BCR-ABL Gene Research Articles

Dynamic prediction of disease progression for leukemia patients by functional principal component analysis of longitudinal expression levels of an oncogene

Patients’ biomarker data are repeatedly measured over time during their follow-up visits. Statistical models are needed to predict disease progression on the basis of these longitudinal biomarker data. Such predictions must be conducted on a real-time basis so that at any time a new biomarker measurement is obtained, the prediction can be updated immediately to reflect the patient’s latest prognosis and further treatment can be initiated as necessary. This is called dynamic prediction. The challenge is that longitudinal biomarker values fluctuate over time, and their changing patterns vary greatly across patients. In this article, we apply functional principal components analysis (FPCA) to longitudinal biomarker data to extract their features, and use these features as covariates in a Cox proportional hazards model to conduct dynamic predictions. Our flexible approach comprehensively characterizes the trajectory patterns of the longitudinal biomarker data. Simulation studies demonstrate its robust performance for dynamic prediction under various scenarios. The proposed method is applied to dynamically predict the risk of disease progression for patients with chronic myeloid leukemia following their treatments with tyrosine kinase inhibitors. The FPCA method is applied to their longitudinal measurements of BCR-ABL gene expression levels during follow-up visits to obtain the changing patterns over time as predictors.

How to detect the rare BCR-ABL (e14a3) transcript: A case report and literature review.

The Philadelphia (Ph; BCR-ABL) chromosome originates from a translocation event between chromosomes 9 and 22, and results in the BCR-ABL fusion gene. In chronic myelogenous leukemia (CML), the BCR-ABL gene is mainly coded for by a major breakpoint cluster region (M-bcr, e13a2 and e14a2). However, in some patients, BCR-ABL genes are encoded by a minor (m)-bcr, e1a2, and a micro (µ)-bcr region, e19a2. These transcripts revealed a different clinical course. The present study described a CML patient whose cytogenetics and FISH analyses of bone marrow revealed a karyotype of 46, XY t(9,22) (q34;q11), while the commercial kits of quantitative PCR (qPCR) failed to detect the BCR-ABL fusion gene. Further multiplex Reverse transcription-PCR (RT-PCR) and sequencing analyses identified a rare e14a3 (b3a3) fusion transcript.

Coexistence of p210BCR-ABL and CBFβ-MYH11 fusion genes in myeloid leukemia: A report of 4 cases.

Numerous acquired molecular and cytogenetic abnormalities are strongly associated with hematological malignancies. The breakpoint cluster region-ABL proto-oncogene 1 (BCR-ABL) rearrangement leads to a p210 chimeric protein in typical chronic myeloid leukemia (CML), whereas 17–25% of patients with acute lymphocytic leukemia and 0.9–3% patients with de novo acute myeloid leukemia (AML) carry a p190BCR-ABL fusion protein. Cases of patients with AML/CML carrying two specific primary molecular changes, BCR-ABL and core binding factor-β-myosin heavy chain 11 (CBFβ-MYH11) fusion genes have been rarely reported. The present study aimed to understand the nature and mechanism of this particular type of leukemia through case reports and literature review. A total of four patients who were diagnosed as AML/CML with BCR-ABL and CBFβ-MYH11 fusion genes in the First Affiliated Hospital of Soochow University (Suzhou, China) between January 2004 and December 2012 were examined. Morphological analysis of bone marrow cells, flow cytometry, quantitative polymerase chain reaction of p210BCR-ABL and CBFβ-MYH11 transcripts as well as cytogenetic and fluorescence in situ hybridization analyses were performed. A total of 4 patients who exhibited fusion of p210BCR-ABL and CBFβ-MYH11 were identified. A single patient (case 1) was first diagnosed CML-acute phase (AP), which progressed rapidly to CML-blast crisis (BC), and three patients (cases 2, 3 and 4) were diagnosed with AML with bone marrow eosinophilia at first presentation with no evidence of previous onset of CML. All cases achieved remission following conventional chemotherapy/hematological stem cell transplantation combined with the inhibitor of tyrosine kinase (TKI) maintenance therapy. The patients with CML carrying and expressing BCR-ABL and CBFβ-MYH11 fusion genes appeared more likely to rapidly progress to AP or BC. Therefore, the product of the CBFβ-MYH11 fusion gene may serve an important role in the transformation of CML. The co-expression of p210BCR-ABL and CBFβ-MYH11 fusion genes in myeloid leukemia may be a molecular event occurring not only during the development of CML, but also in AML.

