Chronic Myeloid Leukemia Cell Lines Research Articles

Tyrosine kinase inhibitorsand interferon-α increase tunneling nanotube (TNT) formation and cell adhesion in chronic myeloid leukemia (CML) cell lines.

Chronic myeloid leukemia (CML) is a stem cell disease of the bone marrow where mechanisms of inter-leukemic communication and cell-to-cell interactions are proposed to be important for optimal therapy response. Tunneling nanotubes (TNTs) are novel intercellular communication structures transporting different cargos with potential implications in therapy resistance. Here, we have investigated TNTs in CML cells and following treatment with the highly effective CML therapeutics tyrosine kinase inhibitors (TKIs) and interferon-α (IFNα). CML cells from chronic phase CML patients as well as the blast crisis phase cell lines, Kcl-22 and K562, formed few or no TNTs. Treatment with imatinib increased TNT formation in both Kcl-22 and K562 cells, while nilotinib or IFNα increased TNTs in Kcl-22 cells only where the TNT increase was associated with adherence to fibronectin-coated surfaces, altered morphology, and reduced movement involving β1integrin. Ex vivo treated cells from chronic phase CML patients showed limited changes in TNT formation similarly to bone marrow cells from healthy individuals. Interestingly, in vivo nilotinib treatment in a Kcl-22 subcutaneous mouse model resulted in morphological changes and TNT-like structures in the tumor-derived Kcl-22 cells. Our results demonstrate that CML cells express low levels of TNTs, but CML therapeutics increase TNT formation in designated cell models indicating TNT functionality in bone marrow derived malignancies and their microenvironment.

A regulatory role for CHD2 in myelopoiesis

ABSTRACT The transcriptional program that dictates haematopoietic cell fate and differentiation requires an epigenetic regulatory and memory function, provided by a network of epigenetic factors that regulate DNA methylation, post-translational histone modifications and chromatin structure. Disturbed epigenetic regulation causes perturbations in the blood cell differentiation program that results in various types of haematopoietic disorders. Thus, accurate epigenetic regulation is essential for functional haematopoiesis. In this study, we used a CRISPR-Cas9 screening approach to identify new epigenetic regulators in myeloid differentiation. We designed a Chromatin-UMI CRISPR guide library targeting 1092 epigenetic regulators. Phorbol 12-myristate 13-acetate (PMA) treatment of the chronic myeloid leukaemia cell line K-562 was used as a megakaryocytic myeloid differentiation model. Both previously described developmental epigenetic regulators and novel factors were identified in our screen. In this study, we validated and characterized a role for the chromatin remodeller CHD2 in myeloid proliferation and megakaryocytic differentiation.

Berries and leukemia: a systematic review of experimental studies and the in vitro effect of ellagic acid on chronic myeloid leukemia cells

Diet is directly involved in cancer etiology, influencing positively or negatively. The aim of this study was to conduct a systematic review on the effect of berries on leukemia, identifying the main bioactive compounds involved in biochemical and molecular mechanisms through which they act and also to test the in vitro effect of ellagic acid, one of the phytochemicals found in berries, on leukemia cells. The work was subdivided into two parts: the first consisted of analysis of abstracts in Pubmed / Medline databases using MeSH (Medical Subject Headings) terms, synonyms, related and free terms, such as: leukemia, berries, anthocyanins, ellagic acid and flavonoids resulting in 274 articles. After abstract analysis, based on the eligibility criteria, this number was reduced to 21 articles. The second part was the in vitro investigation of the effect of ellagic acid by the MTT reduction in chronic myeloid leukemia cell lines (CML) resistant or not to chemotherapeutic agents. In vitro , in vivo and ex vivo studies associated the bioactive compounds present in berries with anticancer effect, acting mainly by induction of metabolic enzymes, modulation of gene expression and cell proliferation, chemotherapic resistance, free radical scavenging and induction of apoptosis. The present work also highlights the induction of collateral sensitivity by ellagic acid, since this compound was able to preferentially act on chemotherapeutic resistant cells. However, it is emphasized that animal models and clinical trials are for establishing the main mechanisms of action and possible dosages of berries intake daily or by dietary supplementation that could contribute to leukemia's treatment.

