentiviral vector (Cellecta). Transduced parental and K562-R cells were treated with/out imatinib (2.5 mM) in the presence or absence of doxycycline. Parental and K562-R cells were treated with various concentrations of the oral XPO1 inhibitor Selinexor (KPT-330), which is now being evaluated in Phase 1 clinical trials in patients with solid and hematological malignancies. Following 72 hours of treatment, proliferation was measured by MTS assay (Promega). Additionally, CD34+ cells from newly diagnosed CML patients were lentivirally transduced with RAN shRNA and treated with/out imatinib (2.5 mM) in the presence or absence of doxycycline (n=3) or KPT-330 (n=1). Survival was assessed by colony formation assays. Normal cord blood CD34+cells were used as controls. ResultsTransduction of parental or K562-R cells with the inducible RAN shRNA and treatment with doxycycline reduced RAN mRNA levels by 57 and 76% respectively compared to the untreated control. This level of RAN suppression was sufficient to reduce the proliferation of parental K562 by 54%. It also reduced the IC50 of imatinib by > 3-fold in K562-R cells. In CD34+ cells from newly diagnosed CML patients, RAN shRNA alone had no significant effect on colony formation, whereas RAN shRNA in combination with imatinib (2.5 µM) reduced colony formation by 46% compared to imatinib alone. The IC50 of KPT-330 was 30-35% less in K562-R cells compared to parental controls suggesting a higher sensitivity of K562-R cells to XPO1 inhibition. We observed that 25 nM KPT-330 reduced colony formation by 52% in primary CML cells without reducing cord blood CD34+colonies. Combination of 2.5 µM imatinib and 25 nM KPT-330 reduced the colony formation significantly compared to imatinib alone in primary CML cells with no change in the control. ConclusionAn unbiased, shRNA library screen and subsequent validations with primary CML cells suggested that the activity of nucleocytoplasmic transport is crucial for the survival of CML cells. Genetic and pharmacologic disruption revealed that nucleocytoplasmic transport is a target in both TKI sensitive and resistant CML cells. A deeper understanding of this pathway in CML will be important for understanding BCR-ABL1 independent resistance to TKI treatment Disclosures:Kauffman:Karyopharm Therapeutics Inc.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties. Schacham:Karyopharm: Membership on an entity's Board of Directors or advisory committees. Chenchik:Cellecta: Membership on an entity's Board of Directors or advisory committees. Bonneau:Cellecta: Membership on an entity's Board of Directors or advisory committees. Deininger:Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

Direct Contact With Bone Marrow Stromal Cells Protects CML Progenitors From Imatinib Through Cytoplasmic Stabilization Of β-Catenin

