PKC‐β/Alox5 axis activation promotes Bcr‐Abl‐independent TKI‐resistance in chronic myeloid leukemia

2021 ◽  
Author(s):  
Dan Ma ◽  
Ping Liu ◽  
Ping Wang ◽  
Zhen Zhou ◽  
Qin Fang ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Tki Resistance ◽  
Pkc Β

scholarly journals Metabolic Plasticity Is an Essential Requirement of Acquired Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukemia

Cancers ◽  
2020 ◽  
Vol 12 (11) ◽  
pp. 3443
Author(s):  
Miriam G. Contreras Mostazo ◽  
Nina Kurrle ◽  
Marta Casado ◽  
Dominik Fuhrmann ◽  
Islam Alshamleh ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Mitochondrial Respiration ◽  
Myeloid Leukemia ◽  
Chemotherapeutic Agents ◽  
Response To Therapy ◽  
Metabolic Reprogramming ◽  
Metabolic Inhibitors ◽  
Metabolic Plasticity ◽  
Tki Resistance

Tyrosine kinase inhibitors (TKIs) are currently the standard chemotherapeutic agents for the treatment of chronic myeloid leukemia (CML). However, due to TKI resistance acquisition in CML patients, identification of new vulnerabilities is urgently required for a sustained response to therapy. In this study, we have investigated metabolic reprogramming induced by TKIs independent of BCR-ABL1 alterations. Proteomics and metabolomics profiling of imatinib-resistant CML cells (ImaR) was performed. KU812 ImaR cells enhanced pentose phosphate pathway, glycogen synthesis, serine-glycine-one-carbon metabolism, proline synthesis and mitochondrial respiration compared with their respective syngeneic parental counterparts. Moreover, the fact that only 36% of the main carbon sources were utilized for mitochondrial respiration pointed to glycerol-phosphate shuttle as mainly contributors to mitochondrial respiration. In conclusion, CML cells that acquire TKIs resistance present a severe metabolic reprogramming associated with an increase in metabolic plasticity needed to overcome TKI-induced cell death. Moreover, this study unveils that KU812 Parental and ImaR cells viability can be targeted with metabolic inhibitors paving the way to propose novel and promising therapeutic opportunities to overcome TKI resistance in CML.

High Density SNP Array Analysis of Tyrosine Kinase Inhibitor (TKI) Resistant Chronic Myeloid Leukemia (CML) Shows Secondary Genomic Alterations

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3182-3182
Author(s):  
Daniel Nowak ◽  
Norihiko Kawamata ◽  
Tadayuki Akagi ◽  
Ryoko Okamoto ◽  
Nils Thoennissen ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Snp Array ◽  
High Density ◽  
Array Analysis ◽  
Snp Arrays ◽  
Tki Resistance ◽  
Snp Array Analysis

Abstract Despite the success story of tyrosine kinase inhibitors (TKIs) for the treatment of Chronic Myeloid Leukemia (CML), patients can develop resistances against the drugs. The main known causes for resistance are mutations or over-expression of the BCR/ABL fusion protein, reduced bioavailability of the drugs and activation of compensatory molecular pathways. It is hypothesized that during disease progression, genomic instability of CML cells increases, which may lead to new genomic lesions harboring additional mechanisms of resistance. In this context, we studied genomic DNA profiles of 32 Imatinib resistant CML patients with high density 250K SNP arrays (Affymetrix). Molecular allelokaryotyping for allele specific copy number and loss of heterozygosity analysis was performed with the CNAG software. Single DNA samples from 27 patients were extracted after they had acquired resistance to Imatinib or alternative TKIs such as Nilotinib or Dasatinib. DNA from 12 patients could be analyzed in sequential samples from the initial diagnosis timepoint and a second timepoint upon the emergence of TKI resistance. All patients were positive for BCR/ABL by PCR and FISH. 10 relapse patient samples had known BCR/ABL mutations of which two were T315I mutations. High density allelokaryotyping confirmed pre-existent data on unbalanced translocations, amplifications and deletions from routine cytogenetics: 5 samples displayed a genomic duplication of the BCR/ABL fusion gene, 4 samples had trisomy 8, 1 sample showed deletion of chromosome 17p, 1 sample had heterozygous deletion of chromosome 9. Apart from this, SNP array analysis revealed numerous new submicroscopic genomic lesions. After exclusion of genomic copy number polymorphisms (CNPs) by comparison to recorded CNPs in the UCSC Genome Browser (http://genome.ucsc.edu/) the following results were obtained: Two patients displayed common heterozygous microdeletions of the reciprocal ABL/BCR fusion product. Furthermore, single samples displayed heterozygous micro-deletions on chromosomes 1, 2, 10, 12, 15, 17, and 22 or microduplications on chromosomes 2,3,6, 8, 9, 11, 12, 14, 15, 22. The affected regions contained potentially interesting genes in respect to resistance to therapy such as tumor suppressor candidate MBP-1, apoptosis related protein RERE, metastasis associated gene MTA3, nuclear body associated gene SP100, alpha-T-catenin (CTNNA3), Cbl-interacting protein Sts-1 and the DNA repair associated gene RAD51. As a new genomic alteration in CML, we detected acquired uniparental disomy (UPD) in 5 samples with a common site of UPD on chromosome 19q in 2 patients. In conclusion, in 14 out of 39 TKI resistant cases, high density SNP arrays enabled us to identify submicroscopic copy number lesions and regions of UPD containing promising candidate genes, which merit further research as sites conferring TKI resistance.