Advances of ecotropic viral integration site 1 gene in acute lymphoblastic leukemia

As an proto-oncogene, ecotropic viral integration site 1 (EVI1) gene is over-expressed in acute myeloid leukemia (AML) because of chromosomal translocation or other genetic abnormalities, finally cause poor prognosis. In recent years, an increasing research of the expression of EVI1 in acute lymphoblastic leukemia (ALL), especially in pediatric ALL. The overexpression of EVI1 gene also suggest a poor prognosis, which is closely relate to the age. But the relationship between the expression of EVI1 gene and mixed lineage leukemia (MLL) and BCR-ABL gene rearrangement needs to be further studied. Researching the expression of EVI1 gene in ALL and exploring targeted therapeutic agents are important research directions in the future. Key words: Proto-oncogene; Leukemia, lymphoid; Gene expression; Ecotropic viral integration site 1 gene

Gas6/AXL Signaling Regulates Self-Renewal of Chronic Myelogenous Leukemia Stem Cells by Stabilizing β-Catenin.

Purpose: Quiescent leukemia stem cells (LSC) are important resources of resistance and relapse in chronic myelogenous leukemia (CML). Thus, strategies eradicating CML LSCs are required for cure. In this study, we discovered that AXL tyrosine kinase was selectively overexpressed in primary CML CD34+ cells. However, the role of AXL and its ligand Gas6 secreted by stromal cells in the regulation of self-renewal capacity of LSCs has not been well investigated.Experimental Design: The function of CML CD34+ cells was evaluated by flow cytometer, CFC/replating, long-term culture-initiating cells (LTC-IC), CML mouse model driven by human BCR-ABL gene and NOD-scid-IL2Rg-/- (NSI) mice.Results: AXL was selectively overexpressed in primary CML CD34+ cells. AXL knockdown reduced the survival and self-renewal capacity of human CML CD34+ cells. Pharmacologic inhibition of AXL reduced the survival and self-renewal capacity of human CML LSCs in vitro and in long-term grafts in NSI mice. Human CML CD34+ cells conscripted bone marrow-derived stromal cells (BMDSC) and primary mesenchymal stem cells (MSC) to secrete Gas6 to form a paracrine loop that promoted self-renewal of LSCs. Suppression of AXL by shRNA and inhibitor prolonged survival of CML mice and reduced the growth of LSCs in mice. Gas6/AXL ligation stabilizes β-catenin in an AKT-dependent fashion in human CML CD34+ cells.Conclusions: Our findings improve the understanding of LSC regulation and validate Gas6/AXL as a pair of therapeutic targets to eliminate CML LSCs. Clin Cancer Res; 23(11); 2842-55. ©2016 AACR.

A rare e13a3 (b2a3) BCR-ABL1 fusion transcript with normal karyotype in chronic myeloid leukemia: The challenges in diagnosis and monitoring minimal residual disease (MRD)

Patients with chronic myeloid leukemia (CML) have a t (9;22)(q34;q11.2) or variant translocation that results in a BCR-ABL1 fusion gene. For many years, conventional karyotyping has been used as the standard diagnostic tool for t (9;22) (q34;q11.2). However, it has several limitations that may lead to failure for detecting BCR-ABL1 gene rearrangements in around 5% of all CML patients. Although reverse transcription polymerase chain reaction (RT-PCR) has evolved as a sensitive method for detecting BCR-ABL1 translocation, this method fail to detect certain BCR-ABL1 fusion transcript type, such as e13a3 (also known as b2a3), as a result of many commercially available and laboratory-developed primer sets. Fortunately, these two rare situations rarely appear at the same time, therefore, the combination of two methods rarely misdiagnosed the patients with CML. In this study, we report a patient with CML who tested both negative by RT-PCR and cytogenetic analysis at the time of diagnosis. She was diagnosed as atypical CML (aCML) and allogeneic hematopoietic stem cell transplantation was suggested. Further fluorescence in situ hybridization (FISH) showed cryptic insertion of ABL into BCR gene on chromosome 22, and DNA sequencing with alternative primer sets demonstrated the presence of an e13a3 BCR-ABL1 fusion. She was diagnosed as CML and received imatinib 400 mg/day. A follow-up BCR-ABL1 FISH analysis demonstrated a markedly reduced BCR-ABL1 fusion rate of 0 after 6months treatment, indicating a complete cytogenetic response.

