Imatinib-resistant Chronic Myelogenous Leukemia Research Articles

Unveiling IL6R and MYC as Targeting Biomarkers in Imatinib-Resistant Chronic Myeloid Leukemia through Advanced Non-Invasive Apoptosis Detection Sensor Version 2 Detection.

The management of chronic myelogenous leukemia (CML) has seen significant progress with the introduction of tyrosine kinase inhibitors (TKIs), particularly Imatinib. However, a notable proportion of CML patients develop resistance to Imatinib, often due to the persistence of leukemia stem cells and resistance mechanisms independent of BCR::ABL1 This study investigates the roles of IL6R, IL7R, and MYC in Imatinib resistance by employing CRISPR/Cas9 for gene editing and the Non-Invasive Apoptosis Detection Sensor version 2 (NIADS v2) for apoptosis assessment. The results indicate that Imatinib-resistant K562 cells (K562-IR) predominantly express IL6R, IL7R, and MYC, with IL6R and MYC playing crucial roles in cell survival and sensitivity to Imatinib. Conversely, IL7R does not significantly impact cytotoxicity, either alone or in combination with Imatinib. Further genetic editing experiments confirm the protective functions of IL6R and MYC in K562-IR cells, suggesting their potential as therapeutic targets for overcoming Imatinib resistance in CML. This study contributes to understanding the mechanisms of Imatinib resistance in CML, proposing IL6R and MYC as pivotal targets for therapeutic strategies. Moreover, the utilization of NIADS v2 enhances our capability to analyze apoptosis and drug responses, contributing to a deeper understanding of CML pathogenesis and treatment options.

Hydroxychavicol sensitizes imatinib-resistant chronic myelogenous leukemia cells to TRAIL-induced apoptosis by ROS-mediated IAP downregulation: Erratum.

Hydroxychavicol sensitizes imatinib-resistant chronic myelogenous leukemia cells to TRAIL-induced apoptosis by ROS-mediated IAP downregulation: Erratum.

NDRG3 regulates imatinib resistance by promoting β‑catenin accumulation in the nucleus in chronic myelogenous leukemia.

Imatinib resistance in chronic myelogenous leukemia (CML) is a clinical problem. The present study examined the role of N‑Myc downstream regulatory gene3 (NDRG3) in imatinib resistance in CML. Quantitative PCR demonstrated that NDRG3 was highly expressed in patients with CML. Cell Counting Kit (CCK)‑8 experiments proved that NDRG3 promoted the proliferation of K562 CML cells and enhanced imatinib resistance. Dual‑luciferase assay showed that microRNA (miR)‑204‑5p inhibited expression of NDRG3 and immunofluorescence experiments showed that NDRG3 promoted accumulation of β‑catenin in the nucleus, thereby increasing the expression of downstream drug resistance‑ and cell cycle‑associated factors (c‑Myc and MDR1). At the same time, cell proliferation experiments showed that β‑catenin played a role in cell proliferation and drug resistance. Co‑transfection with small interfering (si)‑β‑catenin partially reversed the effect of NDRG3. This finding indicated that NDRG3 plays an important role in imatinib resistance and miR‑204‑5p and β‑catenin are involved in the biological behavior of NDRG3. The present results provide theoretical support for overcoming drug resistance in CML.

BCR-ABL triggers a glucose-dependent survival program during leukemogenesis through the suppression of TXNIP

sImatinib is highly effective in the treatment of chronic myelogenous leukemia (CML), but the primary and acquired imatinib resistance remains the big hurdle. Molecular mechanisms for CML resistance to tyrosine kinase inhibitors, beyond point mutations in BCR-ABL kinase domain, still need to be addressed. Here, we demonstrated that thioredoxin-interacting protein (TXNIP) is a novel BCR-ABL target gene. Suppression of TXNIP was responsible for BCR-ABL triggered glucose metabolic reprogramming and mitochondrial homeostasis. Mechanistically, Miz-1/P300 complex transactivates TXNIP through the recognition of TXNIP core promoter region, responding to the c-Myc suppression by either imatinib or BCR-ABL knockdown. TXNIP restoration sensitizes CML cells to imatinib treatment and compromises imatinib resistant CML cell survival, predominantly through the blockage of both glycolysis and glucose oxidation which results in the mitochondrial dysfunction and ATP production. In particular, TXNIP suppresses expressions of the key glycolytic enzyme, hexokinase 2 (HK2), and lactate dehydrogenase A (LDHA), potentially through Fbw7-dependent c-Myc degradation. In accordance, BCR-ABL suppression of TXNIP provided a novel survival pathway for the transformation of mouse bone marrow cells. Knockout of TXNIP accelerated BCR-ABL transformation, whereas TXNIP overexpression suppressed this transformation. Combination of drug inducing TXNIP expression with imatinib synergistically kills CML cells from patients and further extends the survival of CML mice. Thus, the activation of TXNIP represents an effective strategy for CML treatment to overcome resistance.

