P210 Cells Research Articles

HDAC inhibitor chidamide overcomes drug resistance in chronic myeloid leukemia with the T315i mutation through the Akt-autophagy pathway.

Currently, therapy for Chronic Myeloid Leukemia (CML) patients with the T315I mutation is a major challenge in clinical practice due to its high degree of resistance to first- and second-generation Tyrosine Kinase Inhibitors (TKIs). Chidamide, a Histone Deacetylase Inhibitor (HDACi) drug, is currently used to treat peripheral T-cell lymphoma. In this study, we investigated the anti-leukemia effects of chidamide on the CML cell lines Ba/F3 P210 and Ba/F3 T315I and primary tumor cells from CML patients with the T315I mutation. The underlying mechanism was investigated, and we found that chidamide could inhibit Ba/F3 T315I cells at G0/G1 phase. Signaling pathway analysis showed that chidamide induced H3 acetylation, downregulated pAKT expression and upregulated pSTAT5 expression in Ba/F3 T315I cells. Additionally, we found that the antitumor effect of chidamide could be exerted by regulating the crosstalk between apoptosis and autophagy. When chidamide was used in combination with imatinib or nilotinib, the antitumor effects were enhanced compared with chidamide alone in Ba/F3 T315I and Ba/F3 P210 cells. Therefore, we conclude that chidamide may overcome T315I mutation-related drug resistance in CML patients and works efficiently if used in combination with TKIs.

TPEN/TPGS (T2) combo dramatically reduces Philadelphia chromosome-positive pro-lymphoblastic B leukemia in BALB/c mice

Acute lymphoblastic leukemia (ALL) is hematological neoplasia that affects human beings from early life to adulthood. Although ALL treatment has been effective, an important percentage of ALL patients are resilient to treatment. Therefore, there is an urgent need for testing a new combination of compounds for the treatment of this disease. Recently, combined TPEN and TPGS (T2 combo) have shown selective cytotoxic effects in vitro leukemia cells such as Jurkat, K562, and Ba/F3 cells. In this study, we aimed to test the effect of combined TPEN and TPGS agents (T2 combo) at a fixed dose (TPEN 5 mg/kg: TPGS 100 mg/kg) on leukemic Ba/F3-BCR-ABL P210 BALB-c mice model. We found that 4 successive 2-day apart intravenous injections of T2 combo showed a statistically significant reduction of Ba/F3 BCR-ABL leukemia cells (− 69%) in leukemia BALB/c mice (n = 6) compared to untreated leukemia group (n = 6). Moreover, the T2 combo was innocuous to non-leukemia BALB/c mice (n = 3) compared to untreated non-leukemia mice (control, n = 3). After treatments (day 42), all mice were left to rest until day 50. Outstandingly, the leukemia BALB/c mice treated with the T2 combo showed a lower percentage of Ba/F3-BCR-ABL P210 cells (− 84%) than untreated leukemia BALB/c mice. Furthermore, treatment of leukemia and non-leukemia mice with T2 combo showed no significant tissue alteration/damage according to the histopathological analysis of brain, heart, liver, kidney, and spleen samples; however, T2 combo significantly reduced the number of leukocytes in the bone marrow of treated leukemia mice. We conclude that the T2 combo specifically affects leukemia cells but no other tissue/organs. Therefore, we anticipate that the T2 combo might be a potential pro-oxidant combination for the treatment of leukemia patients.