Expression of CAMKIIγ, HSP70 and HSP90 Genes Are Differentially Associated with the Mutational Status of Tirosine Kinase Domain in Chronic Myeloid Leukemia Resistant Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4877-4877
Author(s):  
Mariana Selena Gonzalez ◽  
Carlos Daniel De Brasi ◽  
Michele Bianchini ◽  
Cristian Ferri ◽  
Raquel Bengió ◽  
...  
Keyword(s):  
Heat Shock ◽  
Chronic Myeloid Leukemia ◽  
Heat Shock Protein ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Kinase Domain ◽  
Chromosome 9 ◽  
Common Mechanism ◽  
Resistance Mutations ◽  
Tki Resistance

Abstract Abstract 4877 Chronic Myeloid Leukemia (CML) is a clonal myeloproliferative disorder characterized by the reciprocal chromosomal translocation between chromosome 9 and 22, t(9;22)(q34;q11), known as Philadelphia chromosome. At molecular level, this cytogenetic aberration associates with a chimeric BCR-ABL fusion gene responsible for the pathogenesis of the disease. Targeting BCR-ABL with tyrosine kinase inhibitors (TKIs) has led to a rapid clinical response in most CML cases; however, harboring of point mutations within the kinase domain (KD) of BCR-ABL is the most common mechanism responsible for acquired resistance to therapy. The objective of this work was to evaluate the expression profiles of 4 different genes involved in proliferation, differentiation or cell survival, in order to study their association with acquired missense mutations within the KD of BCR-ABL. Due to its clear involvement in cell proliferation and apotosis the following genes were analyzed. CAMKIIγ (Ca2+ /calmodulin dependent protein kinase IIγ) is a critical regulator of multiple signalling networks regulating the proliferation in myeloid leukaemia. HSP70 (Heat Shock protein 70) is a chaperone protein that protects cells from apoptotic and necrotic stimuli. HSP90 (Heat Shock protein 90) is a chaperone that facilitates folding of client proteins such as BCR-ABL. Ki-67 is a nuclear antigen associated with cellular proliferation and ribosomal RNA transcription. Total RNA was extracted from 50 TKI-resistant CML-patient's PBMC samples; by quantitative real time-PCR (QRT-PCR) (SybrGreen method, melting analysis and β2-microglobulin as an endogenous control reference gene) we measured the gene expression of CaMKIIγ, Ki67, HSP70, HSP90 and BCR-ABL. All patients were treated with TKIs (26 imatinib, 13 nilotinib, 11 dasatinib) and showed primary lack or loss of haematological and/or cytogenetic response according to the ELN (European Leukaemia Net) criteria. To characterize TKI-resistant mutations within the KD of the chimeric BCR-ABL gene, ABL exons 4, 5, 6 and 7 were subjected to automated DNA sequencing following a nested- BCR-ABL-specific PCR. Mutations were observed in 27/50 cases at 15 different residues: 1 L248V, 1 G250E, 1 Y253H, 5 E255K/V, 1 E279K, 1 V289F, 4 T315I, 2 F317L, 1 L348M, 3 M351T, 1 E355G, 1 N358S, 2 F359V/C, 2 L387M and 1 L389V. Remaining 23 CML cases did not show mutations (detection limit 10–20%). Our results showed a significant increase in the expression of CaMKIIγ and HSP70 and decrease of HSP90 in mutated patients (MT) with respect to cases without mutations (WT) (p<0.01). On the contrary, transcript levels of Ki67 and BCR-ABL did not show significant differences between MT and WT, likely due to the resistant status of both groups. Taking into account these results we design a score (TKI-MT) to estimate the likelihoods for a patient to harbor TKI-resistance mutations using the transcript normalised expression of CaMKIIγ, HSP70 and HSP90 as follows: [Log10(CaMKIIγ) + Log10(HSP70) – Log10(HSP90)]. TKI-MT scores from 27 and 23 patients from the MT and WT groups respectively were analysed by use of ROC curves in order to find an optimal cut-off value to classify new unstudied cases. We found that patients with TKI-MT scores over a cut-off value of −0.79 showed 4.8 times more probabilities to present TKI-resistance mutations than those below −0.79 (OR 4.8, CI95% 1.3–17.6, P<0.02). We concluded that the expression of CaMKIIγ, HSP70 and HSP90 allowed prediction of mutations in the ABL tyrosine kinase domain with 82% of specificity in CML patients treated with TKIs and associated with lack or loss of response. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Leukemia Stem Cells as a Potential Target to Achieve Therapy-Free Remission in Chronic Myeloid Leukemia