Long-term durability of improved glycemic control in type 2 diabetes mellitus treated with dasatinib, a second-generation tyrosine kinase inhibitor, for chronic myeloid leukemia: A report of two cases

Dasatinib, a second-generation tyrosine kinas inhibitoris effective against most cases of imatinib-resistant Chronic Myelogenous Leukemia (CML), and this drug has also shown significant glucose-lowering effects. Here, we report two diabetes patients with sudden onset of CML. In Case 1, dasatinib produced mild but clear improvement of hyperglycemia with the same anti-diabetic regimen together with rapid control of CML. In Case 2, initiation of dasatinib resulted inmarked potentiation of insulin-induced glucose-lowering effects in parallel with rapid improvements of CML. In both cases, this favorable diabetes control was well maintained for 5 years under leukemia management with dasatinib

An Unexpected Finding on Screening Colonoscopy in a Patient With Chronic Myelogenous Leukemia

Question: A 64-year-old woman presented for screening colonoscopy. Her only medical history included chronic myelogenous leukemia (CML) diagnosed 1 year prior that was improving and in the chronic phase with dasatinib 50 mg/d. She denied taking any other medications. The patient denied having any prior colonoscopies or any family history of colorectal cancer. She also denied any melena or hematochezia. She did report weight loss, which she attributed to her CML. On colonoscopy, multiple small polyps were seen throughout the colon ranging from 3 to 6 mm that were too numerous to count (Figure A). The polyps were friable and seemed to have mucous caps. A few of these polyps were removed from the transverse and sigmoid colon for pathological analysis (Figure B and C). What is the diagnosis? Look on page 58 for the answer and see the Gastroenterology web site (www.gastrojournal.org) for more information on submitting your favorite image to Clinical Challenges and Images in GI. Inflammatory reactive polyposis caused by dasatinib is a rare phenomenon and has only been described once in the Japanese literature.1Kobayashi M. Ikezawa K. Yamaura M. et al.[Inflammatory reactive polyposis caused by dasatinib: a case report].Nihon Shokakibyo Gakkai Zasshi. 2018; 115 ([In Japanese]): 977-984PubMed Google Scholar Our patient had polyps with mucoid discharge similar in appearance to those described in the Japanese paper. It was determined that the patient’s polyposis was caused by inflammation that was triggered by dasatinib. Four months after discontinuing dasatinib in the Japanese patient, a repeat colonoscopy revealed significant improvement in the polypoid lesions. In the case of our patient, we did not advise discontinuing dasatinib because she was on a low dose and did not tolerate other therapies for CML. The patient remained asymptomatic on follow-up. To our knowledge, this is the first case of dasatinib-induced inflammatory polyposis described in the English literature. Cases of hemorrhagic colitis secondary to dasatinib already exist. With dasatinib becoming a widely used therapy for Imatinib resistant CML, it is prudent to monitor for complications. Dasatinib is taken orally, making the gastrointestinal tract particularly vulnerable to complications. It is important to be aware of the inflammatory effects of dasatinib and this warrants further laboratory and clinical studies.2Nakamura S. Kino I. Akagi T. Inflammatory myoglandular polyps of the colon and rectum. A clinicopathological study of 32 pedunculated polyps, distinct from other types of polyps.Am J Surg Pathol. 1992; 16: 772-779Crossref PubMed Scopus (63) Google Scholar It is unknown if prolonged polyposis owing to dasatinib is a benign process or may eventually result in malignancy3Fei F. Yu Y. Schmitt A. et al.Dasatinib inhibits the proliferation and function of CD4+CD25+ regulatory T cells.Br J Haematol. 2009; 144: 195-205Crossref PubMed Scopus (60) Google Scholar The mechanism of polyposis has also not yet been elucidated and it is necessary to accumulate more cases to further our understanding. Therefore, it is important to offer screening colonoscopies to these patients at regular intervals.