Biologic profile evaluation of mesenchymal stem cells in co-culture with K562 cells

Background: Mesenchymal stem cells (MSCs) are among the most essential components of bone marrow (BM) microenvironment. Any infiltration of malignant cells or malignancy of BM cells could affect the fate of other cells in the BM microenvironment. Several studies have assessed the function and phenotype of BM-derived MSCs in leukemia patients, which have presented different results. Our goal in this research was to examine the cytogenetic and flow cytometric profiles as well as the growth of human umbilical cord MSCs (hUC-MSC) after co-culture with a chronic myeloid leukemia cell line, namely K562. Subjects and Methods: MSCs were isolated as a primary culture from hUC, co-cultured with K562 cells and examined in two groups of control (MSCs) and test (hUC-MSCs + K562 cells). Using karyotypic and flow cytometric techniques, cytogenetic and surface markers, as well as growth patterns of MSCs, were investigated in the two groups by plotting the growth curves. Results: MSCs cultured in the test group (together with K562 cells) were morphologically similar to those in the control medium. Cytogenetic analysis of MSCs in the test group indicated no chromosomal abnormalities; however, there were significant differences in the expressions of surface markers as well as in MSCs growth curves between control and test groups. Discussion/Conclusion: K562 cells do not have the ability to induce cytogenetic changes in MSCs, but they are capable of altering the expressions of surface markers as well as growth rates of MSCs.

IGF-1R Pathway Mediates LQB-118 Antitumoral Activity in Leukemia Resistant Cells

Chronic myeloid leukemia (CML), a myeloproliferative disorder characterized by the BCR-ABL oncoprotein, presents its treatment based on tyrosine kinase inhibitors (TKIs), mainly imatinib. However, despite its clinical success, almost 30% of all CML patients demand alternative therapy. In this context, the development of drugs capable of overcoming TKIs resistance is imperative. LQB-118 is a compound with anti-tumour effect in two CML cell lines (K562, sensitive and K562-Lucena, resistant) whose mechanism of action is being elucidated. Here, we demonstrate that combined treatment of CML cell lines with imatinib and LQB-118 increased cell death and that microarray analysis of CML cells treated with LQB-118 presented several differentially expressed genes. Also, IGF-1R, AKT and mTOR protein levels are decreased after LQB-118 treatment and the compound alters the expression of all members of miR-29 family. CML cells silenced for IGF-1R were less sensitive to LQB-118 treatment than negative control and protein levels of IGF-1R and AKT were no longer altered after LQB-118 or imatinib treatment. Taken together, our results highlight IGF-1R/AKT/mTOR as a critical axis for LQB-118 activity and demonstrate that LQB-118 is effective in sensitizing CML cells to TKI, contributing to the development of a more efficient therapy.

Avaliação Citotóxica do Óleo Essencial de Siparuna cymosa Frente a Células de Leucemia Mielóide

This work presents the chemical characterization of the essential oil obtained from the Siparuna cymosa (Siparunaceae), an endemic species of the Atlantic Rainforest, as well as its cytotoxicity assessment in the presence of tumor cells. The oil was extracted from dry leaves by hydrodistillation and characterized (GC-MS); and its cytotoxicity was assessed by 3-(4,5-dimethyl-2-thyazolyl)-2,5-diphenyl-tetrazolium bromide (MTT) colorimetric method in AML, Acute Myeloid Leukemia (THP-1) and CML, Chronic Myeloid Leukemia (K652) cell lines. The average oil content was 2.11%, and the majority compound was a-bisabolol (68.9%) and, in significant amounts, p -cimen-9-ol (7.9%) and spathulenol (3.7%). The IC 50 obtained were the 25.44 ± 1.55 μg mL -1 (CML), and 30.88 ± 2.45 μg mL -1 (AML). The essential oil exhibits moderate cytotoxic action in the presence of leukemia cells, especially AML, for which the selectivity index was higher. The oil's cytotoxic action in the presence of the essential oil’s antitumor cells may be associated with the presence of the component a-bisabolol, a substance with recognized antitumor action. Keywords: Siparunaceae; GC-MS analysis; bisabolol; THP-1; K562

