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Ouabain has shown powerful anti-proliferation activities in various cancers, but its effect on imatinib)resistant chronic myeloid leukemia and toxicity on normal mice has not been investigated. Cell Counting Kit-8 assay was used to detect cytotoxicity and reversal effect of ouabain with different concentration (0.01 µM, 0.1 µM, 1.0 µM, 10 µM) on drug resistance of imatinib-resistant cell line of chronic myeloid leukemia (K562/G01 cell line). Flow cytometry was used to detect the apoptosis effect and cell cycle arrest. Hematological examination, biochemical examination and histological examination were used to detect sub-chronic toxicity of ouabain on healthy mice. In our present study, ouabain showed greatly inhibitory effect and significantly reduced half minimal inhibitory concentration of imatinib in K562/ G01 cells, an imatinib-resistant cell line of chronic myeloid leukemia, in a dose- and time- dependent manner, which implied that ouabain increased cell sensitivity to imatinib. Ouabain enhanced apoptosis induced by imatinib in K562/G01 cells not through cell cycle arrest. Animal experiments showed that there were no significant variances in hematological, liver function, kidney function parameters and organ histopathology of all mice groups. These data suggested that ouabain could be a potential agent to treat imatinib-resistant chronic myeloid leukemia for its powerful cytotoxicity as well as reversal effect, but further study is needed to find out its specific mechanism. [ABSTRACT FROM AUTHOR]

Purpose: Imatinib is the standard treatment for unresectable and metastatic GIST. In the late stages, patients undergoing imatinib show drug resistance. Surgical intervention has been occasionally performed for resistant lesions. However, the clinical significance of such intervention remains unclear. Methods: Between 2006 and 2015, 37 patients were diagnosed with imatinib-resistant GISTs. We performed surgical intervention only for localized resistant lesions. We retrospectively investigated the background characteristics, data on surgical intervention and subsequent treatment, progression-free survival (PFS), and overall survival (OS). Results: Eighteen patients diagnosed with localized resistance received surgical intervention (S-group) and 19 patients diagnosed with generalized resistance were received other TKIs (M-group). In S-group, no serious complications occurred, and all patients restarted imatinib after resection. The median PFS was 14.5 months. Five patients underwent surgical intervention multiple times followed by the continuation of imatinib, and the median duration of imatinib continuation was 22.2 months. Second-line TKIs were administered to 93% of the patients and the dose-intensity and outcome were similar in both groups. The median OS was 47.2 months after surgery. Conclusions: Surgical intervention could be performed safely and therefore could be followed by the continuation of TKI therapy. Surgical intervention based on the appropriate criteria of resistance might thus be useful for imatinib-resistant GISTs. [ABSTRACT FROM AUTHOR]

Abstract 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. Highlighits • ZNF224 expression inversely correlates with Axl expression in imatinib-sensitive and imatinib-resistant CML cell lines. • The receptor tyrosine kinase Axl is a novel target of ZNF224 transcriptional repression. • ZNF224 expression contributes to restore imatinib sensitivity of imatinib-resistant CML cells, through the repression of Axl. • ZNF224 could represent a new and promising therapeutic target in CML. [ABSTRACT FROM AUTHOR]

The Sonic hedgehog (Shh) signaling pathway may be interrelated with other signaling pathways, such as the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways in gastrointestinal stromal tumor (GIST). The present study investigated the interaction among Shh, PI3K and MAPK signaling pathways in GIST cells. The expression of PI3K, MAPK and Shh signaling pathways in GIST-H1 cells were upregulated by endothelial growth factor (EGF) and recombinant Shh (N-shh) stimulation, and were downregulated by specific inhibitors of each signaling pathway. The proliferation rate of GIST-H1 cells were significantly increased under EGF or N-shh treatment (P<0.01). In addition, this effect was partially prevented by the pretreatment of the inhibitors of these signaling pathways. In summary, a cross regulation exists among the Shh, PI3K and MAPK signaling pathways in GIST-H-1 cells. The combined use of the inhibitors of these signaling pathways is a potentially novel option for GIST targeted therapy. [ABSTRACT FROM AUTHOR]