Dynein axonemal heavy chain 9 M4374I variation may have an effect on imatinib mesylate resistance in CML

Chronic myelogenous leukemia (CML) is a myeloproliferative neoplasm caused by a translocation between the breakpoint cluster region (BCR) and Abelson murine leukemia 1 (ABL1) genes. Tyrosine kinase inhibitors (TKIs) are used in the treatment of CML. TKIs, bind the ABL1 kinase domain of hybrid BCR‑ABL1 protein and inhibit its function. However, resistance can occur due to the pathogenic variations in the ABL kinase domain or BCR‑ABL1‑independent mechanisms. In the present study, genetic variations possibly related to imatinib resistance in CML were explored. A total of five single nucleotide polymorphisms [SNPs; MORN2 rs3099950, PTCRA rs9471966, ANKRD35 rs11579366, dynein axonemal heavy chain 9 (DNAH9) rs1990236 and MAGEC1 rs176037] were investigated in imatinib sensitive and in resistant CML patients. Additionally, sequencing of the ABL1 kinase domain was also performed. The frequency of DNAH9 M4374I (NP_001363.2)/M686I (NP_004653.2) (rs1990236) was found to be significantly higher in the imatinib‑resistant group. However, the other SNPs did not exhibit any statistically significant differences and no new variant was detected in the ABL1 kinase domain. Considering the frequency difference of the DNAH9 rs1990236 between imatinib‑sensitive and imatinib‑resistant groups, DNAH9 gene may play a role in TKI resistance. Due to the limited amounts of literature available on this subject, further studies on DNAH9 and related genes may prove to be beneficial for the elucidation of the association between DNAH9 and TKI resistance. Moreover, further larger studies are required to support the current findings. This may aid in the development of novel treatment protocols for patients with CML with DNAH9 genetic polymorphisms.

JKST6, a novel multikinase modulator of the BCR-ABL1/STAT5 signaling pathway that potentiates direct BCR-ABL1 inhibition and overcomes imatinib resistance in chronic myelogenous leukemia

Chronic myelogenous leukemia (CML) is a hematological malignancy that highly depends on the BCR-ABL1/STAT5 signaling pathway for cell survival. First-line treatments for CML consist of tyrosine kinase inhibitors that efficiently target BCR-ABL1 activity. However, drug resistance and intolerance are still therapeutic limitations in Ph+ cells. Therefore, the development of new anti-CML drugs that exhibit alternative mechanisms to overcome these limitations is a desirable goal. In this work, the antitumoral activity of JKST6, a naphthoquinone-pyrone hybrid, was assessed in imatinib-sensitive and imatinib-resistant human CML cells. Live-cell imaging analysis revealed JKST6 potent antiproliferative activity in 2D and 3D CML cultures. JKST6 provoked cell increase in the subG1 phase along with a reduction in the G0/G1 phase and altered the expression of key proteins involved in the control of mitosis and DNA damage. Rapid increases in Annexin V staining and activation/cleavage of caspases 8, 9 and 3 were observed after JKST6 treatment in CML cells. Of interest, JKST6 inhibited BCR-ABL1/STAT5 signaling through oncokinase downregulation that was preceded by rapid polyubiquitination. In addition, JKST6 caused a transient increase in JNK and AKT phosphorylation, whereas the phosphorylation of P38-MAPK and Src was reduced. Combinatory treatment unveiled synergistic effects between imatinib and JKST6. Notably, JKST6 maintained its antitumor efficacy in BCR-ABL1-T315I-positive cells and CML cells that overexpress BCR-ABL and even restored imatinib efficacy after a short exposure time. These findings, together with the observed low toxicity of JKST6, reveal a novel multikinase modulator that might overcome the limitations of BCR-ABL1 inhibitors in CML therapy.