A β-catenin expression signature is associated with primary resistance to tyrosine kinase inhibitors (TKIs) (McWeeney et al. 2010), but its role in TKI resistance is not completely understood. To assess the role of β-catenin in TKI resistance, we used shRNA targeting β-catenin (shβcat) in in vitro models of BCR-ABL1 kinase-independent resistance. To model resistance in the absence of bone marrow (BM)-derived factors, we used TKI-resistant K562R cells that are adapted for growth in the presence of imatinib, as well as primary CD34+ progenitors from CML patients who had failed treatment with two or more TKIs. These cells are cultured in regular medium (RM), proliferate in 1.0-2.5 μM imatinib, and exhibit β-catenin expression that is independent of BCR-ABL1 kinase activity. To model resistance mediated by the BM microenvironment, we cultured TKI-sensitive parental K562 cells and CD34+ progenitors from newly diagnosed CML patients in direct contact (DC) with HS-5 BM stromal cells. HS-5 co-culture increases β-catenin protein levels and clonogenic potential by >3-fold despite continued suppression of BCR-ABL1 kinase activity. All cells lack BCR-ABL1 kinase domain mutations and undergo TKI-mediated kinase inhibition as detected by immunoblot analyses.CML cell lines and primary cells with BCR-ABL1-independent resistance were lentivirally transduced with shβcat or a scrambled control (shSCR), and knockdown was confirmed by immunoblot and/or qRT-PCR. In RM, shβcat reduced the clonogenicity of TKI-sensitive K562 cells and CD34+ cells from newly diagnosed CML patients by 49% and 39%, respectively, compared to shSCR controls. In TKI-resistant K562R cells and CD34+ cells from TKI-resistant CML patients, shβcat reduced clonogenicity by 60% and 50%, respectively, in the presence or absence of imatinib (0-10 μM), suggesting a role for β-catenin in the development or maintenance of TKI resistance. In contrast to cells grown in RM, clonogenicity of cell lines and patient samples cultured in HS-5 DC was unaffected by shβcat compared to imatinib alone. Immunoblot analyses revealed that β-catenin protein levels were fully restored in HS-5 DC, despite the continued presence of shβcat. qRT-PCR revealed that while cells in HS-5 DC have high amounts of β-catenin protein, the mRNA levels remained similar to shβcat-expressing cells cultured in RM, consistent with post-translational stabilization of β-catenin. Importantly, increased β-catenin was not observed when cells were cultured in HS-5 conditioned medium, indicating that stabilization requires DC with the bone marrow stroma. These data are consistent with a role for β-catenin in TKI resistance mediated by DC with the BM microenvironment, similar to a recent report (Zhang et al., 2013).To understand nuclear versus cytoplasmic distribution following HS-5 DC, CD34+ cells from newly diagnosed CML patients were cultured in RM or HS-5 DC for 36 hours and analyzed for β-catenin localization by immunfluorescence. As expected, cells cultured in RM had low levels of β-catenin in the nucleus and cytoplasm that decreased upon treatment with imatinib (2.5 μM). In contrast, cells cultured in HS-5 DC had a marked increase of β-catenin that was unaffected by treatment with imatinib. While detectable in the nucleus, the majority of β-catenin protein was localized in the cytoplasm and at the cell membrane, consistent with its role in cell-cell junctions. Accordingly, in CD34+ cells from newly diagnosed CML patients, an N-cadherin blocking antibody impaired the clonogenic potential of cells cultured in HS-5 DC, with no significant effect on cells grown in RM. Affymetrix Human Gene 1.0 ST arrays revealed high levels of genes encoding the CDH2 and CDH13 cadherins, which may be involved in N-cadherin-mediated β-catenin stabilization, even in the presence of shβcat. Preliminary data also suggests that HS-5 DC reduces luciferase reporter activity from a construct harboring sequential β-catenin binding elements (pGF1-Lef/Tcf-eFGP-luc), further supporting a role for cytoplasmic β-catenin in TKI resistance. These data demonstrate a critical role for β-catenin in BCR-ABL1 kinase-independent TKI resistance, and suggest new strategies for targeting TKI resistance in the absence of BCR-ABL1 kinase domain mutations. Disclosures:Deininger:Bristol-Myers Squibb: Advisory Boards Other, Consultancy, Research Funding; Ariad Pharmaceuticals: Advisory Boards, Advisory Boards Other, Consultancy; Novartis: Advisory Boards, Advisory Boards Other, Consultancy, Research Funding; Celgene: Research Funding; Gilead Sciences: Research Funding.

MDR1 Gene Expression Predicts Response and Progression-Free Survival Of Ph+ CML Patients On Second-Line Nilotinib Therapy After Imatinib Failure - 4-Year Follow-Up