Lyn regulates creatine uptake in an imatinib-resistant CML cell line

BackgroundImatinib mesylate (imatinib) is the first-line treatment for newly diagnosed chronic myeloid leukemia (CML) due to its remarkable hematologic and cytogenetic responses. We previously demonstrated that the imatinib-resistant CML cells (Myl-R) contained elevated Lyn activity and intracellular creatine pools compared to imatinib-sensitive Myl cells. MethodsStable isotope metabolic labeling, media creatine depletion, and Na+/K+-ATPase inhibitor experiments were performed to investigate the origin of creatine pools in Myl-R cells. Inhibition and shRNA knockdown were performed to investigate the specific role of Lyn in regulating the Na+/K+-ATPase and creatine uptake. ResultsInhibition of the Na+/K+-ATPase pump (ouabain, digitoxin), depletion of extracellular creatine or inhibition of Lyn kinase (ponatinib, dasatinib), demonstrated that enhanced creatine accumulation in Myl-R cells was dependent on uptake from the growth media. Creatine uptake was independent of the Na+/creatine symporter (SLC6A8) expression or de novo synthesis. Western blot analyses showed that phosphorylation of the Na+/K+-ATPase on Tyr 10 (Y10), a known regulatory phosphorylation site, correlated with Lyn activity. Overexpression of Lyn in HEK293 cells increased Y10 phosphorylation (pY10) of the Na+/K+-ATPase, whereas Lyn inhibition or shRNA knockdown reduced Na+/K+-ATPase pY10 and decreased creatine accumulation in Myl-R cells. Consistent with enhanced uptake in Myl-R cells, cyclocreatine (Ccr), a cytotoxic creatine analog, caused significant loss of viability in Myl-R compared to Myl cells. ConclusionsThese data suggest that Lyn can affect creatine uptake through Lyn-dependent phosphorylation and regulation of the Na+/K+-ATPase pump activity. General significanceThese studies identify kinase regulation of the Na+/K+-ATPase as pivotal in regulating creatine uptake and energy metabolism in cells.

A Novel Inhibitor of STAT5 Signaling Overcomes Chemotherapy Resistance in Myeloid Leukemia Cells.

Signal transducers and activators of transcription 5A and 5B (STAT5A and STAT5B) are crucial downstream effectors of tyrosine kinase oncogenes (TKO) such as BCR-ABL in chronic myeloid leukemia (CML) and FLT3-ITD in acute myeloid leukemia (AML). Both proteins have been shown to promote the resistance of CML cells to tyrosine kinase inhibitors (TKI) such as imatinib mesylate (IM). We recently synthesized and discovered a new inhibitor (17f) with promising antileukemic activity. 17f selectively inhibits STAT5 signaling in CML and AML cells by interfering with the phosphorylation and transcriptional activity of these proteins. In this study, the effects of 17f were evaluated on CML and AML cell lines that respectively acquired resistance to IM and cytarabine (Ara-C), a conventional therapeutic agent used in AML treatment. We showed that 17f strongly inhibits the growth and survival of resistant CML and AML cells when associated with IM or Ara-C. We also obtained evidence that 17f inhibits STAT5B but not STAT5A protein expression in resistant CML and AML cells. Furthermore, we demonstrated that 17f also targets oncogenic STAT5B N642H mutant in transformed hematopoietic cells.

Utilizing Stimulated Raman Scattering Microscopy To Study Intracellular Distribution of Label-Free Ponatinib in Live Cells.

Stimulated Raman scattering (SRS) microscopy represents a powerful method for imaging label-free drug distribution with high resolution. SRS was applied to image label-free ponatinib with high sensitivity and specificity in live human chronic myeloid leukemia (CML) cell lines. This was achieved at biologically relevant, nanomolar concentrations, allowing determination of ponatinib uptake and sequestration into lysosomes during the development of acquired drug resistance and an improved understanding of target engagement.

Morin Inhibits Proliferation and Induces Apoptosis by Modulating the miR-188-5p/PTEN/AKT Regulatory Pathway in CML Cells.

Increased activity of the PI3K/AKT/mTOR pathway has been observed in chronic myeloid leukemia (CML). Morin, a kind of flavonoid, exhibits a significant anticancer activity by suppressing the PI3K/AKT signaling pathway. However, the effect of morin on CML and its underlying mechanisms is poorly understood. Here, we found that morin dose dependently inhibited the proliferation of CML cell lines K562 and KCL22 and induced their apoptosis, with a significant increase in cell apoptosis upon exposure of cells to 50 μmol/L morin. Moreover, morin significantly reduced CML xenograft growth in nude mice. Mechanically, morin attenuated phosphorylated AKT level by upregulating PTEN expression, thus leading to the inhibition of AKT signaling. Knockdown of PTEN by its siRNA completely abrogated morin-induced cell apoptosis, indicating that PTEN mediates the inductive effect of morin on CML cell apoptosis. More importantly, we found that miR-188-5p was significantly upregulated in CML patients and CML cell lines. Treating CML cells with morin markedly downregulated the miR-188-5p expression level. Further, we demonstrated that miR-188-5p repressed PTEN expression by directly targeting its 3'-UTR. miR-188-5p downregulation induced by morin enhanced CML cell apoptosis by relieving miR-188-5p repression of PTEN expression. In summary, morin exerts significant anticancer efficacy in CML by regulating the miR-188-5p/PTEN axis and thus repressing the PI3K/AKT signaling.