The Novel H19/miR‐675‐5p/LNC000093‐Mediated Non‐Coding RNA Pathway Regulates Treatment Response in BCR‐ABL1‐Positive CML Cells

Introduction In recent years, considerable research has been done on the mechanisms of long non-coding RNAs (lncRNAs) modulating various cellular processes, especially in pathological condition such as tumorigenesis. Currently, however, little is known about the functional characteristics of these non-coding elements in the genome. In the present study, we aim to explore any mechanisms that involve novel lncRNA and microRNA (miRNA) axes participating in drug response of chronic myelogenous leukemia (CML). Methods Novel lncRNAs were identified via mRNA sequencing (RNA-seq) in CML cell lines with or without imatinib (IM) resistance. The sequence of a selected novel lncRNA candidate named LNC000093 was confirmed by cycle sequencing in several cell types. The expression level of LNC000093 in different cell lines and human samples was detected by RT-qPCR. In silico analysis and luciferase reporter assays were performed to identify miRNA binding regions on LNC000093. Knockdown and overexpression of H19/miR-675 were performed to examine their regulation on LNC000093 and cell viability upon IM treatment in CML cells. The potential interaction of H19/miR-675 and LNC000093 with RUNX1 was also investigated to study their interrelationship in IM-resistant of CML. Results A significant downregulation of newly identified LNC000093 was found in IM-resistant CML cells while an inverse expression pattern was observed in blood cells from IM-sensitive CML cases. Conversely, H19 and its derived miR-675 were upregulated in IM-resistant CML cells. Deletion of H19 led to upregulation of LNC000093 while miR-675-5p overexpression but not miR-675-3p reversed the elevated expression of LNC000093. In silico analysis revealed LNC000093 may contain three putative binding regions for miR-675-5p close to the 3’ end of its transcript. Subsequent luciferase reporter assays showed the interaction between LNC000093 and miR-675-5p via direct binding through the seed sequence GGUGCG on miR-675-5p. CRISPR-Cas9-mediated deletion of H19 caused downregulation of miR-675 and facilitated CML cell death upon IM treatment especially in IM-resistant cells, while overexpression of miR-675-5p reversed the enhancement of cell death in H19-deleted cells. Western blot assay revealed a downregulation of RUNX1 expression in IM-resistant cells. Overexpression of miR-675-5p could also result in RUNX1 downregulation and this was rescued by co-expression of LNC000093. Ectopic expression of LNC000093 could further increase RUNX1 expression and enhance cell death of IM-resistant cells. Conclusions Increased H19/miR-675-5p levels could contribute to IM resistance in CML cells through regulation of RUNX1 expression, and the newly identified lncRNA LNC000093 may serve as a ceRNA for miR-675-5p. This finding supports a possible linkage between the LNC000093-H19/miR-675-RUNX1 axis and IM resistance in CML.

Screening and identification of key genes in imatinib-resistant chronic myelogenous leukemia cells: a bioinformatics study

ABSTRACT Background Chronic myelogenous leukemia (CML) is one of the most common cancers in the world. Imatinib is one of the most effective therapeutic strategies to inhibit the BCR-ABL tyrosine Kinase in patients with CML, but resistance is increasingly encountered. Material and Methods Microarray data GSE7114, GSE92624 and GSE97562 were downloaded and analyzed from Gene Expression Omnibus (GEO) to identify the candidate genes in the imatinib-resistant CML cells. The differentially expressed genes (DEGs) were appraised, and the protein–protein interaction (PPI) network was created by using STRING and Cytoscape. Results We screened a total of 217 DEGs, including 151 upregulated genes and 66 downregulated genes. The enriched functions and pathways of genes include insulin-like growth factor I binding, cysteine-type endopeptidase inhibitor activity involved in apoptotic process, cell adhesion, positive regulation of nitric oxide biosynthetic process and hematopoietic cell lineage. Nine hub genes were appraised and Gene Ontology enrichment analysis revealed that these genes are mainly enriched in cell cycle, peptidase inhibitor activity and cell division. Several genes such as BIRC5, CCNE2 and MCM4 were identified in survival analysis and these genes alteration are significantly associated with worse overall survival and disease-free survival. Conclusions These genes have the potential to become surrogate markers for a clinical evaluation of imatinib-resistant CML patients. Our results provide potential target genes for diagnosis and treatment of imatinib-resistant CML patients.