IntroductionThe activity of the drug efflux transporter protein MDR1 reduces the intracellular concentration of nilotinib and thereby impairs its efficacy. Nilotinib has been shown to be efficacious in imatinib-resistant patients. In the face of competing second-generation tyrosine-kinase inhibitors (TKI), early identification of favorable responders is crucially important. We have reported the unexpected positive prognostic impact of high MDR1 gene expression at time of imatinib resistance for subsequent 2nd line nilotinib treatment. Here, we present (i) a 48-month follow-up of our clinical data and (ii) additional functional analyses studied in an established in vitro, transposon-based vector system with stable siRNA mediated knockdown of MDR1. AimWe sought to assess (i) whether high MDR1 expression remains associated with improved cumulative rates of major molecular remission (MMR), complete cytogenetic remission (CCyR) and patients' outcome (progression-free survival, PFS) after 48 months and (ii) the impact of MDR1 expression on nilotinib in vitro sensitivity. Methods(i) We analyzed 83 patients resistant to imatinib frontline treatment in chronic phase CML treated with nilotinib 400 mg bid within the CAMN107A2101 trial. MDR1 and BCR-ABL mRNA expression levels were determined by qRT-PCR using LightCycler™ technology, normalized against beta-glucuronidase (GUS) and standardized according to the international scale (IS). Log-rank tests were performed to compare PFS and the cumulative incidences (CI) of MMR and CCyR at 48 months. (ii) MDR1high overexpressing (K562-DoxH1) and MDR1low knockdown (K562-DoxMM) cell lines were used for in vitro testing (Rumpold et al., Exp Hematol 2005). Results(i) (a) At 24 and 48 months, patients with MDR1/GUS ratios ≥2.0 attained MMR in 39% and 41%, CCyR in 58% (at both time points), and PFS rates of 75% and 67%, whereas patients with initial MDR1/GUS ratios <2.0 had significantly worse response and PFS rates, i.e. MMR in 13% and 16% (p=0.014), CCyR in 35% and 39% (p=0.044), and PFS of 50% and 46% (p=0.032). (b) BCR-ABL tumor burden prior to nilotinib revealed a significant impact on molecular response rates. BCR-ABLIS <28% separated best concerning cumulative incidences of MMR by 24 and 48 months (41% vs 21% and 48% vs 21%, p=0.009). (c) Nilotinib in vitro sensitivity of BCR-ABL kinase domain mutations at time of imatinib resistance was associated with improved PFS under nilotinib therapy: patients without any mutation showed PFS rates of 71% and 63% at 24 and 48 months, whereas those with either sensitive mutations, intermediately sensitive mutations or mutations with unknown IC50attained PFS rates of 67% and 61%; patients with mutations resistant to nilotinib achieved PFS rates of 23% at both time points (p=0.01). (ii) Even though MDR1high K562 cells are less sensitive than MDR1low expressing cells, nilotinib (applying doses from 0.01 µM up to 0.5 µM) was still able to significantly impede proliferation of both MDR1high and MDR1low, whereas imatinib-mediated growth inhibition was only seen in MDR1low, but not in MDR1highcells. Conclusion(i) At the time of imatinib-resistance, a high MDR1 gene expression predicts favorable MMR, CCyR, and PFS on consecutive 2nd line nilotinib treatment. As shown earlier, single nucleotide polymorphisms (SNPs) within MDR1 (1236CT/TT and 2677GT/TT) were significantly associated with higher MDR1 expression. (Agrawal et al., ASH 2010) (ii) Our functional data support our clinical observation that nilotinib remains efficacious in MDR1 overexpressing cells, whereas even dose-escalated imatinib does not reverse resistance. High MDR1 gene expression might select patients whose mode of resistance is essentially determined by increased efflux activity of MDR1 and not by other pathways of resistance that cannot be overcome by nilotinib. Altogether, our data might be used for the clinical risk stratification in case of imatinib resistance before switching to nilotinib and are undergoing prospective validation within the ENEST1st study. Disclosures:Saussele:Novartis: Honoraria, Research Funding, Travel Other; BMS: Honoraria, Research Funding, Travel, Travel Other; Pfizer: Honoraria. Purkayasatha:Novartis: Employment. Woodman:Novartis: Employment. Hehlmann:BMS: Consultancy, Research Funding; Novartis: Research Funding. Hochhaus:Novartis: Consultancy, Honoraria, Research Funding, Travel Other; BMS: Consultancy, Honoraria, Research Funding; Pfizer: Consultancy, Honoraria; Ariad: Consultancy, Honoraria. Müller:Novartis: Consultancy, Honoraria, Research Funding; BMS: Consultancy, Honoraria, Research Funding; Ariad: Consultancy, Honoraria, Research Funding.

Philadelphia-Positive Leukemia Patients With The Y253H Or F359V Mutations Have a High Risk Of Developing New Mutations In The Setting Of Dasatinib Resistance