Inhibition of KPNB1 Inhibits Proliferation and Promotes Apoptosis of Chronic Myeloid Leukemia Cells Through Regulation of E2F1.

BackgroundKaryopherin-β1 (KPNB1) belongs to the karyopherin superfamily, which functions as shuttling proteins from the cytoplasm to nuclear. A high level of KPNB1 has been reported in various cancers which promotes cell proliferation and inhibits apoptosis. However, the role of KPNB1 in chronic myeloid leukemia (CML) remains uncertain.MethodsExpression level of KPNB1 in CML patient samples and cell lines was analyzed by Western blotting. The proliferation assays and colony formation assay were used to study the CML cell proliferation when KPNB1 knockdown in vitro. Next, Western blotting was used to evaluate the effects of KPNB1 on E2F1 and other cell cycle regulators. Then, the location of E2F1 was detected by immunofluorescence. Finally, flow cytometry was used to detect the effect of KPNB1 inhibitor importazole (IPZ) on CML cells.ResultsIn this study, we firstly showed that KPNB1 is over-expressed in CML cells. Targeting KPNB1 with small interfering RNA (siRNA) and IPZ reduced proliferation and induced apoptosis of CML cells. The underlying mechanisms were also investigated that E2F1 nuclear transport was blocked after inhibiting KPNB1 with siRNA, suggesting KPNB1 over-expression mediates the excessive nuclear transport of E2F1 in CML cells. Moreover, the expression of the E2F1 targeted molecule such as c-Myc and KPNA2 was markedly reduced. The IPZ arrested CML cells at G2/M phase and induced cell apoptosis.ConclusionIn summary, our results clearly showed that KPNB1 is over-expressed in CML cells and mediates the translocation of E2F1 into the nucleus of CML cells, thereby inhibition of KPNB1 reduced proliferation and induced apoptosis of CML cells which provides new insights for targeted CML therapies.

Inhibition of eukaryotic initiation factor 3B suppresses proliferation and promotes apoptosis of chronic myeloid leukemia cells.

Eukaryotic translation initiation factor 3B (eIF3b) has been reported to be overexpressed in colon, bladder and prostate cancers as well as in glioblastoma. However, there is no report on any correlation of eIF3b gene expression with cell proliferation and apoptosis in chronic myeloid leukemia (CML). In this study, we evaluated the role of eIF3b in cell proliferation and apoptosis in CML. Samples from patients with CML and CML cell lines were used. Quantitative RT-PCR, siRNA transfection, flow cytometry, and western blot analysis were performed. Quantitative RT-PCR revealed that the expression of eIF3b mRNA in CML patients was higher than that in the non-malignant controls. The proliferation of CML cells decreased after transfection of the cells with siRNA. The proportion of cells in the G1 and S phases in the experimental group decreased after transfection, while the number of cells in the G2/M phase increased, as compared with the control group. The total cell apoptosis percentage in the sheIF3b group (transduction with lentivirus-anti-eIF3b in K562 cells) was higher than the shCtrl group (transduction with empty-vector lentivirus in K562 cells) after transfection. Caspase 3/7 activity was higher and the expression of anti-apoptotic protein BCL-2 was lower in the sheIF3b group than in the shCtrl group after transfection. Our results suggest that downregulation of eIF3b expression inhibits proliferation and induces apoptosis in CML cells.