Identification and development of non-cytotoxic cell death modulators: Impact of sartans and derivatives on PPARγ activation and on growth of imatinib-resistant chronic myelogenous leukemia cells

4’-((2-Propyl-1H-benzo[d]imidazol-1-yl)methyl)-[1,1′-biphenyl]-2-carboxylic acid derived from telmisartan was identified as lead for the design of cell death modulators. In this study, we evaluated the efficacy of telmisartan itself and other sartans in combination with imatinib against K562-resistant cells. The findings were directly used to further optimize the lead structure. Telmisartan and candesartan cilexetil represented the most effective sartans, thus the influence of carboxyl/methyl carboxylate groups at positions 7 (compounds 6, 7) or 4 (compounds 12–14) at the benzimidazole core was studied. Additionally, according to the results of a former structure-activity study, telmisartan was transformed to the related amide (1). Telmisartan amide 1, as well as the esters 6 and 12 markedly sensitized the resistant CML cells to imatinib treatment. Correlation with their potency to activate PPARγ is not given. Candesartan cilexetil, telmisartan and 1 showed the profile of partial agonists at PPARγ with EC50 values of 4.2, 4.3 and 9.1 μM, respectively, while 6 and 12 caused only marginal intrinsic activation at 10 μM (Amax = 22% and 13%). However, the repression of the STAT5 phosphorylation relates with the possibility to sensitize K562-resistant CML cells to imatinib treatment. It is worth mentioning that all compounds were per se non-cytotoxic at relevant concentrations.

Homoharringtonine promotes BCR‑ABL degradation through the p62‑mediated autophagy pathway.

Drug resistance to tyrosine kinase inhibitors (TKIs) is currently a clinical problem in patients with chronic myelogenous leukemia (CML). Homoharringtonine (HHT) is an approved treatment for adult patients with chronic- or accelerated-phase CML who are resistant to TKIs and other therapies; however, the underlying mechanisms remain unclear. In the present study, HHT treatment demonstrated induction of apoptosis in imatinib-resistant K562G cells by using MTS assay and western blotting, and BCR-ABL protein was reduced. CHX chase assay revealed that HHT induced degradation of the BCR-ABL protein, which could be reversed by autophagy lysosome inhibitors Baf-A1 and CQ. Next, HHT treatment confirmed the induction of autophagy in K562G cells, and silencing the key autophagic proteins ATG5 and Beclin-1 inhibited the degradation of the BCR-ABL protein and cytotoxicity. In addition, autophagic receptor p62/SQSTM1(p62) participated during the autophagic degradation of BCR-ABL induced by HHT, and this was confirmed by co-immunoprecipitation, in which HHT enhanced the ubiquitination of the BCR-ABL protein and promoted its binding to p62. In conclusion, HHT induced p62-mediated autophagy in imatinib-resistant CML K562G cells, thus promoting autophagic degradation of the BCR-ABL protein and providing a novel strategy for the treatment of TKI-resistant CML.