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5822
Author(s):  
Kyoko Ito ◽  
Keisuke Ito
Keyword(s):  
Stem Cells ◽  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Drug Binding ◽  
Leukemia Stem Cells ◽  
Phase Point ◽  
Hematopoietic Stem ◽  
Wide Range ◽  
Tki Resistance

Leukemia stem cells (LSCs, also known as leukemia-initiating cells) not only drive leukemia initiation and progression, but also contribute to drug resistance and/or disease relapse. Therefore, eradication of every last LSC is critical for a patient’s long-term cure. Chronic myeloid leukemia (CML) is a myeloproliferative disorder that arises from multipotent hematopoietic stem and progenitor cells. Tyrosine kinase inhibitors (TKIs) have dramatically improved long-term outcomes and quality of life for patients with CML in the chronic phase. Point mutations of the kinase domain of BCR-ABL1 lead to TKI resistance through a reduction in drug binding, and as a result, several new generations of TKIs have been introduced to the clinic. Some patients develop TKI resistance without known mutations, however, and the presence of LSCs is believed to be at least partially associated with resistance development and CML relapse. We previously proposed targeting quiescent LSCs as a therapeutic approach to CML, and a number of potential strategies for targeting insensitive LSCs have been presented over the last decade. The identification of specific markers distinguishing CML-LSCs from healthy HSCs, and the potential contributions of the bone marrow microenvironment to CML pathogenesis, have also been explored. Nonetheless, 25% of CML patients are still expected to switch TKIs at least once, and various TKI discontinuation studies have shown a wide range in the incidence of molecular relapse (from 30% to 60%). In this review, we revisit the current knowledge regarding the role(s) of LSCs in CML leukemogenesis and response to pharmacological treatment and explore how durable treatment-free remission may be achieved and maintained after discontinuing TKI treatment.

scholarly journals Modeling of Chronic Myeloid Leukemia: An Overview ofIn VivoMurine and Human Xenograft Models

2016 ◽  
Vol 2016 ◽  
pp. 1-12 ◽  
Author(s):  
Pallavi Sontakke ◽  
Jenny Jaques ◽  
Edo Vellenga ◽  
Jan Jacob Schuringa
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Inhibitor ◽  
Therapeutic Approaches ◽  
Retroviral Transduction ◽  
Murine Bone Marrow ◽  
Tki Resistance ◽  
Xenograft Models ◽  
Human Xenograft Models

Over the past years, a wide variety ofin vivomouse models have been generated in order to unravel the molecular pathology of Chronic Myeloid Leukemia (CML) and to develop and improve therapeutic approaches. These models range from (conditional) transgenic models, knock-in models, and murine bone marrow retroviral transduction models followed by transplantation. With the advancement of immunodeficient xenograft models, it has become possible to use human stem/progenitor cells forin vivostudies as well as cells directly derived from CML patients. These models not only mimic CML but also have been instrumental in uncovering various fundamental mechanisms of CML disease progression and tyrosine kinase inhibitor (TKI) resistance. With the availability of iPSC technology, it has become feasible to derive, maintain, and expand CML subclones that are at least genetically identical to those in patients. The following review provides an overview of all murine as well as human xenograft models for CML established till date.

scholarly journals Shikonin Overcomes Drug Resistance and Induces Necroptosis By Regulating the Mir-92a-1-5p/Mlkl Axis in Chronic Myeloid Leukemia Cells