Arsenic Sulfide Nanoformulation Induces Megakaryocytic Differentiation through Histone Deacetylase Inhibition

AbstractArsenic sulfide (As4S4) is a mineral drug that can be administrated orally and has been applied in several Chinese medicine formulations for the treatment on some kinds of myeloid leukemia. However, mechanisms of As4S4 are not demonstrated adequately due to the poor water solubility of As4S4 in its original crystal form. The authors previously fabricated water dissolvable arsenic sulfide nanoformulation (ee‐As4S4), which downregulated intracellular reactive oxygen species (ROS). As ROS is closely associated with the histone deacetylase (HDAC), in this study the effect of ee‐As4S4 on the HDAC activity in chronic myeloid leukemia (CML) cells and whether the effect can induce subsequent megakaryocytic differentiation and underlying mechanisms are investigated. Results show that ee‐As4S4 inhibits HDAC activity in CML cells, resulting in the suppression of cellular respiration. The inhibition of HDAC activity also leads to the transcriptional activation of RUNX1, which induces megakaryocytic differentiation in CML cell lines, evidenced by the significantly multiple nuclei and giant nuclear structure, upregulation of CD41a and the increased phosphorylation of ERK1/2. In conclusion, ee‐As4S4 is able to inhibit HDAC activity, and therefore induce significant megakaryocytic differentiation in CML cells through RUNX1‐dependent pathways, which suggests its clinical potential as a complementary drug option for CML patients.

Efficacy of OR21, a Novel Oral Demethylating Agent in Chronic Myeloid Leukemia

Chronic myeloid leukemia (CML) is a clonal hematopoietic stem cell disease induced by t(9;22)(q34;q11) translocation. The prognosis of patients with CML has dramatically improved since tyrosine kinase inhibitors (TKIs) were introduced, and recent studies show that approximately 40 to 50 % of CML patients achieved deep molecular response (DASISION and ENESTnd trial) within several years. However, therapeutic options for patients with CML who are resistant for TKIs are limited. Besides BCR-ABL kinase domain mutation, somatic mutations associated with epigenetic gene alteration (e.g. TET, DNMT3A) are reportedly involved in TKI resistance and disease progression. Thus, we investigated the efficacy of DNA demethylating agents in CML. Azacitidine (AZA) and decitabine (DAC), currently available as DNA demethylating agents, have low bioavailability for oral administration because they are easily degraded by cytidine deaminase. Then, we are developing a novel demethylating agent, OR21, with possible oral absorbability as a prodrug of DAC. In vivo analysis using cynomolgus monkeys, the area under the curve of DAC after intraduodenally administration of DAC (1.5mg/kg, 6.6µmol/kg) or OR21 (2.25mg/kg, 6.6µmol/kg) was 0.01 µM·h, 0.298 µM·h respectively, indicating OR21 have high oral absorbability. To assess the demethylating activity of OR21 for CML, we performed western blot analysis and bisulfite pyrosequencing assay to measure LINE1 methylation, using CML cell-lines (K562, BV173). OR21 decreased DNMT1 protein level as a result of demethylating and LINE1 methylation in K562 and BV173 comparable to DAC. Next, we performed cell growth inhibition and cell apoptosis assay after 72 hours exposure of OR21 to assess anti-tumor effect in vitro. OR21 inhibited cell growth comparable to DAC in CML cell-lines (K562, BV173, KCL22, MYL) in a dose-dependent manner. Notably, OR21 inhibited the cell growth and apoptosis against BV173 with an extremely low concentration (IC50; 5nM) than that of AZA (IC50; 122nM) via significant accumulation of reactive oxygen species. Whereas, OR21 weakly induced cell apoptosis against K562. OR21 induced G2/M phase cell-cycle arrest in CML cell-lines except for BV173 via pRb downregulation. These results indicated mechanisms of anti-tumor effect in OR21 were induction of cell apoptosis (BV173) or cell cycle arrest (K562, KCL22, MYL). Because of the different mechanism of action, we assessed whether OR21 and TKIs combination can enhance the anti-tumor effect of CML. We investigated the combination effects of OR21 with imatinib (IM) or dasatinib (DAS) in K562. Combination index values at IC80 calculated by Calucusyn software showed 0.642±0.129 (with IM), 1.182±0.2 (with DAS), respectively. Accordingly, OR21 combined with TKIs showed at least additive or synergistic effect for K562. TKI resistance, which can be associated with somatic mutations leading to epigenetic gene alteration or loss of function of p53, is an obstacle for molecular remission in patients with CML, thus we examined the effects of OR21 in TKI resistant cell lines or efficacy on p53 mutational status. OR21 inhibited the cell growth in IM-resistant cell-line MYL-R, a derivative of MYL, which had overexpression of Lyn, and Ba/F3 BCR-ABLT315I, which exogenously expressed Bcr-Abl (T315I), indicating OR21 could overcome TKI resistance in CML. OR21 or cytarabine did not enhance cell apoptosis against K562, MYL and KCL22 (p53 deficient or mutant cell lines) combination with nutlin-3a (MDM-2 inhibitor), while increased cell apoptosis was observed in BV173 (p53 wild type) treated with cytarabine and nutlin-3a, but not with OR21 and nutlin-3a. These results suggested the effects of OR21 did not depend on p53 mutational status. Finally, we used a mouse xenograft model to evaluate anti-tumor effect of OR21 in vivo. BALB/c Rag-2/JAK3 double-deficient (BRJ) mice were injected intravenously via tail vein with 5 ×106 BV173 cells. OR21 were administered at a dose of 2.7mg/kg (equivalent to DAC 1.0mg/kg in AUC) and PBS (vehicle) twice weekly. OR21 significantly prolonged survival in a xenograft mice model (median 35 days vs not reached, P<0.01). In conclusion, a novel orally available demethylating agent OR21 is effective for CML cells including TKI resistant clones. The efficacy and safety of OR21 for CML is expected to be verified by early-phase clinical trials. Disclosures Kamachi: Ohara Pharmaceutical Co.: Research Funding. Ureshino:OHARA Pharmaceutical Co.: Research Funding. Yoshida:OHARA Pharmaceutical Co., Ltd.: Research Funding. Kurahashi:Ohara Pharmaceutical Co.: Employment. Watanabe:Ohara Pharmaceutical Co.: Research Funding. Okada:Japan Agency for Medical Research and Development: Research Funding; Bristol-Myers Squibb: Research Funding. Kimura:Ohara Pharmaceutical Co.: Research Funding; Novartis: Honoraria, Research Funding.