Abstract 1327: UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia

Abstract Purpose: Long-term treatment of chronic myelogenous leukemia (CML) with the Bcr-Abl inhibitor imatinib, is initially successful but gives rise to several drug-resistant mutations. The most prevalent of these is the T315I ‘gatekeeper’ mutation that is sensitive to the second-line therapy, ponatinib. However, ponatinib causes serious vascular adverse events, including fatalities, in over 25% of patients, in part due to its target promiscuity. We have designed a novel Bcr-Abl inhibitor (UM-9107) that shows significant in vitro and in vivo anti-CML activity against wild-type and drug-resistant mutants, including the gatekeeper mutant. Furthermore, UM-9107 has excellent selectivity across the kinome and in vitro surrogate toxicity assessment models demonstrate a low likelihood of vascular adverse events as compared to ponatinib. Experimental Design: We designed a series of compounds to specifically interact with a specific kinked conformation of the phosphate-binding loop of Abl. Since only a small proportion of kinases display this kinked conformation, we hypothesized that our compounds would be selective for Abl. We have analyzed the compound series biochemically and in vitro. The most potent compound, UM-9107, has been comprehensively characterized including: biochemical inhibition, cellular target engagement, kinome-wide profiling, in vitro cell proliferation, pharmacokinetic studies, and in vivo activity in xenograft models of CML. Results: We demonstrate that UM-9107 potently inhibits all clinical mutants of Bcr-Abl, including the T315I gatekeeper mutant. UM-9107 shows robust activity against CML cell lines, comparable to ponatinib, with significantly fewer off-targets, as determined by kinome-wide profiling. Phenotypic profiling of UM-9107 in human primary cells shows low risk of vascular adverse events as indicated by specific biomarker readouts. UM-9107 demonstrates good oral bioavailability and in vivo activity in xenograft models of CML, with low toxicity. Conclusions: The success of imatinib has been revolutionary and has led to an increase in the number of patients on long-term therapy. This inevitably gives rise to drug resistance through point mutations in the Bcr-Abl protein. Our results suggest that UM-9107 is a safe and efficacious preclinical candidate for the treatment of imatinib-resistant CML. Citation Format: Sameer Phadke, Lluis Lopez-Barcons, Taylor K. Johnson, Eric J. Lachacz, Sofia D. Merajver, Matthew B. Soellner. UM-9107: A selective wild-type and T315I Bcr-Abl inhibitor with in vivo activity against chronic myelogenous leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1327.

Andrographolide and its potent derivative exhibit anticancer effects against imatinib-resistant chronic myeloid leukemia cells by downregulating the Bcr-Abl oncoprotein

Chronic myelogenous leukemia (CML) is clinically treated with imatinib, which inhibits the kinase activity of the Bcr-Abl oncoprotein. However, imatinib resistance remains a common clinical issue. Andrographolide, the major compound of the medicinal plant Andrographis paniculata, was reported to exhibit anticancer activity. In this study, we explored the therapeutic potential of andrographolide and its derivative, NCTU-322, against both imatinib-sensitive and imatinib-resistant human CML cell lines. Both andrographolide and NCTU-322 downregulated the Bcr-Abl oncoprotein in imatinib-resistant CML cells through an Hsp90-dependent mechanism similar to that observed in imatinib-sensitive CML cells. In addition, NCTU-322 had stronger effects than andrographolide on downregulation of Bcr-Abl oncoprotein, induction of Hsp90 cleavage and cytotoxicity of CML cells. Notably, andrographolide and NCTU-322 could induce differentiation, mitotic arrest and apoptosis of both imatinib-sensitive and imatinib-resistant CML cells. Finally, the anticancer activity of NCTU-322 against imatinib-resistant CML cells was demonstrated in vivo. In summary, our data demonstrated that andrographolide and NCTU-322 inhibit Bcr-abl function via a mechanism different from that of imatinib, and they induced multiple anticancer effects in both imatinib-sensitive and resistant CML cells. Our findings demonstrate that andrographolide and NCTU-322 are potential therapeutic agents again CML.

Hydroxychavicol sensitizes imatinib-resistant chronic myelogenous leukemia cells to TRAIL-induced apoptosis by ROS-mediated IAP downregulation.

The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of cytokine superfamily, induces apoptosis in a number of tumor cells through the activation of extrinsic apoptotic pathway but shows little or no cytotoxicity toward normal cells. However some tumor cells are inherently resistant to TRAIL-mediated apoptosis, which needs to be addressed to establish TRAIL as a potential chemotherapeutic drug. In this study, our aim was to manipulate TRAIL-apoptosis pathway by hydroxychavicol (HCH), a polyphenol from Piper betel leaf, for the induction of apoptosis in TRAIL resistant chronic myeloid leukemia cell. When imatinib-resistant K562 cells were treated with HCH, it made these K562 cells sensitive to TRAIL. It was observed that HCH downregulated antiapoptotic proteins XIAP and FLIP, whereas the expression of TRAIL receptors, DR4 and DR5, remains unchanged. Moreover, we observed that reactive oxygen species or ROS played a crucial role in the downregulation of FLIP and XIAP because ROS scavenger significantly reversed the decrease of XIAP, and FLIP. Ubiquitin-proteasome pathway was observed to play a crucial role in the downregulation of XIAP and FLIP, as proteasomal inhibitor MG132 significantly reversed the downregulation of XIAP and FLIP. In conclusion, this study demonstrates the combinatorial treatment of TRAIL and HCH as promising alternative therapeutic approach to treat the imatinib-resistant leukemia, which are also resistant to TRAIL.