Most gastrointestinal stromal tumors (GIST) harbor mutated receptor tyrosine kinase (RTK) KIT/PDGFRA, which provides an attractive therapeutic target. However, a majority of GISTs ultimately develop resistance to KIT/PDGFRA inhibitor imatinib, multiple therapeutic targets will be identified as a reasonable strategy in imatinib-resistant GISTs. Biological mechanisms of non-RTK activated CDC42 associated kinase 1 (ACK1) are still unclear, which has been found to be activated in GISTs. In the current report, ACK1 overexpression is demonstrated in GIST cell lines and biopsies. RNA-seq analysis and immunoblotting show that ACK1 expression is dependent on imatinib treatment time in GIST-T1 cell line. The colocalization/complex of KIT and ACK1 in GIST cells are observed, and ACK1 activation is in a partially KIT and CDC42 dependent manner. Treatment with a specific ACK1 inhibitor AIM-100 or ACK1 siRNA, mildly suppresses cell viability, but markedly inhibits cell migration in imatinib sensitive and in imatinib resistant GIST cell lines, which is associated with inactivation of PI3K/AKT/mTOR and RAF/MAPK signaling pathways, and inhibition of epithelial-mesenchymal transition, evidencing upregulation of E-cadherin and downregulation of ZEB1, N-cadherin, vimentin, snail, and/or β-catenin after treatment with AIM-100 or ACK1 / CDC42 shRNAs. Combination inhibition of ACK1 and KIT results in additive effects of anti-proliferation and pro-apoptosis as well as cell cycle arrest, and inhibition of invasiveness and migration in vitro and in vivo, compared to either intervention alone through dephosphorylation of KIT downstream intermediates (AKT, S6, and MAPK). Our data suggest that co-targeting of ACK1 and KIT might be a novel therapeutic strategy in imatinib-resistant GIST. • ACK1 overexpression is demonstrated in GISTs. • ACK1 activation is in a partially KIT and CDC42-dependent manner. • ACK1 inhibition suppresses GIST migration associated with the inactivation of PI3K/AKT and RAF/MAPK signaling pathways and inhibition of EMT markers. • Co-targeting of ACK1 and KIT might be a novel therapeutic strategy in imatinib-resistant GIST. [ABSTRACT FROM AUTHOR]

After an initial benefit from tyrosine kinase inhibitors (TKI), most gastrointestinal stromal tumors (GISTs) eventually develop disease progression or secondary resistance. An altered tumor (immune)phenotype with anaplasia and morphological changes secondary to therapy have occasionally been described in the literature. We present a 52-year old patient, diagnosed with high risk, spindle-cell, GIST (CD117 positive, Pankeratin negative) in 2003, showing a c-Kit exon 11 mutation. After TKI therapy, he developed drug resistance and disease progression. Pathological assessment of the last surgical specimen showed a pure epithelioid/clear cell histology, without evidence of cellular anaplasia. Tumor cells were CD117 positive, DOG1 positive but also E-cadherin positive and Pankeratin positive, whereas molecular analysis confirmed the presence of the c-Kit exon 11 mutation, with no additional mutations. We describe an unusual case of GIST showing peculiar (immuno)phenotypic changes under therapy, different from the vast majority of therapy-driven changes, which include marked cellular pleomorphisms and KIT immunonegativity. Possible molecular explanations to understand these phenomena and a brief review of the literature are also addressed. [ABSTRACT FROM AUTHOR]

Gain-of-function mutations of receptor tyrosine kinase KIT play a critical role in the pathogenesis of systemic mastocytosis (SM) and gastrointestinal stromal tumors. D816V KIT mutation, found in ∼80% of SM, is resistant to the currently available tyrosine kinase inhibitors (TKIs) (e.g. imatinib mesylate). Therefore, development of promising TKIs for the treatment of D816V KIT mutation is still urgently needed. We synthesized thiazole amine compounds and chose one representative designated 126332 to investigate its effect on human mast cells expressing KIT mutations. We found 126332 inhibited the phosphorylation of KIT and its downstream signaling molecules Stat3 and Stat5. 126332 inhibited the proliferation of D816V KIT expressing cells. 126332 induced apoptosis and downregulated levels of Mcl-1 and survivin. Furthermore, 126332 inhibited the tyrosine phosphorylation of β-catenin, inhibited β-catenin-mediated transcription and DNA binding of TCF. Moreover, 126332 also exhibited in vivo antineoplastic activity against cells harboring D816V mutation. Our findings suggest thiazole amine compounds may be promising agents for the treatment of diseases caused by KIT mutation. [ABSTRACT FROM AUTHOR]