BCR-ABL and Src Family Kinases Induce YAP Tyrosine Phosphorylation Resulting in Survivin and Cyclin D1 Expression in Chronic Myeloid Leukemia Cells

Chronic myeloid leukemia (CML) is caused by the BCR-ABL fusion protein. To date, several downstream signaling pathways of BCR-ABL have been reported to underlie the leukemogenesis of CML such as the JAK/STAT pathway, the PI3K/AKT pathway, and the Grb2/MAPK pathway. Furthermore, the Src family kinases (SFKs), especially Hck, Lyn and Fyn, have been suggested to be involved in BCR-ABL-induced transformation. Although these studies have revealed important aspects of the downstream signals of BCR-ABL, the detailed molecular mechanism of CML has not been thoroughly elucidated. Yes-associated protein (YAP) is a transcriptional cofactor that functions as an effector of the Hippo pathway which regulates cell growth and survival. In the classical Hippo pathway, YAP phosphorylated at serine 127 (S127) by LATS1/2 is bound to 14-3-3 and prevented from nuclear translocation. Apart from this serine/threonine phosphorylation, YAP undergoes phosphorylation at several tyrosine residues by various kinases to be activated. SFKs can phosphorylate and activate YAP, which has been demonstrated in some tumors. Among the several possible phosphorylated tyrosine residues, the phosphorylation at Y357 (p-Y357) has been demonstrated to be the most important for tumorigenesis. Therefore, it is possible that BCR-ABL directly or indirectly phosphorylates YAP through SFKs and thus activated YAP is translocated into the nucleus and together with TEAD induces expression of genes necessary for cell growth and survival. In the present study, we investigated the effects of imatinib and an SFK-specific inhibitor RK-20449 on viable cell number, YAP p-Y357 and expression of Survivin as well as Cyclin D1 in CML-derived cell lines in comparison with AML-derived cell lines. Furthermore, we established BCR-ABL stable transfectants and the control lines derived from TF-1, a factor dependent human erythroleukemia cell line, in order to verify our results obtained with CML-derived cell lines. We first checked the phosphorylation status of YAP and found that it was constitutively phosphorylated at tyrosine 357in CML-derived cell lines (TCC-S and K562) but not in AML-derived cell lines (HL-60 and KG-1a). Treatment with imatinib or RK-20449 inhibited cell growth and decreased tyrosine phosphorylation of YAP in both CML lines. Expression of Survivin or Cyclin D1 was decreased at least in TCC-S but not in AML cell lines. Furthermore, we established BCR-ABL stable transfectant and control empty vector transfectant from TF-1, a factor dependent human erythroleukemia cell line, in order to verify our results obtained with CML cell lines. YAP was phosphorylated at Y357 constitutively in BCR-ABL stable transfectant but not in control transfectant, and treatment with imatinib or RK-20449 inhibited cell growth, YAP tyrosine phosphorylation, and expression of Cyclin D1 in BCR-ABL stable transfectant. These results suggest that BCR-ABL induces tyrosine phosphorylation of YAP presumably through Src family kinases, which results in expression of Survivin and Cyclin D leading to leukemogenesis in CML cells. We have not determined which SFK is involved in the downstream signaling of BCR-ABL. Overexpression experiments in HEK293T have indicated that Lck, Lyn and Fyn can phosphorylate YAP at Y357 without BCR-ABL. However, it remains to be determined which SFK is activated by BCR-ABL and practically involved in YAP phosphorylation resulting in leukemogenesis. Further studies are required to specify the relevant SFKs and disclose the downstream signaling from activated YAP. Disclosures No relevant conflicts of interest to declare.