Fluorescence In situ hybridization signal patterns and intrachromosomal breakpoint cluster region-abelson murine leukemia viral oncogene homolog 1 amplification analysis in imatinib-resistant chronic myelogenous leukemia patients using tricolor dual fusion probe

BACKGROUND: Cytogenetic evaluation is required till a complete cytogenetic remission is achieved in chronic myelogenous leukemia (CML) patients on tyrosine kinase inhibitor (TKI) therapy. The routine dual colour fluorescence in situ hybridization (FISH) probes are less sensitive in identifying der(9) abnormalities. BCR/ABL/ASS1 tri-colour dual fusion (TCDF) probe is highly sensitive and specific in identifying der(9) deletions and random signal overlaps. METHODS: Peripheral blood interphase FISH analysis was performed on imatinib-resistant CML patients using TCDF probe. RESULTS: On analyzing 37 adult patients, all had residual Philadelphia (Ph) chromosome. Classic Ph fusion pattern was seen in 33 (89%), derivative chromosome 9 [der(9)] abnormalities in 25 (67.5%) and supernumerary Ph chromosomes in 11 (30%) patients. Coexistence of classical fusion and der(9) abnormalities was seen in 21 patients (57%); and classical fusion, der(9) abnormalities and supernumerary Ph chromosome in 8 patients (22%). None of the patients had BCR-ABL1 gene amplification. There was significant difference in the der(9) abnormal cell percentages between patients with e13a2 and e14a2 transcripts (P = 0.008) and patients with disease transformation (P = 0.007). CONCLUSION: A high frequency of der(9) abnormalities and absence of BCR-ABL1 gene amplification was seen in imatinib-resistant CML patients analyzed. The use of TCDF probe for cytogenetic follow-up in CML patients was found to be useful in identifying BCR-ABL1 related aberrations. The identified patterns in this study, can serve as a reference material for I-FISH signal interpretation using TCDF probe.

Lncrna RP1-68D18.2 Promotes K562 Cell Line Resistance to Imatinib through CD44 and Its Downstream Signal Molecules

As the first-generation tyrosine kinase inhibitor specifically targeting the BCR-ABL oncoprotein, imatinib has been successfully used in the therapy for chronic myelogenous leukemia (CML) with a significant response and good compliance. However, imatinib resistance becomes the main reason for failure of the treatment of CML. In addition to the common BCR-ABL dependence of imatinib resistance, e.g. BCR-ABL kinase domain mutations and BCR-ABL gene amplification, BCR-ABL independence also plays important roles, which involves alteration of dynamics of drug import and efflux, persistent or compensatory activation of signaling pathways etc. Recently, several studies have reported the correlation of lncRNA with resistance of chemotherapeutic drugs in many cancers. Here, we report, for the first time, discernible overexpression of lncRNARP1-68D18.2 and CD44 mRNA in the imatinib resistant chronic myelogenous leukemia cell line K562R, using lncRNA and mRNA microarray, and further verify it using qPCR. Knockdown of RP1-68D18.2 in K562R cell lines increased their sensitivity to imatinib, and simultaneously caused a decrease in CD44 and c-Myc protein levels. Correspondingly, overexpression of RP1-68D18.2 in K562, a sensitive cell line, led to a slightly decrease in imatinib sensitivity and partially reverse downregulation of c-Myc, p-ERK and p-MEK caused by imatinib treatment. Analysis of correlations revealed positive correlation between RP1-68D18.2 and CD44 mRNA expression in the primary CML blasts and hematological malignant cell lines. We conclude that overexpression of RP1-68D18.2 could promote imatinib resistance in the K562R cell line through the regulation of CD44 and its downstream signal molecules. DisclosuresNo relevant conflicts of interest to declare.