Gold Nanoparticles for BCR-ABL1 Gene Silencing: Improving Tyrosine Kinase Inhibitor Efficacy in Chronic Myeloid Leukemia.

Introduction of tyrosine kinase inhibitors for chronic myeloid leukemia treatment is associated with a 63% probability of maintaining a complete cytogenetic response, meaning that over 30% patients require an alternative methodology to overcome resistance, tolerance, or side effects. Considering the potential of nanotechnology in cancer treatment and the benefits of a combined therapy with imatinib, a nanoconjugate was designed to achieve BCR-ABL1 gene silencing. Gold nanoparticles were functionalized with a single-stranded DNA oligonucleotide that selectively targets the e14a2 BCR-ABL1 transcript expressed by K562 cells. This gold (Au)-nanoconjugate showed great efficacy in gene silencing that induced a significant increase in cell death. Variation of BCL-2 and BAX protein expression, an increase of caspase-3 activity, and apoptotic bodies in cells treated with the nanoconjugate demonstrate its aptitude for inducing apoptosis on K562 BCR-ABL1-expressing cells. Moreover, the combination of the silencing Au-nanoconjugate with imatinib prompted a decrease of imatinib IC50. This Au-nanoconjugate was also capable of inducing the loss of viability of imatinib-resistant K562 cells. This strategy shows that combination of Au-nanoconjugate and imatinib make K562 cells more vulnerable to chemotherapy and that the Au-nanoconjugate alone may overcome imatinib-resistance mechanisms, thus providing an effective treatment for chronic myeloid leukemia patients who exhibit drug tolerance.

Impact of Chromosomal Rearrangement upon DNA Methylation Patterns in Leukemia.

Genomic instability, including genetic mutations and chromosomal rearrangements, can lead to cancer development. Aberrant DNA methylation occurs commonly in cancer cells. The aim of this study is to determine the effects of a specific chromosomal lesion the BCR-ABL translocation t(9:22), in establishing DNA methylation profiles in cancer. Materials and methods We compared DNA methylation of 1,505 selected promoter CpGs in chronic myelogenous leukemia (CML), acute lymphoblastic leukemia (ALL) with and without the Philadelphia chromosome t(9:22), CD34+ hematopoietic stem cells transfected with BCR-ABL, and other tumors without BCR-ABL (acute promyelocytic leukemia (APL) and gastrointestinal stromal tumors (GIST). In this study, the DNA methylation profile of CML was more closely related to APL, another myeloid leukemia, than Ph+ ALL. Although DNA methylation profiles were consistent within a specific tumor type, overall DNA methylation profiles were no influenced by BCR-ABL gene translocation in the cancers and tissues studied. We conclude that DNA methylation profiles may reflect the cell of origin in cancers rather than the chromosomal lesions involved in leukemogenesis.

The use of Imatinib resistance mutation analysis to direct therapy in Philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukaemia patients failing Imatinib treatment, in Patan Hospital, Nepal.

Philadelphia chromosome/BCR-ABL1 positive chronic myeloid leukaemia (CML) can be successfully treated with Glivec (Imatinib), which is available free of cost through the Glivec International Patient Assistance programme (GIPAP) to patients with proven CML without means to pay for the drug. We review the acquired mutations in the tyrosine kinase encoded by the BCR-ABL1 gene underlying Glivec failure or resistance in a cohort of 388 imatinib-treated CML patients (149 Female and 239 male) registered between February 2003 and June 2016 in Nepal. Forty-five patients (11 female 34 male) were studied; 18 different BCR-ABL1 mutations were seen in 33 patients. P-loop mutation, Kinase domain and A-loop mutations were seen in 9, 16 and 4 patients respectively. Other mutations were seen in five patients. A T315I mutation was the most common mutation, followed by F359V and M244V. Sixteen mutations showed intermediate activity to complete resistance to Glivec. Among the 45 patients evaluated for BCR-ABL1 mutations, 4 were lost to follow-up, 14 died and 27 are still alive. Among the surviving patients, 16 are receiving Nilotinib, 5 Dasatinib and 3 Ponatinib, while 3 patients were referred to India, one of who received allogenic bone marrow transplantation. Understanding the spectrum of further acquired mutations in BCR-ABL1 may help to choose more specific targeted tyrosine kinase inhibitors that can be provided by GIPAP.