Abstract: Due to the mutations of the Bcr/Abl oncogene that obstacle the binding of the protein with imatinib, the resistance to imatinib has developed in a significant portion of chronic myeloid leukemia (CML) patients. It stimulated the search for novel molecules for treatment of imatinib-resistance CML. Inhibiting the amplification of Bcr/Abl oncogene is believed to be a new effective strategy to override the imatinib resistance on CML cells. In present research, we demonstrated that dihydroartemisinin (DHA), a safe and effective antimalarial analog of artemisinin, could significantly inhibit the Bcr/Abl fusion gene at the mRNA level in CML cells sensitive or resistant to imatinib (including the primary CML cells with T315I mutation) and induce cell death. Moreover, dihydroartemisinin could also lead to the inhibition of the Bcr/Abl protein expression and tyrosine kinase activity, and strongly suppress on the downstream signals of Bcr/Abl, which included inhibition of tyrosine kinase activity of AKT and ERK, promotion of cytochrome c release from the mitochondria and the consequential activation of caspase-9/3 in imatinib-resistant CML cells. These results suggest for the first time that Dihydroartemisinin might be a potential novel drug candidate for treatment of imatinib-resistant CML and worthy of further study. [Copyright &y& Elsevier]

Abstract: We investigated the effect of a novel Wnt/β-catenin signaling inhibitor, AV65 on imatinib mesylate (IM)-sensitive and -resistant human chronic myeloid leukemia (CML) cells in vitro. AV65 inhibited the proliferation of various CML cell lines including T315I mutation-harboring cells. AV65 reduced the expression of β-catenin in CML cells, resulting in the induction of apoptosis. Moreover, AV65 inhibited the proliferation of hypoxia-adapted primitive CML cells that overexpress β-catenin. The combination of AV65 with IM had a synergistic inhibitory effect on the proliferation of CML cells. These findings suggest that AV65 could be a novel therapeutic agent for the treatment of CML. [Copyright &y& Elsevier]

Abstract: Imatinib (STI571) is the frontline targeted-therapeutic agent for patients with chronic myelogenous leukemia (CML). However, resistance to imatinib due to point mutations in Bcr-Abl kinase domain is an emerging problem. We recently reported that triptolide (compound 1) could effectively kill CML cells including those harboring T315I mutant Bcr-Abl. In the present study, we designed a series of C-14 triptolide derivatives with C-14-hydroxyl substituted by different amine esters (3–18): 3–6 and 13 (by aliphatic chain amine esters); 7–9, 11, 12 and 15–18 (by alicyclic amine esters with different size), and 10 and 14 (by aralkylamine esters).The compounds were examined for their antineoplastic activity against CML cells (including KBM5-T315I cells) in terms of proliferation inhibition, apoptosis and signal transduction. Nude mouse xenograft model was also used to evaluate the in vivo activity. Compounds 2–9, 11–14, 17 and 18 exhibited a potent inhibitory activity against KBM5 and KBM5-T315I cells. This series of derivatives down-regulated Bcr-Abl mRNA. Compounds 4, 5, 8 and 9 were further examined for their impact on signaling and apoptosis with immunoblotting. Compound 5 was chosen for evaluation in a nude mouse xenograft model. The stereo-hindrance of C-14 group appeared to be responsible for the antitumor effect. The computational small molecule-protein docking analysis illustrated the possible interaction between compound 9 and RNA polymerase II. Our results suggest that this series of derivatives may be promising agents to overcome imatinib-resistance caused by the Bcr-Abl-T315I mutation. [Copyright &y& Elsevier]

The t(9;22) translocation leads to the formation of the chimeric bcr/abl fusion gene, which encodes the BCR/ABL fusion protein. In contrast to its physiological counterpart c-ABL, the BCR/ABL kinase is constitutively activated, inducing the leukemic phenotype. The N-terminus of c-ABL (Cap region) contributes to the regulation of its kinase function. It is myristoylated, and the myristate residue binds to a hydrophobic pocket in the kinase domain known as the myristoyl-binding pocket in a process called ‘capping’, which results in an auto-inhibited conformation. Because the cap region is replaced by the N-terminus of BCR, the BCR/ABL ‘escapes’ this auto-inhibition. Allosteric inhibition by myristate ‘mimics’, such as GNF-2, is able to inhibit unmutated BCR/ABL, but not the BCR/ABL that harbors the ‘gatekeeper’ mutation T315I. In this study, we analyzed the possibility of increasing the efficacy of allosteric inhibition by blocking BCR/ABL oligomerization. We showed that inhibition of oligomerization was able to not only increase the efficacy of GNF-2 on unmutated BCR/ABL, but also overcome the resistance of BCR/ABL-T315I to allosteric inhibition. These results strongly suggest that the response to allosteric inhibition by GNF-2 is inversely related to the degree of oligomerization of BCR/ABL. In summary, our observations establish a new approach for the molecular targeting of BCR/ABL and its resistant mutants represented by the combination of oligomerization and allosteric inhibitors. [ABSTRACT FROM AUTHOR]

In Philadelphia chromosome-positive (Ph+) leukemia BCR/ABL induces the leukemic phenotype. Targeted inhibition of BCR/ABL by kinase inhibitors leads to complete remission. However, patients with advanced Ph+ leukemia relapse and acquire resistance, mainly due to point mutations in BCR/ABL. The ‘gatekeeper mutation’ T315I is responsible for a general resistance to small molecules. It seems not only to decrease the affinity for kinase inhibitors, but to also confer additional features to the leukemogenic potential of BCR/ABL. To determine the role of T315I in resistance to the inhibition of oligomerization and in the leukemogenic potential of BCR/ABL, we investigated its influence on loss-of-function mutants with regard to the capacity to mediate factor independence. Here, we show that T315I (i) requires autophosphorylation at tyrosine 177 in the BCR-portion to mediate resistance against the inhibition of oligomerization; (ii) restores the capacity to mediate factor-independent growth of loss-of-function mutants due to an increase in or activation of ABL-kinase; (iii) leads to phosphorylation of endogenous BCR, suggesting aberrant substrate activation by BCR/ABL harboring the T315I mutation. These data show that T315I confers additional leukemogenic activity to BCR/ABL, which might explain the clinical behavior of patients with BCR/ABL–T315I-positive blasts. [ABSTRACT FROM AUTHOR]

Imatinib was the first treatment for chronic myeloid leukemia (CML) that specifically targeted the causative BCR-ABL oncoprotein, and represented a major therapeutic advance in this disease; however, some patients develop resistance or intolerance. Resistance can be classified as BCR-ABL-dependent (e.g., mutation in the BCR-ABL gene) or BCR-ABL-independent (alternative pathways of disease progression, e.g., SRC-family tyrosine kinases). The investigation of therapeutic options post-imatinib failure resulted in the development and regulatory approval of dasatinib, a BCR-ABL and SRC-family kinase inhibitor. Dasatinib is active across all phases of CML and Philadelphia chromosome-positive acute lymphoblastic leukemia, and demonstrates activity in almost all imatinib-resistant mutations. Other therapeutic options are also under investigation, with nilotinib being the most clinically advanced. Nilotinib is an analog of imatinib with similar multiple kinase targets, but without inhibition of SRC, and reduced in vitro activity against BCR-ABL P-loop mutations compared with dasatinib. Similar to dasatinib, nilotinib has no activity against T315I mutations. The availability of dasatinib and development of other tyrosine kinase inhibitors provide positive prospects for patients with imatinib-resistant or -intolerant CML. Here, we discuss several of these new strategies for treating patients after imatinib failure. [ABSTRACT FROM AUTHOR]

Chronic myelogenous leukaemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukaemia (ALL) are caused by the BCR-ABL oncogene. Imatinib inhibits the tyrosine kinase activity of the BCR-ABL protein and is an effective, frontline therapy for chronic-phase CML. However, accelerated or blast-crisis phase CML patients and Ph+ ALL patients often relapse due to drug resistance resulting from the emergence of imatinib-resistant point mutations within the BCR-ABL tyrosine kinase domain. This has stimulated the development of new kinase inhibitors that are able to over-ride resistance to imatinib. The novel, selective BCR-ABL inhibitor, AMN107, was designed to fit into the ATP-binding site of the BCR-ABL protein with higher affinity than imatinib. In addition to being more potent than imatinib (IC50< 30 nM) against wild-type BCR-ABL, AMN107 is also significantly active against 32/33 imatinib-resistant BCR-ABL mutants. In preclinical studies, AMN107 demonstrated activity in vitro and in vivo against wild-type and imatinib-resistant BCR-ABL-expressing cells. In phase I/II clinical trials, AMN107 has produced haematological and cytogenetic responses in CML patients, who either did not initially respond to imatinib or developed imatinib resistance. Dasatinib (BMS-354825), which inhibits Abl and Src family kinases, is another promising new clinical candidate for CML that has shown good efficacy in CML patients. In this review, the early characterisation and development of AMN107 is discussed, as is the current status of AMN107 in clinical trials for imatinib-resistant CML and Ph+ ALL. Future trends investigating prediction of mechanisms of resistance to AMN107, and how and where AMN107 is expected to fit into the overall picture for treatment of early-phase CML and imatinib-refractory and late-stage disease are discussed. [ABSTRACT FROM AUTHOR]