ObjectiveThe clinical significance of individual mutations in Philadelphia-positive (Ph+) leukemia patients is not well-characterized on dasatinib therapy. The treatment outcomes and development of new mutations in imatinib- and/or nilotinib-failure Ph+ leukemia patients with highly nilotinib-resistant mutations (Y253H, E255K/V and F359V/C) were assessed during dasatinib therapy. MethodsOne hundred and eight Ph+ leukemia patients, including 36 with chronic myeloid leukemia in the chronic phase, 18 in the accelerated phase, 40 in the blastic phase and 14 with Ph+ acute lymphoblastic leukemia, who failed imatinib (n=79) or imatinib and nilotinib (n=29) were treated with dasatinib. Those harbored highly dasatinib-resistant mutations (T315I/A, V299L and F317L/V/I/C) were excluded. The patients were grouped into 3 cohorts by baseline BCR-ABL kinase domain (KD) mutation status: no mutation (n=42), non-nilotinib-resistant mutation (any mutation except highly nilotinib-resistant mutations; n=25), or nilotinib-resistant mutation (Y253H, E255K/V or F359V/C; n=41). BCR-ABL KD mutation analysis was detected by direct sequencing at the time of dasatinib resistance. ResultsWith a median follow-up of 6 months (range 2-60 months), the frequencies of hematological and cytogenetic responses and clinical resistance to dasatinib, failure-free survival, progression-free survival and alternative treatment-free survival were comparable by baseline BCR-ABL KD mutation status. Sixty of sixty-four patients (93.8%) underwent BCR-ABL KD mutation analysis at the time of developing clinical resistance to dasatinib. Among the 60 evaluated patients, 33 new mutations were first identified in 29 (48.3%) patients at a median of 3 months (range, 2-26 months) of dasatinib therapy, including 8 of 28 (28.6%) with no mutation, 4 of 9 (44.4%) with non-nilotinib-resistant mutations and 17 of 23 (73.9%) with nilotinib-resistant mutations at baseline. New mutations were most frequently observed in the cohort with nilotinib-resistant mutations (P=0.031). Specially, the subset with the Y253H (12/13, 92.3%) or F359V (5/5, 100%) in the cohort with nilotinib-resistant mutations was more likely to acquire new mutations. Multivariate analyses revealed that harboring the Y253H (HR=9.0, 95%CI: 2.7-30.5; P<0.001) or F359V (HR=7.6, 95%CI: 1.5-39.4; P=0.016) mutation at baseline and a lack of any hematologic response to dasatinib (HR=2.1, 95%CI: 1.4-3.1; P<0.001) were identified as independent predictors of developing new mutations during dasatinib therapy. ConclusionsOur study suggested that Ph+ leukemia patients with the Y253H or F359V mutation had a high likelihood of developing new mutations in the setting of dasatinib resistance. Our data highlight the importance of closer monitoring and mutation follow up for therapeutic choices, especially alternative therapies, which should be considered for patients with the Y253H or F359V mutation during dasatinib therapy. Larger studies are needed to assess the significance of various BCR-ABL KD mutations prior to and during sequential TKI therapy for Ph+ leukemia patients. Disclosures:No relevant conflicts of interest to declare.

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

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

Algorithms and Processing Pipeline For Error Correction and Detection Of Significant Mutations In The Kinase Domain Of BCR-ABL Analyzed By Next-Generation Sequencing: Implications For Clinical Practice Of Chronic Myeloid Leukemia