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 1633-1633
Author(s):  
Xianbo Huang ◽  
Jie Jin ◽  
Wenbin Qian ◽  
Xiujin Ye
Keyword(s):  
Cell Death ◽  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Metabolic Reprogramming ◽  
Luciferase Reporter ◽  
Wide Range ◽  
Tki Resistance ◽  
Poor Outcomes

BACKGROUND: Resistance to cell death and metabolic reprogramming are common features of tumor cells. Although the introduction of selective BCR/ABL tyrosine kinase inhibitors (TKIs) has dramatically improved the outcomes and survival rates of chronic myeloid leukemia (CML) patients, some patients (20-30%) develop TKI resistance. The most aggressive and treatment-resistant CML is the subtype harboring BCR/ABL with the T315I mutation, and this subtype is refractory to nearly all TKI-induced apoptosis. Thus, alternative approaches that induce apoptosis-independent cell death are thought to compensate for apoptotic-resistant cells. Recently, necroptosis (also called programmed necrosis), which is generally driven by RIPK1/RIPK3/MLKL activation, has been demonstrated to be a new type of programmed cell death mode that is different from apoptosis. Thus, a deeper understanding of the molecular mechanisms regulating necroptosis might lead to the development of new therapeutic strategies that could remarkably improve the treatment-responses and outcomes of patients with TKI-resistant CML. RESULTS: Shikonin, a compound purified from traditional Chinese medicine, has been reported to induce cell death in various tumor cell lines via a wide range of mechanisms. In our current study, we found that shikonin can effectively inhibit proliferation and induce necrosis-like morphological alterations (Fig. A and B) accompanied by RIPK1/RIPK3/MLKL signaling activation ((Fig. C) in CML cell lines, including the T315I mutant type (32Dp210-T315I). The effects of shikonin can be attenuated by the necroptosis-specific inhibitor (essentially a RIPK1 inhibitor) Nec-1, but not by the pan-apoptosis inhibitor z-VAD-fmk, indicating the occurrence of necroptosis in these cells ((Fig. B and C). Our data also show that shikonin has in vivo anti-CML activity via necroptosis induction in 32Dp210-T315I cells xenografted into NOD/SCID mice via subcutaneous injection ((Fig. D). miRNAs play an important role in tumorigenesis mainly via regulation of gene expression. Our next generation sequencing-based microRNA expression profiling showed significant dysregulation of miR-92a-1-5p expression in a shikonin-treated CML cell line (K562) (Fig. E). We then measured the miR-92a-1-5p expression levels in bone marrow samples from CML patients and patients with nonhematologic malignant diseases. The data showed that the miR-92a-1-5p expression level was higher in primary cells obtained from CML-BC patients than in those from non-CML-CP patients, suggesting that miR-92a-1-5p upregulation is correlated with poor outcomes (Fig. F). Bioinformatics analyses and a dual luciferase reporter gene assay proved that MLKL, a downstream factor in the necroptosis pathway that usually acts as the necroptosis executor, is a direct target of miR-92a-1-5p (Fig. G). Overexpression of miR-92a-1-5p in vitro led to decreased MLKL protein abundance in CML cells (Fig. G). Inhibition of miR-92a-1-5p via use of a specific antago-miRNA could inhibit CML xenograft tumor growth and induce necroptosis via MLKL upregulation in vivo (Fig. H). Hence, we believe that miR-92a-1-5p plays a role in promoting the proliferation and survival of CML via downregulating the abundance of MLKL, the necroptosis executor. CONCLUSIONS: In conclusion, our study proves that shikonin can overcome TKI resistance and induce necroptosis in CML cells, mainly via a mechanism involving RIPK1/RIPK3/MLKL activation. Our study also suggests that miR-92a-1-5p is frequently overexpressed in CML patients with poor outcomes and that it can promote tumor survival by inhibiting MLKL expression. For the first time, we demonstrated that miR-92a-1-5p inhibition via antago-miRNA can potentially be used to treat CML via necroptosis induction. Since necroptosis has not yet been considered to be a therapeutic strategy for tumor treatment, our research confirms that it might indeed serve as a new modality to better control drug-resistant CML. Figure Disclosures No relevant conflicts of interest to declare.

scholarly journals CRISPR/Cas9-mediated microRNA-21 Cleavage Sensitizes the Treatment Response to Dasatinib in Chronic Myeloid Leukemia Cell Line K562

2020 ◽  
Author(s):  
Yun Zhang ◽  
Lingyan Wang ◽  
Jiazheng Li ◽  
Peifang Jiang ◽  
Minhui Lin ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Gene Editing ◽  
Molecular Mechanisms ◽  
Target Genes ◽  
Leukemia Cell ◽  
K562 Cells ◽  
Leukemia Cell Line ◽  
Number Of Patients ◽  
Tki Resistance