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

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

The Role of the BMP Pathway in Sustaining CML Stem Cells in the Bone Marrow Niche

Despite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukemia (CML) patients require life-long treatment due to leukemic stem cell (LSC) persistence. LSCs reside together with mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) in the bone marrow (BM) niche, which they modify to their advantage whilst impairing normal hematopoiesis. To date it has proved difficult to understand how LSCs both dominate and alter the niche, and to effectively target LSCs with current therapies. Recent studies have shown intrinsic and extrinsic deregulation of the bone morphogenetic protein (BMP) pathway in CML. These cells show altered stem cell fate, persistence, and response to BMP receptor (BMPR) antagonists, which affect cell behavior including cell cycle, apoptosis, and expansion (Laperrousaz et al. 2013; Grockowiak et al. 2017; Zylbersztejn et al. 2018; Toofan et al. 2018). We compared TKIs alone and in combination with a BMPR inhibitor to gain improved insights into BCR-ABL1 dependent and independent regulatory mechanisms within the niche environment using this therapeutic approach. K562 CML cell line and CML CD34+ primary cells were used in this study in combination with HS5 stromal cell co-culture. CML cells were treated with single or combination treatments of imatinib, the dual SRC-ABL1 TKI saracatinib, and the BMPR inhibitor dorsomorphin for 4h or 72h with and without BMP4 stimulation/co-culture. HSC and LSC interactions were also investigated using our artificial 3D BM niche model which comprises of magnetically levitated MSC spheroids embedded in medical-grade collagen type I, mimicking the BM biological and mechanical microenvironment, along with a high throughput microfluidic MSC spheroid formation system for drug testing. Kinase screens were performed on CML CD34+ cells using a chip-based microarray assay (PamGene) following 4h drug treatments. Phosphorylation data were then utilized for upstream kinase and pathway analysis using the metacore platform. This analysis facilitated identification of proteins showing a change in expression of ≥0.5-fold across all (n=3) patient samples, which was validated by qPCR and immunoblotting. Fluidigm multiplex qPCR was utilized to assess changes in expression of early response, self-renewal and differentiation genes. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays of primary CD34+CP-CML samples (n=3) following treatment. We demonstrated a synergistic mode of action upon inhibition of the BMP pathway in combination with TKI treatment, resulting in increased apoptosis (p< 0.001), altered cell cycle (G2-M, p< 0.01), fewer cell divisions, and a reduction in CD34+cells. Primary patient samples displayed differential gene expression in relation to response for genes involved in cell cycle (CDKN1A, CDKN2B, RB1; p< 0.05), self-renewal (PBX1; p< 0.01) and cell survival (GATA1, CKIT, p< 0.05). Kinase prediction analysis identified kinases involved in cell growth, development, differentiation, apoptosis, and cell-cell adhesion in treated sample lysates, with consequent pathway analysis highlighting transcription factors, ETS1, TP53 and C-MYC as main regulators across all common pathways. QPCR identified significant changes in expression of ETS1 (p< 0.001) and C-MYC (p< 0.01) following treatment. Within the top 10 deregulated pathways based on PamGene TK profiling we also identified GAB1 and GAB2 as common effectors, known to play an important role in growth and differentiation of myeloid cells, and which were demonstrated by immunoblotting to be considerably downregulated following 72h single and dual treatments in K562. Co-culture of CD34+ cells on stroma was chemo-protective, however dual treatment was still able to elicit strong anti-proliferative effects. We are currently investigating these promising findings in more detail using our 3D niche model and microfluidic spheroid platform. Taken together, these results provide vital insights into the mechanisms by which CML cells respond to current treatments, which is critical for improving therapeutic approaches and avoiding patient resistance or relapse. A combinatorial approach targeting the BMP pathway with BMP antagonists or small molecule inhibitors together with second-generation TKIs could open up new therapeutic possibilities. Disclosures No relevant conflicts of interest to declare.