FUSION GENE BCR-ABL : FROM ETIOPATHOGENESIS TO THE MANAGEMENT OF CHRONIC MYELOID LEUKEMIA

C hronic Myeloid Leukemia (CML) is a myeloproliferative neoplasm. CML is relative frequent disorder. Most of CML patients have Philadelphia chromosome (Ph),which is derived from a reciprocal translocation between chromosome 9 and 22, t(9;22)(q34;ql1), generating the BCRABLfusion gene. In general, there are 3 breakpoint cluster regions in BCR gene : mayor(M-bcr), minor (m-bcr) and micro (μ-bcr). The BCR-ABL gene encodes proteins that vary in size depending on the breakpoint in the BCR gene. However, these proteins share a high tyrosine kinase activity. In the absence of activating stimuli, BCR-ABL tyrosine kinase able to transferphosphate from ATP (autophosphorilation) to tyrosine residues on various substrates in the cell. It actives intracelluler signaling pathways. These pathways cause increase proliferation or decrease apoptosis and differentiation of a hematopoietic stem cell; and defect in adherence of myeloid progenitors to marrow stroma resulting in CML. These discoveries determined that BCR-ABL fusion gene is critical event in etiopathogenesis of CML and a ideal target for therapy. Therapy of CML patients with BCRABL fusion gene-positif is by block autophosphorilation mechanism by Tyrosine Kinase Inhibitor (TKI), example imatinib. Molecular method to detect BCR-ABL transcript is necessary for monitoring response to TKI in CML pateints.

Germline mutations in ABL1 cause an autosomal dominant syndrome characterized by congenital heart defects and skeletal malformations.

ABL1 is a proto-oncogene well known as part of the fusion gene BCR-ABL in the Philadelphia chromosome of leukemia cancer cells1. Inherited germline ABL1 changes have not been associated with genetic disorders. Here we report ABL1 germline variants co-segregating with an autosomal dominant disorder characterized by congenital heart disease, skeletal abnormalities, and failure to thrive. The variant c.734A>G (p.Tyr245Cys) was found as de novo or co-segregating with disease in five individuals (families 1-3). Additionally, a de novo c.1066G>A (p.Ala356Thr) variant was identified in the sixth individual (family 4). We overexpressed the mutant constructs in HEK 293T cells and observed increased tyrosine phosphorylation, suggesting increased ABL1 kinase activities associated with both p.Tyr245Cys and p.Ala356Thr substitutions. Our clinical and laboratory findings, together with previously reported teratogenic effects of selective BCR-ABL inhibitors in humans2-5 and developmental defects in Abl1 knock-out mice6,7, suggest ABL1 plays an important role during organismal development.

The Genomic Landscape of PAX5, IKZF1, and CDKN2A/B Alterations in B-Cell Precursor Acute Lymphoblastic Leukemia

We present a comprehensive comparison of PAX5,IKZF1, and CDKN2A/B abnormalities in 21 B-cell precursor acute lymphoblastic leukemia (B-ALL) patients studied by aCGH and gene-specific FISH assays. In our cohort of B-ALL patients, alterations of IKZF1, PAX5, and CDKN2A/B were detected by aCGH analysis in 43, 52, and 57% of samples, respectively. Deletions of IKZF1 were present in 9 samples, including 5 cases positive for both PAX5 and IKZF1 deletions, implying digenic impairment. Furthermore, all cases with IKZF1 deletions also had additional genomic alterations, including BCR-ABL1 gene fusions, PAX5 deletions, CDKN2A/B deletions, and FLT3 amplification. Deletions of CDKN2A/B represented the most frequent abnormalities in our group of patients. Our study demonstrates the high incidence of PAX5, IKZF1, and CDKN2A/B alterations in B-ALL detected by aCGH analysis. Due to the small size and variability in the deletion breakpoints, FISH studies showed false-negative results in 10, 40, and 28% of the samples tested for the IKZF1,PAX5, and CDKN2A/B gene deletions, respectively. The PAX5 and IKZF1 abnormalities are highly specific to B-ALL and can be used as diagnostic markers. Moreover, IKZF1 alterations frequently coexist with a BCR-ABL gene fusion. Our study revealed multiple additional B-ALL-specific genomic alterations and showed that aCGH is a more sensitive method than FISH, allowing whole genome profiling and identification of aberrations of diagnostic and prognostic significance in patients with B-ALL.