High sequencing depths of NGS may cause false-positive variant calls of minor subclones (up to 10%). Errors inserted into the NGS pipeline during sample preparation and sequencing manifest by erroneous detections of mutations including point mutations. Thus, there is a need for algorithms for filtering data which occur by error from relevant biological information.We developed algorithms and a processing pipeline for error correction and detection of significant mutations after NGS of BCR-ABL kinase domain (KD). We validated our algorithms on the retrospective NGS analysis of 135 samples from 15 CML patients in chronic phase (median 8 samples per patient; range 5-19), who developed resistant mutations (confirmed by Sanger Sequencing, SS) during 2-4 lines of therapy.Amplicon libraries were prepared using reverse transcription and 2-step PCR. The second PCR was performed partly using fusion-primers designed within the IRON-II study research consortium (Roche Applied Science) which tested 4 overlapping amplicons and partly using alternative in-house set of fusion-primers that we have developed upfront and which utilized 3 overlapping amplicons covering the KD coding region. Key concept of our error control algorithm was to apply statistics used for bacterial mutation rate prediction, Lea-Coulson probability distribution (Lea and Coulson, J of Genet 1949), to distinguish sequencing pipeline errors from biologically relevant mutations. We postulated that spontaneous mutations in bacteria are similar phenomena as enzyme errors in vitro. In both processes there are new generations of bacteria or transcripts in which mutations or errors replicate exponentially. The error rate distributions based on analysis of c-ABL kinase domain of healthy donors (n=24) were fitted to Lea-Coulson distribution. From this analysis we derived, for each type of single nucleotide substitution, estimated thresholds based on which a particular mutation may be called significant by a self-developed statistical test. We cross-checked our results with results of standard Roche pipeline including GS Amplicon Variant Analyzer.Table 1 summarizes the estimated thresholds to be applied for transitions and transversions. Higher frequency of errors was found in case of using a 3-amplicon assay in comparison to a 4-amplicon assay. The PCR products in the 3-amplicon assay are 71 bp longer on average than in the 4-amplicon assay, thus the error frequency distribution may be dependent on the length of the sequence amplified. Using our algorithm we processed NGS data and reported significant mutations. Overall, no significant mutation that caused resistance during the treatment was detected at the time of diagnosis. During 1st line imatinib treatment 10 resistant mutations in 9 patients were detected as significant 2-5 months earlier than by SS. At the time of therapy switchover, in 3 patients the algorithm already detected minor populations of one of significant mutations F317L, T315I and M351T, while SS did not. These mutations manifested after the therapy switchover and caused treatment failure. After the therapy switch, baseline mutations were still significantly detectable by our algorithm in NGS data, but not by SS in 7 patients who achieved at the time of the analysis PCgR and MMR. In 5 patients, who subsequently failed therapy after switchover, resistant mutations were significantly detected by our algorithm in NGS data 2-9 months earlier than by SS. New minor mutations were revealed by NGS after the therapy switch in 8 patients.Table 1TRANSITIONS (%)TRANSVERSIONS (%)P valueA/GG/AT/CC/TA/C+C/A+T/A+A/T+T/G+G/T+C/G+G/C3-amplicon assay0.0112.24.5311.84.770.570.053.031.032.931.100.134-amplicon assay0.015.171.934.501.930.130.051.200.431.030.430.03Since enzymes create errors during reverse transcription, 2-step PCR and sequencing process, the error correction is an essential part of the bioinformatics pipeline for relevant interpretation of BCR-ABL KD mutations detected with the highly sensitive NGS assay. Our validated algorithm and processing pipeline for significant mutation evaluations from NGS data is helpful for future clinical practice as it filters errors and allows reporting only significant mutations. This avoids false-positive results and misleading interpretations which may negatively influence treatment management of CML patients.Supported by IGANT11555 and NT13899 Disclosures:Machova Polakova:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Soverini:Novartis: Consultancy; Bristol-Myers Squibb: Consultancy; ARIAD: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:NOVARTIS, BMS(Consultancy and speaker bureau), PFIZER, ARIAD ( Consultancy): Consultancy, Speakers Bureau. Kohlmann:MLL Munich Leukemia Laboratory: Employment; Roche Diagnostics: Honoraria. Klamova:Novartis: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding.

Bosutinib As Therapy For Chronic Phase Chronic Myeloid Leukemia Following Resistance Or Intolerance To Imatinib: 48-Month Update