Abstract Background: Tyrosine kinase inhibitors (TKIs) targeting BCR-ABL1 kinase are effective in treating chronic myeloid leukemia (CML), but TKI resistance occurs in a significant number of patients, and the underlying molecular mechanisms of this resistance remain largely unknown. As an oncomiR, microRNA-21(miR-21) functions directly in drug resistance, but its relationship with TKI resistance in CML is rarely reported. As a novel and effective gene editing tool, clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9(CRISPR/Cas9) has certain advantages in completely knocking out target genes at the gene level. Methods: We successfully constructed lentiviruses LV-VMP1-sgRNA (045001) and human chronic myeloid leukemia K562 cells were transducted with them. Single-cell-derived clones were screened for miR-21 deletion by genomic DNA PCR and Sanger sequence. RQ-PCR assays was used to confirm the knockout of miR-21. 3-(4,5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide(MTT) and colony formation assays were applied to assess the cell growth inhibition. Imatinib and dasatinib sensitivity was determined by MTT assay and Annexin-V APC/7-AAD double staining flow cytometry. Western blot assay was performed to measure the levels of Phosphatase and tensin homolog(PTEN), Phosphatidylinositol 3-kinase(PI3K), Serine/threonine Kinase(AKT), p-AKT, BCR-ABL(P210), p-BCR-ABL (p-P210) . Result: miR-21 knockout inhibited proliferation of K562 cells, promoted their apoptosis and increased their sensitivity to dasatinib. Further mechanism studies suggest that this is achieved by inhibiting the PI3K/AKT signaling pathway and destroying BCR-ABL. Conclusions: Our study reveals the efficacy of CRISPR/Cas9 gene editing to miR-21 in K562 and indicates miR-21 as a potential target in sensitizing dasatinib treatment for CML patients with pool response to the TKI targeting therapy.

scholarly journals Resistance to Tyrosine Kinase Inhibitors in Chronic Myeloid Leukemia—From Molecular Mechanisms to Clinical Relevance

Cancers ◽  
2021 ◽  
Vol 13 (19) ◽  
pp. 4820
Author(s):  
Raquel Alves ◽  
Ana Cristina Gonçalves ◽  
Sergio Rutella ◽  
António M. Almeida ◽  
Javier De Las De Las Rivas ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Tyrosine Kinase ◽  
Myeloid Leukemia ◽  
Molecular Mechanisms ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
De Novo ◽  
Fusion Gene ◽  
Computational Prediction ◽  
Tki Resistance

Resistance to targeted therapies is a complex and multifactorial process that culminates in the selection of a cancer clone with the ability to evade treatment. Chronic myeloid leukemia (CML) was the first malignancy recognized to be associated with a genetic alteration, the t(9;22)(q34;q11). This translocation originates the BCR-ABL1 fusion gene, encoding the cytoplasmic chimeric BCR-ABL1 protein that displays an abnormally high tyrosine kinase activity. Although the vast majority of patients with CML respond to Imatinib, a tyrosine kinase inhibitor (TKI), resistance might occur either de novo or during treatment. In CML, the TKI resistance mechanisms are usually subdivided into BCR-ABL1-dependent and independent mechanisms. Furthermore, patients’ compliance/adherence to therapy is critical to CML management. Techniques with enhanced sensitivity like NGS and dPCR, the use of artificial intelligence (AI) techniques, and the development of mathematical modeling and computational prediction methods could reveal the underlying mechanisms of drug resistance and facilitate the design of more effective treatment strategies for improving drug efficacy in CML patients. Here we review the molecular mechanisms and other factors involved in resistance to TKIs in CML and the new methodologies to access these mechanisms, and the therapeutic approaches to circumvent TKI resistance.

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

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4233-4233 ◽  
Author(s):  
Matthieu Lewis ◽  
Valerie Prouzet-Mauleon ◽  
Elodie Richard ◽  
Beatrice Turcq ◽  
Richard Iggo ◽  
...  
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Cell Lines ◽  
Myeloid Leukemia ◽  
Kinase Inhibitors ◽  
Knock Out ◽  
Individual Gene ◽  
Oncogenic Pathways ◽  
A Genome ◽  
Tki Resistance ◽  
Genome Scale

Abstract 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. Disclosures Mahon: ARIAD: Honoraria; PFIZER: Honoraria; BMS: Consultancy, Honoraria; NOVARTIS PHARMA: Consultancy, Honoraria, Research Funding.

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