Quantitative real-time monitoring of RCA amplification of cancer biomarkers mediated by a flexible ion sensitive platform

Ion sensitive field-effect transistors (ISFET) are the basis of radical new sensing approaches. Reliable molecular characterization of specific detection of DNA and/or RNA is vital for disease diagnostics and to follow up alterations in gene expression profiles. Devices and strategies for biomolecular recognition and detection should be developed into reliable and inexpensive platforms. Here, we describe the development of a flexible thin-film sensor for label free gene expression analysis. A charge modulated ISFET based sensor was integrated with real-time DNA/RNA isothermal nucleic acid amplification: Loop-mediated isothermal amplification (LAMP) and Rolling Circle Amplification (RCA) techniques for c-MYC and BCR-ABL1 genes, allowing for the real-time quantification of template. Also, RCA allowed the direct quantification of RNA targets at room temperature, eliminating the requirement for external temperature controllers and overall complexity of the molecular diagnostic approach. This integration between the biological and the sensor/electronic approaches enabled the development of an inexpensive and direct gene expression-profiling platform.

BCR-ABL1 transcript types showed distinct laboratory characteristics in patients with chronic myeloid leukemia.

In chronic myeloid leukemia (CML) two main types of messenger RNA (e14a2 and e13a2) can be produced by BCR-ABL1 gene rearrangement. Due to conflicting results, the clinical value of these transcripts remains controversial. The aim of this study was to identify associations of e14a2 and e13a2 transcripts with laboratory variables and also the response to treatment. This study included 203 adult patients with CML treated with Imatinib as first-line drug in a reference hematology center in Northeast Brazil. Clinical and laboratory data were obtained after informed consent. Samples were collected for RNA extraction and analyzed by reverse transcription-polymerase chain reaction (PCR), according to the international protocol BIOMED-1. The LeukemiaNet 2013 criteria were used to establish the molecular response. The frequency distribution of the BCR-ABL1 transcripts was e14a2 (64%), e13a2 (34%), and double positives (2%). The results showed a statistically significant association of the e14a2 transcript type with thrombocytosis (P = 0.0005) and the e13a2 with higher leukocyte count (P = 0.0491). In a subgroup of 44 patients, the molecular response to treatment with Imatinib was assessed by quantitative PCR at 3 months (BCR-ABL1 ≤ 10%), 6 months (BCR-ABL1 ≤ 1%), or 12 months (BCR-ABL1 ≤ 0.1%). Although patients with the transcript e14a2 showed higher frequency of good responses than patients with the transcript e13a2, this difference was not statistically significant. In agreement with published data, our results showed association of the BCR-ABL1 transcript e14a2 with thrombocytosis and the BCR-ABL1 transcript e13a2 with higher leukocytosis in patients with chronic myeloid leukemia.