Bosutinib (BOS) is an oral dual Src/Abl tyrosine kinase inhibitor (TKI). This open-label, phase 1/2 study evaluated BOS in patients (pts) aged ≥18 y with chronic-phase (CP) chronic myeloid leukemia (CML) following imatinib resistance (IM-R) or intolerance (IM-I).IM-R (n=196) or IM-I (n=90) CP-CML pts received BOS as 2nd-line TKI therapy starting at 500 mg/d. Median (range) age was 53 (18–91) y; time from CML diagnosis was 3.7 (0.1–15.1) y. Treatment duration was 24.8 (0.2-83.4) mo and follow-up duration 47.3 (0.6–90.6) mo. BOS dose was escalated to 600 mg/d in 13% of pts. For the last enrolled pt, time from first BOS dose was ≥48 mo; 40% of pts are still receiving BOS.Confirmed complete hematologic responses (CHR) and major cytogenetic responses (MCyR, including complete cytogenetic response [CCyR]) newly attained or maintained from baseline are summarized (Table).Of 210 pts with known mutation status at baseline, 79 (38%) had ≥1 BCR-ABL kinase domain mutation (n=42 unique), most commonly T315I (n=9), M351T (n=9), F359V (n=8), G250E (n=6), M244V (n=6), and L248V (n=5). Among pts with T315I, CHR and MCyR rates were low (22% each). Of 67 pts evaluated for mutations before and during therapy, 18 had ≥1 new BCR-ABL mutation (T315I, n=8; V299L, n=3; and E255V, E450A, E450G, G250E, K378E, L273M, and M244V, n=1 each); 17 of these 18 pts discontinued due to disease progression (n=12), lack of efficacy (n=4), or death (n=1).The cumulative incidence of on-treatment progression (transformation to accelerated phase [AP] or blast phase [BP] CML, increasing white blood cell count [doubling over ≥1 mo with second count >20×109/L and confirmed ≥1 wk later], or loss of confirmed CHR or unconfirmed MCyR) or death at 4 y was 22% for IM-R and 10% for IM-I pts; 40% of pts discontinued BOS without an event. Kaplan-Meier (KM)–estimated overall survival (OS) at 2 y was 88% for IM-R and 98% for IM-I pts (4-y OS not reliable; pts were followed for OS for only 2 y after BOS discontinuation). The cumulative incidence of on-treatment transformation to AP/BP CML at 4 y was 4%; 57% of pts discontinued treatment without transformation. No new transformations occurred after 2 y. Overall, 173 (60%) pts discontinued BOS, most common primary reasons were adverse event (AE; n=64 [22%]) and disease progression (n=51 [18%]). Forty (14%) deaths occurred on study, 7 within 30 d of last BOS dose. Most deaths were due to disease progression (n=24 [8%; 22 and 2 in IM-R and IM-I pts, respectively]) or AE unrelated to BOS (n=13 [5%; 11 and 2]). 3 deaths occurring ≥99 d after last BOS dose were due to unknown causes; no deaths were assessed as BOS-related.The most frequent hematologic treatment-emergent AEs (TEAEs; all grades/grade 3/4) were thrombocytopenia (42%/26%) and anemia (27%/11%); the most frequent non-hematologic TEAEs were diarrhea (86%/10%), nausea (46%/1%), vomiting (37%/4%), rash (36%/9%), abdominal pain (26%/2%), fatigue (26%/1%), pyrexia (26%/1%), increased alanine aminotransferase (ALT; 22%/9%), cough (22%/0), and upper abdominal pain (20%/<1%). Cardiac events occurred in 16% of pts (7% grade 3/4). Grade 3/4 on-treatment hematologic and non-hematologic laboratory abnormalities in ≥10% of pts included thrombocytopenia (25%), neutropenia (17%), lymphopenia (15%), anemia (14%), alanine transaminase elevation (11%), and hypermagnesemia (11%). Toxicities were managed by ≥1 BOS dose reduction in 44% of IM-R and 58% of IM-I pts and by ≥1 dose delay in 66% of IM-R and 83% of IM-I pts. AEs led to BOS discontinuation in 32 (16%) IM-R and 35 (39%) IM-I pts; the most common reason was thrombocytopenia (3% of IM-R and 12% of IM-I pts).In conclusion, BOS demonstrates durable efficacy and manageable toxicity in CP-CML pts following IM-R or IM-I after ≥48 mo of follow-up, highlighting the therapeutic potential of BOS in these pts. Disclosures:Brümmendorf:Patent on the use of imatinib and hypusination inhibitors: Patents & Royalties; Ariad: Consultancy; Novartis: Consultancy, Honoraria, Research Funding; Bristol Myer Squibb: Consultancy, Honoraria; Pfizer Inc: Consultancy, Honoraria. Cortes:Pfizer: Consultancy, Research Funding; Novartis: Research Funding; Ariad: Consultancy, Research Funding; Teva: Consultancy, Research Funding; Bristol Myer Squibb: Research Funding. Kantarjian:Pfizer Inc: Research Funding. Kim:BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Conlan:Pfizer Inc: Employment. Leip:Pfizer Inc: Employment. Turnbull:Pfizer Inc: Employment. Gambacorti-Passerini:Novartis: Consultancy; Bristol Myers Squibb: Consultancy; Pfizer Inc: Consultancy, Research Funding. Lipton:Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ariad: Equity Ownership, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Bristol Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau.