TKI-resistant Chronic Myelogenous Leukemia Research Articles

The Tyrosine Kinase-Driven Networks of Novel Long Non-coding RNAs and Their Molecular Targets in Myeloproliferative Neoplasms.

Recent research has focused on the mechanisms by which long non-coding RNAs (lncRNAs) modulate diverse cellular processes such as tumorigenesis. However, the functional characteristics of these non-coding elements in the genome are poorly understood at present. In this study, we have explored several mechanisms that involve the novel lncRNA and microRNA (miRNA) axis participating in modulation of drug response and the tumor microenvironment of myeloproliferative neoplasms (MPNs). We identified novel lncRNAs via mRNA sequencing that was applied to leukemic cell lines derived from BCR-ABL1-positive and JAK2-mutant MPNs under treatment with therapeutic tyrosine kinase inhibitors (TKI). The expression and sequence of novel LNC000093 were further validated in both leukemic cells and normal primary and pluripotent cells isolated from human blood, including samples from patients with chronic myelogenous leukemia (CML). Downregulation of LNC000093 was validated in TKI-resistant CML while a converse expression pattern was observed in blood cells isolated from TKI-sensitive CML cases. In addition to BCR-ABL1-positive CML cells, the driver mutation JAK2-V617F-regulated lncRNA BANCR axis was further identified in BCR-ABL1-negative MPNs. Further genome-wide validation using MPN patient specimens identified 23 unique copy number variants including the 7 differentially expressed lncRNAs from our database. The newly identified LNC000093 served as a competitive endogenous RNA for miR-675-5p and reversed the imatinib resistance in CML cells through regulating RUNX1 expression. The extrinsic function of LNC000093 in exosomal H19/miR-675-induced modulation for the microenvironment was also determined with significant effect on VEGF expression.

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.

MiR-217 sensitizes chronic myelogenous leukemia cells to tyrosine kinase inhibitors by targeting pro-oncogenic anterior gradient 2

BCR-ABL1-independent mechanisms had been thought to mediate drug resistance to tyrosine kinase inhibitors (TKIs) in patients with chronic myelogenous leukemia (CML). The pro-oncogenic anterior gradient 2 (AGR2) mediates drug resistance of cancer cells. In this study, we observed an increased level of AGR2 in TKI-resistant CML cells. Silence of AGR2 in dasatinib-resistant K562 (K562DR) cells led to restored sensitivity to dasatinib both in vitro and in vivo. Exposure to dasatinib induced upregulation of AGR2 in K562 cells, which indicated a probable treatment-related drug resistance. We further investigated the potential interaction between microRNA (miRNA) and AGR2 in K562DR cells and found that downregulation of miR-217 was associated with overexpression of AGR2 in K562DR cells. Luciferase reporter assay identified that miR-217 negatively regulated expression of AGR2 through binding the 3'-untranslated region of AGR2. Hypermethylation of the CpG island on the promoter region of the MIR217 gene is a probable reason for the downregulation of miR-217 in dasatinib-treated K562 cells. Forced expression of miR-217 led to decreased expression of AGR2 as well as compromised TKI-resistant potential of K562DR cells. Similarly, overexpression of miR-217 resensitized K562DR cells to dasatinib treatment in a murine xenograft transplantation model. TKI treatment-induced drug resistance is correlated with a decrease of miR-217 and upregulation of AGR2. The miR-217/AGR2 interaction might be a potential therapeutic target in treating CML patients with TKI resistance.

Novel combined Ato-C treatment synergistically suppresses proliferation of Bcr-Abl-positive leukemic cells in vitro and in vivo

Chronic myelogenous leukemia (CML) accounts for 15–20% of all leukemias affecting adults. Despite recent advances in the development of specific Bcr-Abl tyrosine kinase inhibitors (TKIs), some CML patients suffer from relapse due to TKI resistance. Here, we assessed the efficacy of a novel combinatorial arsenic trioxide (ATO) and cisplatin (CDDP) treatment (Ato-C) in human Bcr-Abl-positive leukemic cells. Combination index analyses revealed that a synergistic interaction of ATO and CDDP elicits a wide range of effects in K562, KU-812, MEG-A2, and KCL-22 cells. Notably, Ato-C synergistically enhanced apoptosis and decreased the survival of both acquired TKI-resistant CML cells and the cells expressing mutant Bcr-AblT315I. In addition, Ato-C dramatically decreased the phosphorylation level of forkhead transcription factor FOXO1/3a and STAT5 as well as c-Myc protein level. Interestingly, results of gene set enrichment analysis showed that Ato-C significantly downregulates the expression of MYC- and/or E2F1-target genes. Furthermore, Ato-C significantly suppressed the proliferation of MEG-A2-derived tumor when compared with that following monotherapy in vivo. Collectively, these results suggest that combined Ato-C treatment could be a promising alternative to the current therapeutic regime in CML.

Patient-Derived Induced Pluripotent Stem Cells Revealed ADAM8/CD156 As a Novel Marker of TKI-Resistant Chronic Myeloid Leukemia Cells

Since the emergence of tyrosine kinase inhibitors (TKIs), long-term survival of patients with chronic myelogenous leukemia (CML) has been improved. However, those TKIs have not fully succeeded in curing CML, mainly due to TKI-resistant CML stem cells. CML stem cells are often difficult to analyze because they represent an extremely minor population of CML cells. To overcome this obstacle, we established integration-free induced pluripotent stem cells (iPSCs) from bone marrow (BM) cells of two patients with CML in chronic phase (CML-CP) and obtained CML pre-hematopoietic progenitor cells (CML-pre-HPCs), immature hematopoietic cells phenotypically defined by CD34+/CD45-/CD43+ cells.In semisolid and liquid cultures, CML-pre-HPCs recapitulated the principal features of CML stem cells, multi-potency and the resistance against imatinib. Gene expression enrichment analysis for CML-pre-HPCs demonstrated that several gene sets, including those related to the maintenance of hematopoietic stem cells were enriched. In addition, we found that a disintegrin and metalloprotease 8 (ADAM8), also known as CD156, was highly enhanced in CML-pre-HPCs and the expression level of ADAM8 was even increased after the treatment of imatinib in vitro. To address the significance of ADAM8 in CMP-CP patient, we evaluated purified ADAM8+ cells by fluorescence-activated cell sorting (FACS) in primary samples.First, FACS analysis found that ADAM8+ cells were enriched more in BM samples of patient with newly diagnosed CML-CP than normal or other types of leukemias among CD34+ fraction. ADAM8+ cells were enriched in CD34+/CD38- fraction compaered to CD34+/38+ fraction in BM of CML-CP patients, indicating that ADMA8+ cells represent immature hematopietic cells. In cell viability assays, ADAM8+/CD38+ CML cells in newly diagnosed CML-CP patient enhibited imatinib-resistance and imatinib-induced apoptosis in vitro was strongly suppressed in ADAM8+ CML cells compared to ADAM8- cells. Even in CD34+/CD38+ fraction, which was previously known as TKI-sensitive fraction, ADAM8+ cells exhibited TKI-resistance in both cell viability and apoptosis assay, indicating that ADAM8 would be a useful marker of TKI-resistant CML cells.Finally, to evaluate the significance of ADAM8 as a marker of TKI-resistant CML cells in vivo, we measured the frequency of CML cells in BM samples of CML-CP patients who had achieved major or complete molecular response (MMR; n = 2 or CMR; n = 1) after the administration of TKIs by limiting dilution analysis. In CML patients with MMR, CML cells remained in ADAM8+ cells at higher frequency in spite of steep decline of CML cells in ADAM8- cells The frequency of CML cells was as high in CD34+/CD38+/ADAM8+ fraction as in CD34+/38- CML stem cell fraction. Even in a patient with CMR, residual CML cells were detected in ADAM8+ population among CD34+/CD38+ fraction, whereas CML cells were undetectable in ADAM8- population.In conclusion, we have established a powerful platform with CML-iPSCs to investigate the pathophysiology of TKI-resistant CML stem cells. Using this platform, we have identified ADAM8 as a novel marker of TKI-resistant CML cells. CD34+/CD38+/ADAM8+ fraction, as well as CD34+/CD38- fraction, was an important population that defines residual CML cells even in CML-CP patients with deep molecular response after the treatment of TKI. ADAM8 would become an attractive candidate of novel therapeutic targets against TKI-resistant CML cells. DisclosuresKataoka:Yakult: Honoraria; Boehringer Ingelheim: Honoraria; Kyowa Hakko Kirin: Honoraria.

Chronic Myelogenous Leukemia- Initiating Cells Require Polycomb Group Protein EZH2.

This work defines EZH2 as a selective vulnerability for CML cells and their LICs, regardless of BCR-ABL1 mutational status. Our findings provide an experimental rationale for improving disease eradication through judicious use of EZH2 inhibitors within the context of standard-of-care TKI therapy. Cancer Discov; 6(11); 1237-47. ©2016 AACR.See related article by Scott et al., p. 1248This article is highlighted in the In This Issue feature, p. 1197.

Structure-Activity Relationship Study of Rakicidins: Overcoming Chronic Myeloid Leukemia Resistance to Imatinib with 4-Methylester-Rakicidin A.

Natural product rakicidin A induces cell death in TKI-resistant chronic myelogenous leukemia (CML) cells. Therefore, 14 rakicidin A analogues were synthesized via a highly efficient combinatorial strategy and were evaluated against CML cell lines. The conjugated diene moiety was found to be crucial for the anti-CML activity of rakicidin A, and the changes in the configuration(s) at C-2, C-3, C-14, C-15, and C-16 resulted in lower levels of anti-CML activity. The most promising compound was 4-methylester rakicidin A (1a). Compared with rakicidin A, 1a exhibited 2.8-fold greater potency against the imatinib-resistant cell line K562/G(+) and approximately 100-fold enhanced potency compared with that of imatinib. Furthermore, compound 1a demonstrated a significantly lower resistance index against Ba/F3 cells expressing BCR-ABL(T315I) than bosutinib, dasatinib, nilotinib, and ponatinib, while 1a exhibited less effect on normal hematopoietic cells. Preliminary results indicated that 1a down-regulated caspase-3 and PARP, which contributes to its K562 cell inhibitory activity.

Identification of the Epigenetic Reader BRD4 As a Novel Potential Target in Ph+ CML

Chronic myelogenous leukemia (CML) is a bone marrow-derived hematopoietic neoplasm in which BCR/ABL1 acts as a major driver of proliferation, differentiation and survival of leukemic cells. In a majority of all patients with CML, leukemic cells can be kept under control by BCR/ABL1 tyrosine kinase inhibitors (TKI), including imatinib, nilotinib, dasatinib, bosutinib, and ponatinib. Nevertheless, resistance or intolerance against one or more of these TKI may occur. Therefore, current research is focusing on novel potential drug targets in CML. A promising class of targets may be epigenetic regulators of cell growth, such as members of the bromodomain and extra-terminal domain (BET) family. The epigenetic reader and BET family member BRD4 has recently been identified as a novel potential drug target in acute myeloid leukemia (AML). However, so far, little is known about the expression and function of BRD4 in CML cells. The aims of the present study were to determine the expression of BRD4 and its downstream target MYC in CML cells and to explore whether BRD4 can serve as a novel drug target in this disease. As determined by qPCR, primary CML cells (chronic phase patients, n=7) as well as the CML cell lines KU812 and K562 expressed BRD4 mRNA. In addition, both CML cell lines stained positive for BRD4 in our immunocytochemistry staining experiments. In one patient with accelerated phase CML, putative leukemic (CD34+/CD38-) stem cells were sorted to near homogeneity and found to express BRD4 mRNA by qPCR. In order to examine the functional role of BRD4 in CML cells, a BRD4-specific shRNA was applied. In these experiments, the shRNA-induced knockdown of BRD4 in KU812 cells and K562 resulted in reduced growth compared to a control shRNA. Furthermore, the BRD4-targeting drug JQ1 was found to inhibit 3H-thymidine uptake and thus proliferation in KU812 cells in a dose-dependent manner (IC50: 0.25-0.75 µM). In addition, we were able to show that JQ1 inhibits growth of primary CML cells with variable IC50 values (0.1-5 µM). However, no substantial growth-inhibitory effects of JQ1 were seen in K562 cells (IC50: >5 µM). As determined by Annexin V/PI staining, JQ1 induced apoptosis in KU812 cells whereas no apoptosis-inducing effect of JQ1 was observed in K562 cells. Nevertheless, we were able to show that both CML cell lines as well as primary CML cells express MYC mRNA, and treatment of KU812 cells or K562 cells with JQ1 resulted in a decreased expression of MYC mRNA and MYC protein. Next, we analyzed whether MYC expression in CML cells can be blocked by BCR/ABL1 TKI. We found that imatinib, nilotinib, dasatinib, and ponatinib decrease MYC mRNA- and MYC protein expression in KU812 and K562 cells. Finally, we found that JQ1 cooperates with imatinib, nilotinib, ponatinib and dasatinib in inhibiting the proliferation of KU812 and K562 cells. Together, our data show that BRD4 serves as a potential new target in CML cells, and that the BRD4 blocker JQ1 cooperates with BCR/ABL1 TKI in inducing growth-inhibition. Whether BRD4 inhibition is a pharmacologically meaningful approach in patients with TKI-resistant CML remains to be determined in clinical trials. DisclosuresSperr:Ariad: Consultancy; Celgene: Consultancy. Zuber:Mirimus Inc.: Consultancy, Other: Stock holder; Boehringer Ingelheim: Research Funding. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.

Tyrosine Kinase Inhibitor Resistance and Epigenetic Regulation of SHP1 Phosphatase Expression in Chronic Myeloid Leukemia

Tyrosine kinase inhibitors (TKI) have significantly improved the prognosis for chronic myelogenous leukemia (CML) patients. Several mechanisms of resistance have been identified, however. BCR-ABL-dependent resistance is the most frequently occurring, usually as mutations preventing therapeutic drug interaction. This study addresses a mechanism of resistance observed in the absence of mutations. During TKI challenge, of downstream dephosphorylators such as Protein-Tyrosine Phosphatase, Nonreceptor-Type 6 (PTPN6 or SHP1) may supplement oncogenic activation. We retrospectively examined expression and control of SHP1 in TKI sensitive and resistant cases of CML in 122 North American patients of European and African ancestry employing mutation detection, methylation analysis and gene expression. Mean SHP1 gene expression was lower in CML cases (SHP1/beta2 microglobulin% = 399.4) than in nonleukemic samples (SHP1/beta2 microglobulin% = 704.5). Mutations were detected in 16/28, 26/47 and 5/18 of TKIresistant cases by standard PCR, quantitative PCR and sequencing, respectively. Expression of SHP1 was slightly lower in cases of drug-resistant CML than in drug sensitive CML (SHP1/beta 2 microglobulin% = 382 vs 422). Methylation of the SHP1 promoter at CpG site at -456 was significantly higher in cells from CML patients than in cells from normal patients (p=0.023). Depressed expression and increased methylation of SHP1 in CML cells are consistent with a role of tumor suppressor of SHP1, with its loss contributing to malignant cellular phenotypes. Although no significant differences were observed in SHP1 expression and methylation in TKI-resistant and TKI sensitive CML, expression was consistently lower and methylation higher in cases of TKI-resistant CML without detectable ABL kinase mutations. Methylation as the cause of SHP1 (or other tumor suppressor gene) suppression would offer new therapeutic strategies for treating CML. Keywords: Abl kinase, BCR-ABL1, DNA methylation, drug resistance, leukemia, phosphatase, src kinase, tumor suppressor, tyrosine kinase inhibitors.

Abstract 1717: Downregulation of miR-217 correlates with resistance of ph+ leukemia cells to ABL tyrosine kinase inhibitors

Abstract This study found that long-term exposure of chronic myelogenous leukemia (CML) K562 cells to BCR/ABL thyrosine kinase inhibirors (TKIs) caused drug-resistance in association with an increase in levels of DNA methyltransferases (DNMTs) and a decrease in levels of microRNAs (miRNAs) miR-217. These observations are clinically relevant; an increase in levels of DNMT3A in association with downregulation of miR-217 were noted in leukemia cells isolated from individuals with BCR/ABL TKI-resistant philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) and CML. Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3′-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI. Of note, long-term exposure of K562 cells to dasatinib (10 nM) together with 5-Aza-2′-deoxycytidine (5-AzadC) (0.1 μM) potently inhibited proliferation of these cells in association with upregulation of miR-217 and downregulation of DNMT3A in vitro. In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune deficient mice that were treated by the combination of 5-AzadC and dasatinib. Taken together, Ph+ leukemia cells acquire TKI-resistance via downregulation of miR-217 and upregulation of DNMT3A. Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKIs. Citation Format: Takayuki Ikezoe, Chie Nishioka, Jing Yang, Akihito Yokoyama. Downregulation of miR-217 correlates with resistance of ph+ leukemia cells to ABL tyrosine kinase inhibitors. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1717. doi:10.1158/1538-7445.AM2014-1717

Downregulation of miR‐217 correlates with resistance of ph+ leukemia cells to ABL tyrosine kinase inhibitors

This study found that long-term exposure of chronic myelogenous leukemia (CML) K562 cells to BCR/ABL thyrosine kinase inhibitors (TKI) caused drug-resistance in association with an increase in levels of DNA methyltransferases (DNMT) and a decrease in levels of microRNA miR-217. These observations are clinically relevant; an increase in levels of DNMT3A in association with downregulation of miR-217 were noted in leukemia cells isolated from individuals with BCR/ABL TKI-resistant Philadelphia chromosome positive acute lymphoblastic leukemia (Ph+ ALL) and CML. Further studies with TKI-resistant K562 cells found that forced expression of miR-217 inhibited expression of DNMT3A through a miR-217-binding site within the 3′-untranslated region of DNMT3A and sensitized these cells to growth inhibition mediated by the TKI. Of note, long-term exposure of K562 cells to dasatinib (10 nM) together with 5-Aza-2′-deoxycytidine (5-AzadC) (0.1 μM) potently inhibited proliferation of these cells in association with upregulation of miR-217 and downregulation of DNMT3A in vitro. In addition, a decrease in levels of DNMT3A and an increase in levels of miR-217 were noted in K562 tumors growing in immune-deficient mice that were treated with the combination of 5-AzadC and dasatinib. Taken together, Ph+ leukemia cells acquire TKI resistance via downregulation of miR-217 and upregulation of DNMT3A. Inhibition of DNMT3A by forced expression of miR-217 or 5-AzadC may be useful to prevent drug resistance in individuals who receive TKI.

Resistance to tyrosine kinase inhibition therapy for chronic myelogenous leukemia: a clinical perspective and emerging treatment options.

The development of tyrosine kinase inhibitors (TKIs) has led to extended lifespans for many patients with chronic myelogenous leukemia (CML). However, 20% to 30% of patients fail to respond, respond suboptimally, or experience disease relapse after treatment with imatinib. A key factor is drug resistance. The molecular mechanisms implicated in this resistance include those that involve upregulation or mutation of BCR-ABL kinase and those that are BCR-ABL independent. The clinical consequences of these molecular mechanisms of resistance for disease pathogenesis remain open for debate. This review summarizes the molecular mechanisms and clinical consequences of TKI resistance and addresses the current and future treatment approaches for patients with TKI-resistant CML.

Fingolimod (FTY720) Overcomes the Resistance to Tyrosine Kinase Inhibitors Via Dual Activation of BIM and BID in Chronic Myelogenous Leukemia

Abstract 3744 [Introduction]Tyrosine kinase inhibitors (TKIs) for BCR-ABL have dramatically improved the treatment outcome of chronic myelogenous leukemia (CML) during this decade, however, the resistance to TKIs due to various molecular mechanisms, such as BCR-ABL mutations, dysfunction of cell killing mechanisms like BIM deregulation, or the supports by leukemia microenvironment factors, has remained to be overcome. We have identified that BIM, a member of BH3-only proteins, is the essential pro-apoptotic mediator under the blockade of BCR-ABL signaling (Kuroda J, PNAS, 2006). One of the attractive therapeutic strategies for complete eradication of CML cells by simultaneously targeting both TKI-sensitive and TKI-resistant CML cells may be the use of agents that can activate BIM as well as other pro-apoptotic BH3-only protein which is not utilized in apoptosis by TKIs. In this study, we assessed the therapeutic potential of the protein phosphatase 2A (PP2A) activator Fingolimod (FTY720), an immunosuppressive agent currently used for multiple sclerosis, in CML. [Materials and methods]Human CML-derived cell lines, K562, MYL, KBM5, KCL22, and their various subclones, were utilized in this study. Murine Ba/F3 lymphoid cells were transfected with either wild type or mutated bcr-abl gene. FTY720 was purchased from Cayman Chemical (Ann Arbor, MI). Cell death analyses were performed using flow cytometry. Protein levels were determined by conventional Western blotting. [Result and discussion]Treatment with FTY720 impairs phosphorylation of BCR-ABL and its downstream targets, AKT, ERK and STAT5, and inhibited the growth of CML cell lines through the induction of apoptosis. The apoptosis induced by FTY720 was accompanied by the activation of both caspase-8 and caspase-9, while enforced expression of either BCL2 or dominant-negative protein of FADD (FADD.DN) by gene introduction partly protected K562 cells from apoptosis by FTY720, indicating the involvement of both cell extrinsic and intrinsic apoptosis pathways in FTY720-induced apoptosis. FTY720 treatment resulted in the activation of Bim as well as the processing of Bid into tBid, a BH3-only protein which plays crucial role in accelerating death-receptor-mediated extrinsic apoptosis pathway and is not utilized in apoptosis by TKIs. Importantly, gene knockdown of either BIM or BID partly protected K562 cells from apoptosis by FTY720, but the degrees of cell protection by those were not as much as the degree of cell protection by overexpression of either BCL2 or FADD.DN. Moreover, double knockdown of BIM and BID did not further protect K562 cells from apoptosis by FTY720, when compared with single knockdown of BIM. These results indicate that BIM and BID compensate each other for promoting apoptosis by FTY720 in CML cells, especially when either of them is functionally disturbed. Through these mechanisms, FTY720 overcomes TKI resistance by various mechanisms, such as ABL kinase domain (KD) mutations in BCR-ABL-transfected Ba/F3 cells (T315I, G250E, E255K or H396P) and in KBM5 cells with T315I. FTY720 also overcomes TKI resistance due to dysfunction of BIM by gene deletion polymorphism in KCL22 cells. Also, although galectin-3 overexpression has been shown to mediate bone marrow environment-mediated drug resistance in CML cells (Yamamoto-Sugitani M, PNAS, 2011), the addition of FTY720 to imatinib overcomes resistance to imatinib in galectin-3 overexpressing K562 cells and MYL cells. In conclusion, FTY720 induces apoptotic cell death in CML via dual activation of both BIM and BID, and is potent in overcoming various types of cell intrinsic and tumor microenvironment-mediated resistance to TKI. Disclosures:No relevant conflicts of interest to declare.

New drugs for chronic myelogenous leukemia.

The introduction of tyrosine kinase inhibitors (TKIs) has changed the landscape of therapy for chronic myelogenous leukemia (CML). Once considered an incurable malignancy, CML now has become a manageable chronic condition. Despite the great advances that imatinib has brought to the treatment of CML, some patients still develop resistance to imatinib and other TKIs, such as dasatinib and nilotinib. Furthermore, none of the clinically available TKIs is capable of eradicating leukemia stem cells and therefore curing CML. Several new compounds have been developed in recent years in an attempt to manage TKI-resistant CML. These include third-generation TKIs (ponatinib, danusertib) and even old compounds such as omacetaxine, which were developed before imatinib and now find a possible niche in the treatment of imatinib-resistant CML. We review the current preclinical and clinical data on the most promising new compounds for the treatment of CML.

Rakicidin A effectively induces apoptosis in hypoxia adapted Bcr‐Abl positive leukemic cells

Treatment with Abl tyrosine kinase inhibitors (TKI) drastically improves the prognosis of chronic myelogenous leukemia (CML) patients. However, quiescent CML cells are insensitive to TKI and can lead to relapse of the disease. Thus, research is needed to elucidate the properties of these quiescent CML cells, including their microenvironment, in order to effectively target them. Hypoxia is known to be a common feature of solid tumors that contributes to therapeutic resistance. Leukemic cells are also able to survive and proliferate in severely hypoxic environments. The hypoxic conditions in the bone marrow (BM) allow leukemic cells that reside there to become insensitive to cell death stimuli. To target leukemic cells in hypoxic conditions, we focused on the hypoxia-selective cytotoxin, Rakicidin A. A previous report showed that Rakicidin A, a natural product produced by the Micromonospora strain, induced hypoxia-selective cytotoxicity in solid tumors. Here, we describe Rakicidin A-induced cell death in hypoxia-adapted (HA)-CML cells with stem cell-like characteristics. Interestingly, apoptosis was induced via caspase-dependent and -independent pathways. In addition, treatment with Rakicidin A in combination with the TKI, imatinib, resulted in synergistic cytotoxicity against HA-CML cells. In conclusion, Rakicidin A is a promising compound for targeting TKI-resistant quiescent CML stem cells in the hypoxic BM environment.

The Durable Clearance of the T315I BCR-ABL Mutated Clone in Chronic Phase Chronic Myelogenous Leukemia Patients on Omacetaxine Allows Tyrosine Kinase Inhibitor Rechallenge

The onset of a BCR-ABLT315I mutation during the course of chronic myelogenous leukemia (CML) on tyrosine kinase inhibitors (TKIs) usually results in poor survival, and therapeutic options remain few in the absence of any allogeneic donor. We have investigated the affect of subcutaneous omacetaxine (OMA, or homo-harringtonine) cycles on unmutated and T315I-mutated BCR-ABL transcripts in a series of 8 TKI-resistant chronic-phase CML patients and we have addressed the question of whether the administration of OMA could resensitize patients to TKIs. Patients were regularly monitored for total disease burden and for BCR-ABLT315I transcripts using a new quantitative sensitive technique (sensitivity threshold, 0.05%), for up to 27 cycles of OMA. Overall, patients demonstrated hematologic, cytogenetic, or molecular improvement. An initial rapid decline and a sustained disappearance of T315I-mutated transcripts were observed in 50% of patients, after a median of 10.5 cycles (range, 3-27 cycles) of OMA. As the unmutated leukemic burden reduction was modest, 2 patients were submitted to nilotinib after 9 months of sustained BCR-ABLT315I transcripts negativity on OMA and mutated transcripts remained undetectable after a median follow-up of 12 months on nilotinib challenge. We suggest that OMA (ie, a non-targeted therapy) might provide a better disease control allowing the disappearance of the mutated clone probably elicited by the clone deselection after TKI release, and/or a preferential activity of OMA on the T315I-mutated cells through unknown mechanisms. These observations suggest that OMA could allow a safe TKI rechallenge in patients with resistant chronic-phase CML.

Triptolide induces cell death independent of cellular responses to imatinib in blast crisis chronic myelogenous leukemia cells including quiescent CD34+ primitive progenitor cells

The advent of Bcr-Abl tyrosine kinase inhibitors (TKI) has revolutionized the treatment of chronic myelogenous leukemia (CML). However, resistance evolves due to BCR-ABL mutations and other mechanisms. Furthermore, patients with blast crisis CML are less responsive and quiescent CML stem cells are insensitive to these inhibitors. We found that triptolide, a diterpenoid, at nanomolar concentrations, promoted equally significant death of KBM5 cells, a cell line derived from a Bcr-Abl-bearing blast crisis CML patient and KBM5STI571 cells, an imatinib-resistant KBM5 subline bearing the T315I mutation. Similarly, Ba/F3 cells harboring mutated BCR-ABL were as sensitive as Ba/F3Bcr-Abl(p210wt) cells to triptolide. Importantly, triptolide induced apoptosis in primary samples from blast crisis CML patients, who showed resistance to Bcr-Abl TKIs in vivo, with less toxicity to normal cells. Triptolide decreased X-linked inhibitor of apoptosis protein, Mcl-1, and Bcr-Abl protein levels in K562, KBM5, and KBM5STI571 cells and in cells from blast crisis CML patients. It sensitized KBM5, but not KBM5STI571, cells to imatinib. More importantly, triptolide also induced death of quiescent CD34(+) CML progenitor cells, a major problem in the therapy of CML with TKIs. Collectively, these results suggest that triptolide potently induces blast crisis CML cell death independent of the cellular responses to Bcr-Abl TKIs, suggesting that triptolide could eradicate residual quiescent CML progenitor cells in TKI-treated patients and benefit TKI-resistant blast crisis CML patients.

Predictors of Primary Imatinib Resistance in Chronic Myelogenous Leukemia Are Distinct From Those in Secondary Imatinib Resistance

A subset of patients with chronic myelogenous leukemia (CML) do not respond to the tyrosine kinase inhibitor (TKI) imatinib mesylate. Such primary imatinib resistance is distinguished from secondary resistance which reemerges after attainment of cytogenetic remission. We studied gene expression patterns in total WBCs using a panel of 21 genes previously implicated in TKI handling, resistance, or progression comparing patients who had newly diagnosed TKI-naive CML that had optimal (n = 41), or suboptimal (n = 7) responses to imatinib, or primary resistance (n = 20). Expression patterns were compared to those in secondary TKI-resistant chronic phase CML without ABL1 kinase domain mutations (n = 29), and to lymphoid (n = 15) or myeloid blast phase disease (n = 12). Fifteen genes in the panel distinguished blast phase from chronic phase disease, and 12 genes distinguished newly diagnosed CML from TKI-resistant CML without ABL1 kinase domain mutations, but only a single gene, prostaglandin-endoperoxide synthase 1/cyclooxgenase 1 (PTGS1/COX1; P = .005), differentiated imatinib-responsive from primary imatinib-resistant CML. The association of primary imatinib resistance with higher transcript levels of the drug metabolism gene PTGS1 was confirmed in a separate data set of 68 newly diagnosed, imatinib-treated CML (P = .008). In contrast, up to 11 different genes were identified in a multivariate model that optimally discriminated secondary imatinib resistance lacking ABL1 kinase domain mutation from imatinib-responsive cases, likely related to the more complex pathogenesis of secondary resistance. Gene expression profiling of CML at diagnosis for PTGS1 may be useful in predicting imatinib response and in selecting alternate therapy.

Targeting Autophagy Potentiates Imatinib-Induced Cell Death in Philadelphia Positive Cells Including Primary CML Stem Cells.

Imatinib mesylate (IM), a potent ATP-competitive inhibitor of the BCR/ABL tyrosine kinase, has become standard therapy for patients with chronic myelogenous leukemia (CML). However, the main limitations of IM- and second generation tyrosine kinase inhibitor (TKI)-based therapy are the insurgence of resistance in patients and the intrinsic refractoriness of primitive Philadelphia-positive stem cells. Therefore, there is the need to develop new therapeutic approaches that, in combination with TKI, might be more effective in targeting the stem cell population and preventing the outgrowth of TKI-resistant CML cells. TKI-induced elimination of BCR/ABL-dependent intracellular signals is known to trigger apoptosis, but it is unclear whether this also activates additional cell death and/or survival pathways. We show that IM treatment induces autophagy in CML blast crisis cell lines, CML primary cells and p210BCR/ABL-expressing 32Dcl3 (32D) myeloid precursor cells, but not in 32D cells expressing v-Src or the IM-resistant T315I p210BCR/ABL mutant. IM-induced autophagy does not involve c-Abl, as it is also observed in cells co-expressing p210BCR/ABL and the IM-resistant T315I c-Abl mutant. Induction of autophagy is associated with endoplasmic reticulum-stress and is suppressed by depletion of intracellular calcium. By contrast, ectopic Bcl-2 expression does not block IM-induced autophagy. Suppression of autophagy by pharmacological inhibitors or siRNA-mediated knockdown of essential autophagy genes enhances cell death induced by IM in cell lines and primary CML cells, demonstrating that induction of autophagy has a pro-survival effect. Critically, the combination of TKI with autophagy inhibitors results in near complete elimination of phenotypically (CD34+38−) and functionally (colony forming cells) defined CML stem cells. Together, these findings suggest that autophagy inhibitors may enhance the therapeutic effects of TKI in the treatment of CML.