BCR-ABL GENE KINASE DOMAIN MUTATION FREQUENCY IN IMATINIB RESISTANT CHRONIC MYELOID LEUKEMIA PATIENTS

Background. BCR-ABL gene mutations are the main course of tyrosine kinase 1 st generation inhibitor (TKI-1) imatinib resistance in chronic myelogenous (CML) patients. Some of them are resistant both for imatinib and TKI-2. The nilotinib and dasatinib mutation spectra are not the same. To ensure a correct choice of TKI-2 for imatinib CML resistant patient treatment it is necessary to investigate frequency of BCR-ABL gene mutations. Aim. To evaluate BCR-ABL gene mutation frequency in imatinib resistant CML patients. Materials and methods. Peripheral blood of imatinib resistant CML patients were studied by means of direct PCR product Sanger sequencing in order to reveal BCR-ABL gene mutations. Results. BCR-ABL gene mutations were found in 31 % (n=262/846) imatinib resistant patients. Proportion of resistant mutations were 40,3 % for nilotinib and 21 % for dasatinib. Conclusion. More than a halve of BCR-ABL mutations resistant for imatinib were resistant either against nilotinib or dasatinib. Consequently, prior to replace imatinib for nilotinib or dasatinib it is necessary to perform BCR-ABL mutational analysis. If to prescribe TKI-2 blindly there may be high risk that nilotinib or dasatinib would be ineffective due to improper mutation in BCR-ABL gene.

Significance of bcr-abl molecular detection in chronic myeloid leukemia patients

Important advances in the understanding of the molecular basis of chronic myeloid leukemia have resulted in the development of new therapies and changed the paradigm for managing this myeloproliferative disease. The reciprocal translocation of the abl (Abelson murine leukemia) proto-oncogene on chromosome 9 to the bcr (breakpoint cluster region) gene on chromosome 22 creates a transcriptionally active, chimeric bcr-abl gene and gives rise to the Philadelphia chromosome. 1
 The genetic hallmark of chronic myeloid leukemia (CML) is the Philadelphia chromosome (Ph), which occurs in at least 95% of CML cases as well as some cases of acute lymphocytic leukemia (ALL; approximately 5% for children, 20% for adults). It also has been reported in some other hematologic disorders, albeit rarely. 2-4 JMS 2016; 19(2):95-96

BCR-ABL1 gene rearrangement as a subclonal change in ETV6-RUNX1–positive B-cell acute lymphoblastic leukemia

AbstractWe report here on a case of ETV6-RUNX1–positive B-cell acute lymphoblastic leukemia (B-ALL) that has acquired a BCR-ABL1 gene rearrangement as a subclonal change. The 19-year-old female patient presented with B symptoms, pancytopenia, and circulating blasts. The bone marrow aspirate was hypercellular and was infiltrated by an immature blast population that was confirmed as B-ALL by flow cytometry. Sequential fluorescent in situ hybridization was performed on the patient's leukemic cells, which were shown to contain both ETV6-RUNX1 and BCR-ABL1 gene rearrangements. The majority of nuclei (85%) showed only the ETV6-RUNX1 gene rearrangement; however, an additional 10% also showed a variant BCR-ABL1 gene rearrangement, indicating the ETV6-RUNX1 gene rearrangement was the primary change. A review of the literature has shown that acquisition of a BCR-ABL1 gene rearrangement as a secondary change in B-ALL is a very rare occurrence, and the effect it may have on prognosis is uncertain in the modern therapy age.

Targeting Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia with a Novel Transcriptional Inhibitor

Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ALL) is caused by reciprocal translocation between chromosome 9 (region q34) and chromosome 22 (region q11). This translocation results in oncogenic BCR-ABL fusion gene that encodes a chimeric BCR-ABL protein, continuously activated tyrosine kinase which causes unregulated cell division and leukemia. Although tyrosine kinase inhibitors (TKIs) are the most useful treatment against Ph+ALL patients, a part of them steadily become refractory to current therapy mainly through acquired mutations in ATP binding sites of BCR-ABL, necessitating a novel strategy to treat TKI resistant Ph+ALL. Here we show that our novel small molecule which targets specific transcriptional factor induces Ph+ALL cell death through directly controlling the expression of BCR-ABL gene. We have previously reported the fundamental requirement of transcription factor A (TFA) in the development of acute myeloid leukemia (AML), another form of acute leukemia originating from myeloid progenitor cells. Since previous reports suggest the involvement of TFA family genes in the development of lymphoid cells also, we first addressed the role of TFA in Ph+ALL cells using tetracycline-responsive short hairpin RNA (shRNA)-mediated gene knockdown system. Upon doxycycline treatment, shRNAs targeting TFA (sh_TFA)-transduced SU-Ph2 (Ph+ALL) and SU/SR (SU-Ph2 with BCR-ABL T315I mutation, therefore resistant to Imatinib) cells showed decreased proliferation rate with cell cycle arrest at G0/G1 phase and profound apoptosis. Intriguingly, TFA knockdown suppressed the expression of BCR-ABL fusion gene both at mRNA and protein levels. Concomitant down-regulation of BCR-ABL cytoplasmic gene targets such as mTOR-AKT, STAT and MEK-ERK pathways were also observed in sh_TFA-transduced ALL cells. These results indicate the possible interaction between BCR-ABL and TFA.Since expression of BCR-ABL fusion gene is tightly regulated by gene promoter of BCR andthere exists a TFA-consensus binding site in the proximal promoter region of BCR, we next addressed the TFA-mediated transcriptional regulation of BCR-ABL. In silico data analysis of gene expression array sets extracted from ALL patients revealed the positive correlation of BCR and TFA expressions. Analysis of chromatin immunoprecipitation and sequencing (ChIP-Seq) in mouse ES cells disclosed the potential TFA binding in the proximal regulatory region of BCR, which we have confirmed by ChIP assay in SU/SR cells. These findings suggest that TFA positively and directly controls BCR-ABLexpression through BCR promoter binding in ALL cells.We next sought to explore the effect of TFA inhibition in ALL cells in vivo. Immunodeficient NOG mice transplanted with SU/SR cells that are transduced with sh_TFA or control vector were followed for overall survival and chimerism of ALL cells in the peripheral blood. As expected, TFA inhibition decelerated the dissemination of leukemia and prolonged overall survival period in these mice.We next challenged this ALL-xenografted mice with newly synthesized small compound which irreversibly attenuates the function of TFA as a transcription factor. Existence of T315I mutation in BCR-ABL gene in SU/SR cells naturally provides TKI-therapy resistance to these mice, and oral Imatinib treatment at 100 mg/kg body weight did not prolonged their survivals actually. In contrast, our novel compound was highly effective in these mice at 375 microgram/kg body weight and significantly prolonged the overall survival period without serious side effects, overcoming TKIs resistance. These findings collectively underscore the importance of TFA in the maintenance of ALL cells with resistance to TKIs and is a druggable target in the leukemia therapy. DisclosuresNo relevant conflicts of interest to declare.

Identification of Imatinib-Sensitizing Genes in Chronic Myeloid Leukemia with a Genome-Scale Crispr Knock-out Screen

Background: Resistance to tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML) can either originate from mutations in the BCR-ABL1 gene, which are mostly well characterized, or emerge from unknown alternative mutations elsewhere in the genome. Small hairpin (sh)RNA screens have been used to discover such genes but are becoming limited due to sup-optimal protein depletion and non-reliable off-target effects. More efficient screening techniques in human cells are now available as a result of the increasing understanding of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) /Cas9 system.Aims: Our goal is to uncover imatinib (IM)-sensitizing genes that cause IM resistance when knocked-out. Characterizing these genes may help understand the mechanisms of IM uptake, metabolism, degradation and/or activity in CML cells. Additionally, we also expect to unveil alternative, BCR-ABL1 independent, oncogenic pathways in CML cells.Methods: In order to find other genes involved in IM resistance, we performed a genome scale CRISPR knock-out (GeCKO) screen, which contains 121,413 sgRNAs that target 20,914 protein coding genes and miRNAs. We transduced one sgRNA per cell and challenged the K562-GeCKO cell pool to IM selection. We compared the abundance of sgRNAs between pre/post-IM treatment by next generation sequencing (NGS).Results: After IM selection, the sgRNAs from surviving cells were identified by NGS and unveiled potential IM-sensitizing genes. The most enriched sgRNAs (FDR < 0.01) targeted genes involved in transcriptional (KLF1, MED24) and translational (EIF2AK1, UBE2M) regulation, apoptosis (BAX, BCL2L11) and cell cycle regulation (BAP1, SPRED2). Subsequent screens on LAMA84 cells are currently underway in order to validate our findings. Additionally, the establishment of individual gene knock-out cell lines are in progress in order to fully understand the role of each gene in IM resistance.Summary/Conclusion: Using a CRISPR knock-out screen, we produced a list of 19 genes (FDR < 0.05) that may play a role in IM resistance. Encouragingly, a subset of these genes (BAX, BAP1, BCL2L11 and SPRED2) have already been correlated to CML progression and/or TKI resistance in the past. We aim to bolster our findings by establishing individual gene KO cell lines and study resistance in LAMA84 cells. The utilization of CRISPR libraries may not only help understand TKI resistance in CML, but also help identify numerous novel genes involved in drug resistances for a myriad of different diseases. DisclosuresMahon:ARIAD: Honoraria; PFIZER: Honoraria; BMS: Consultancy, Honoraria; NOVARTIS PHARMA: Consultancy, Honoraria, Research Funding.