Naked antisense double-stranded DNA oligonucleotide efficiently suppresses BCR-ABL positive leukemic cells

Oligonucleotide-based gene silencing, using molecules such as antisense oligonucleotides (ASOs), small interfering RNA, and aptamers, is widely studied. Another approach uses DNA/RNA heteroduplex oligonucleotides (HDOs). Here, we developed an antisense double-stranded DNA oligonucleotide (ADO) by modification of the complementary RNA in an HDO to generate DNA for increasing resistance to nucleases. Naked BCR-ABL-targeting ADO was significantly more potent than siRNA at reducing BCR-ABL chimeric mRNA expression in chronic myeloid leukemia (CML) cell lines. Further, naked BCR-ABL-targeting ADO suppressed BCR-ABL protein levels in a dose-dependent manner, inhibited CML cell proliferation, and augmented the inhibitory effects of imatinib mesylate. In conclusion, ADO technology is an attractive method for therapeutic application.

Chitosan gold nanoparticles induce different ROS-dependent cell death modalities in leukemic cells.

BackgroundNanotechnology proposes the use of gold nanoparticles (AuNPs) for drug delivery, diagnosis, and treatment of cancer. Leukemia is a type of hematopoietic cancer that results from the malignant transformation of white blood cells. Chitosan-coated AuNPs (CH-AuNPs) are cell death inductors in HeLa and MCF-7 cancer cells without affecting peripheral blood mononuclear cells (PBMC). Considering the selectivity and versatile cytotoxicity of CH-AuNPs, we evaluated whether their selectivity is due to the cell lineage or the characteristics of the cancer cells, by assessing its cytotoxicity in leukemic cells. Moreover, we further examined the cell death mechanism and assessed the implication of nuclear damage, autophagosome formation, and the cell death mechanism induced in leukemic cells.Materials and methodsWe synthesized CH-AuNPs by chemical methods and analyzed their cell death capacity in a T-acute lymphocytic leukemia cell line (CEM), in a chronic myeloid leukemia cell line (K562), and in healthy cells from the same lineage (PBMC and bone marrow, BM, cells). Then, we assessed ROS generation and mitochondrial and nuclear damage. Finally, we evaluated whether cell death occurred by autophagy, apoptosis, or necroptosis, and the role of ROS in this mechanism.ResultsWe found that CH-AuNPs did not affect PBMC and BM cells, whereas they are cytotoxic in a dose-dependent manner in leukemic cells. ROS production leads to mitochondrial and nuclear damage, and cell death. We found that CH-AuNPs induce apoptosis in CEM and necroptosis in K562, both undergoing autophagy as a pro-survival mechanism.ConclusionCH-AuNPs are selective cell death inductors in hematologic cancer cells, without affecting their healthy counterparts. Cell death induced by CH-AuNPs is independent of the cancer cell type; however, its mechanism is different depending on the type of leukemic cells.

Histone chaperone HIRA dictate proliferation vs differentiation of chronic myeloid leukemia cells.

Abnormal proliferation and disrupted differentiation of hematopoietic progenitors mark leukemia. Histone cell cycle regulator A (HIRA), a histone chaperone, regulates hemogenic to hematopoietic transition involved in normal hematopoiesis. But, its role remains unexplored in leukemia, a case of dysregulated hematopoiesis. Here, the Cancer Cell Line Encyclopedia database analysis showed enhanced HIRA mRNA expression in cells of hematopoietic and lymphoid origin with maximal expression in the chronic myeloid leukemia (CML) cell line, K562. This observation was further endorsed by the induced expression of HIRA in CML patient samples compared to healthy individuals and Acute Myeloid Leukemia patients. Downregulation of HIRA in K562 cells displayed cell cycle arrest, loss in proliferation, presence of polyploidy with significant increase in CD41+ population thereby limiting proliferation but inducing differentiation of leukemia cells to megakaryocyte fate. Induced megakaryocyte differentiation of mouse Hira‐knockout hematopoietic progenitors in vivo further confirmed the in vitro findings in leukemia cells. Molecular analysis showed the involvement of MKL1/GATA2/H3.3 axis in dictating differentiation of CML cells to megakaryocytes. Thus, HIRA could be exploited for differentiation induction therapy in CML and in chronic pathological conditions involving low platelet counts.