ZNF224 is a transcriptional repressor of AXL in chronic myeloid leukemia cells

ZNF224 is a KRAB-zinc finger transcription factor that exerts a key tumor suppressive role in chronic myelogenous leukemia.In this study, we identify the receptor tyrosine kinase Axl as a novel target of ZNF224 transcriptional repression activity. Axl overexpression is found in many types of cancer and is frequently associated with drug resistance. Interestingly, we also found that sensitivity to imatinib can be partly restored in imatinib-resistant chronic myelogenous leukemia cells by ZNF224 overexpression and the resulting suppression of Axl expression.These results, in accordance with our previous findings, support the role of ZNF224 in imatinib responsiveness and shed new insights into potential therapeutic use of ZNF224 in imatinib-resistant chronic myelogenous leukemia.

Abstract B17: Activating AMPK signaling by SCT-1015 to suppress tumor growth on triple negative breast cancer cells

Abstract AMPK (AMP-activated protein kinase) is one of the central regulators of cellular and organismal metabolism that has been linked to the regulation of tumor progression; however, its biologic functions and signaling cascades remain unclear. Recently, many AMPK activators, including metformin, phenformin, and nilotinib, have shown anticancer efficacy. Nilotinib is a small-molecule tyrosine kinase inhibitor approved for the treatment of imatinib-resistant chronic myelogenous leukemia (CML). In our previous study, we found that nilotinib increased AMPK phosphorylation and led to autophagic cell death in human hepatocellular carcinoma (HCC) cells. To dissect the involvement of Bcr-Abl kinases in nilotinib-mediated cell death in human TNBC cells, we designed a series of new compounds, with structure similar to nilotinib but without hinge-binding activity in the ATP binding pocket of kinases. In this work, we found one of the nilotinib analogues, SCT-1015, showed better efficacy on AMPK activation and cell death induction compared to nilotinib without inhibiting Bcr-Abl kinase in human TNBC cells. Moreover, we demonstrate that SCT-1015 increased AMPK phosphorylation and inhibited cell proliferation in TNBC. Importantly, SCT-1015 suppressed tumor growth in HCC1806 orthotopic mice model. Our study indicates that targeting AMPK signaling pathway may be a useful approach for the development of targeted agents for anti-breast cancer. Citation Format: Jung-Chen Su, Jui-Wen Huang, Chung-Wai Shiau. Activating AMPK signaling by SCT-1015 to suppress tumor growth on triple negative breast cancer cells [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B17.

Benefits of the early detection of M351T mutation, by allele-specific oligonucleotide polymerase chain reaction, in imatinib-resistant chronic myelogenous leukemia (CML) - A retrospective analysis

<p class="s4"><strong>Introduction:</strong> BCR-ABL kinase domain mutations represent the most important disease-related factor in chronic myelogenous leukemia (CML) resistance. Highly resistant clones may pre-exist and emerge rapidly. Patients with CML can acquire more than one BCR-ABL1 mutation, which may result in increased oncogenicity. <strong>Materials and Methods:</strong> Retrospective analysis of 50 patients of imatinib resistance was done in GCRI, from January 2014 to May 2014. Response to imatinib was defined according to the European LeukemiaNet 2009 criteria. Allele-specific oligonucleotide-polymerase chain reaction (ASO-PCR) was performed on genomic DNA, extracted from peripheral blood mononuclear cells. Results: Average age was 40.75 years, 33 were males and 17 females. 47 (94%) were in chronic phase, 2 (4%) in accelerated phase, and 1 (2%) in blastic crisis. 29/50 were having low EUTOS score, whereas SOKAL score was low in 20, intermediate in 21 while only 9 had high SOKAL at presentation. Median duration of imatinib was 48 months. 43/50 had one or more than 1 mutation, T315I mutation in 5 (10%) patients, and M351T in 32% (16/50). <strong>Conclusion:</strong> The presence of M351T mutation in mutant clone leads to the development of T315I mutations development, and the detection of M351T mutation in the initial months of the therapy has a prognostic significance. ASO-PCR is more sensitive method of the detection of such mutations as compared to direct sequencing. We report low cytogenetic response (25%) and durability of response to 600 mg of imatinib, even in M351T mutation, after 400 mg of imatinib for median period of 4 years.</p>