Polymorphic Variant in GATA3 gene Is a Hallmark of PAR1-Deleted BCP-ALL and Associates with Poor Prognosis Among Pediatric Patients Treated with the BFM Backbone Protocols

Abstract Introduction Germline variant at rs3824662 in GATA3 gene was associated with early response to treatment as well as relapse risk in childhood BCP-ALL treated with COG protocols. This effect resulted from the strong link between the GATA3 polymorphism and the presence of somatic defects in leukemic cells including IKZF1 deletions, CRLF2 rearrangements and JAK2 gene mutations, which promote aggressive course of the disease. Aims The study aimed to evaluate an association between GATA3 gene polymorphism and clinical and biological features of pediatric BCP-ALL treated with the BFM backbone protocols. Methods Between November 2010 and June 2016, 957 consecutive children with newly diagnosed BCP-ALL were enrolled into the study. 822 patients treated according to ALL-IC BFM2009 protocol (n=594) and ALL-IC BFM2002 protocol (n=228) in 15 centers of the Polish Pediatric Leukemia/Lymphoma Study Group were enrolled into the study (median age 4.5 yrs, median follow-up time 2.5 yrs). Patients with BCR-ABL1 and MLL gene rearrangements were excluded from the analysis. The rs3824662 polymorphism of the GATA3 gene was genotyped using TaqMan probes. GATA3 mRNA expression level in leukemic cell was evaluated in BCP-ALL cases using qPCR with FAM-MGB probes (n=136). Targeted copy number screening of selected 23 loci was performed using the P335-B2 SALSA MLPA kit (MRC-Holland, Netherlands) in n=807 available DNA leukemia samples. MRD at day 15 and 33 was measured by flow cytometry with EuroFlow 8-color antibody panels. Results In the study group genotypes distribution withinrs3824662 of GATA3 was as follows: AA: n=44 (5.4%); CA: n=266 (32.4%); CC: n=512 (62.3%). Median MRD15 and MRD33 were 0.31% and 0.001% respectively. IKZF1 deletion were found in 18%, PAX5 in 20%, PAR1 in 12%, CDKN2A in 24%, CDKN2B 20%, BTG1 in 6.3%, ETV6 in 21%, EBF1 in 4% and RB1 in 6% of cases. Leukemic cells harbouring AA variant withinrs3824662 showed GATA3 mRNA expression 1.6 and 2.2 times higher compared to cells with CA and CC variants respectively, with the difference close to significant (ANOVA p=0.06). The presence of AA variant was not related to any gene deletion apart from microdeletions of the pseudoautosomal region PAR1 (Xp22 and Yp11) which occurred more frequently in AA carriers as compared to CA and CC carriers (11/41 vs. 34/242 vs. 52/468, p=0.013). We did not find any association of clinical features such as initial WBC, steroid response, sex and age at diagnosis among BCP-ALL patients with GATA3 genotype. However, AA carriers had a higher risk for MRD&gt;10% at day 15 OR (95%CI)=3.93 (1.37-11.23) as well as MRD&gt;0.01% at day 33, OR(95%CI)=2.95 (1.12-7.73) as compared CC carriers. Cox model of survival analysis was done for variables with univariate significance of p&lt;0.15 (risk group, sex). The results of Cox regression showedthe AA genotype remained associated with an increased risk of death regardless of risk group (assigned based on ALL-IC BFM09 protocol), HR(95%C)=2.95(1.12-7.73), P=0.028). Conclusions We showed that carriers of AA genotype in GATA3 are prone to develop PAR1 -deleted BCP-ALL. Moreover our study confirmed an association of GATA3 AA rs3824662 homozygosity with poor early response to treatment as well as risk of death among pediatric BCP-ALL patients treated with the BFM backbone protocols. Disclosures Madzio: National Science Centre: Research Funding.