BCR-ABL1 p190 in CML: A Minor Breakpoint with a Major Impact

Introduction: The oncoprotein Bcr-Abl has two major isoforms, depending on the breakpoint in BCR gene, p190 and p210. While p210 is the hallmark of chronic myeloid leukemia (CML), p190 occurs in the majority of Philadelphia-positive acute lymphoblastic leukemia (Ph+ ALL) patients. p190 occurs as a sole transcript in 1-2% CML patients, associated with distinct features like monocytosis and frequent additional cytogenetic abnormalities (ACA) at diagnosis. It also confers a risk of treatment failure and progression in chronic phase (CP) CML patients. However, the underlying mechanisms are largely unknown. Here we explore the characteristics of p190 and p210 in CML and ALL patients using next generation sequencing, phospho-flowcytometry and high throughput drug testing. Patients and methods: Peripheral blood mononuclear cells (PMNC) were collected at diagnosis from four CP-CML patients harboring p190 isoform from Helsinki University Hospital. Genetic alterations were identified by whole exome sequencing. RNA sequencing was employed to analyze transcriptional profiles of p190 CML (n=3) in contrast to p210 CML patients (n=4). A thorough transcriptional, phosphorylation and drug sensitivity profiling were applied to five p190- and three p210-expressing Ph+ALL patients. Expression alterations were further characterized in two cell line models mimicking BCR-ABL positive leukemia (Ba/F3 and HPCLSK). Phosphorylation profiles were analyzed by flowcytometry and phospho-array (Tyrosine Phosphorylation ProArray, Full Moon Biosystems). For drug sensitivity and resistance testing (DSRT), a custom plate set comprising 75 approved and investigational oncology drugs was used for patient samples and more extensive 528-drugs plates were used for the cell lines. Results: CML patients with p190 had a median age of 72.5 years at the diagnosis (range: 50-80) and all received imatinib as a frontline treatment. Only one patient achieved a fluctuating major molecular response (MMR) by 12 months while the rest of the patients showed primary resistance to treatment and were shifted to a 2nd line TKI, nilotinib (n=2) or proceeded to HSCT (n=1). By exome sequencing we identified 26 variants in p190-CML samples (median per patient=7, range: 2-10), including variants in ASXL1, DNMT3A and KDM4D genes. RNA-sequencing analysis identified 19 and 97 dysregulated genes (Q <0.05) between p190- and p210 in CML and Ph+ ALL cells respectively. In CML, enrichment analysis revealed upregulation of TNF, interferon (IFN), IL1-R and Toll-like receptor (TLR) signaling, TP53-related, cell cycle and apoptosis pathways. Among Ph+ ALL samples, many CML-related genes were upregulated in samples encompassing p210 while IFN-, TP53- and cell cycle-related molecules were upregulated in p190 samples. p190 samples exhibited hyper-phosphorylation of Src kinase compared to p210 samples. DSRT results also revealed increased sensitivity of primary Ph+ ALL-p190 cells to Src-inhibitors (dasatinib and saracatinib), glucocorticoids and MDM2 inhibitors/TP53 activators (SAR405838 and idasanutlin). Regarding cell lines, Ba/F3-p190 showed the upregulation of interferon signaling pathways compared to p210. Src was also hyperphosphorylated in both Ba/F3 and HPCLSK p190 models. In addition to glucocorticoids and Src-inhibitors, compounds blocking the activity of the inhibitors of apoptosis protein (IAP) family were highly effective at reducing the viability of p190 compared to p210 cells in both cell lines. Conclusions: In CML, p190 isoform of BCR-ABL1 is associated with distinct features and should be considered as a high-risk group. Combining clinical, genomic, phosphorylation and drug sensitivity data, we demonstrated that p190 activates specific cancer pathways, notably Src signaling and interferon pathways. Data also suggests that CML patients with p190 could benefit from broad spectrum TKI with Src inhibiting activity or combination of TKI with MDM2- or IAP-inhibitors. Disclosures Heckman: Orion Pharma: Research Funding; Celgene: Research Funding; Novartis: Research Funding; Oncopeptides: Research Funding. Porkka:Celgene: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Daiichi Sankyo: Consultancy, Research Funding. Mustjoki:Novartis: Research Funding; Pfizer: Research Funding; BMS: Honoraria, Research Funding.

Simultaneous Administration of AMN107 and Imatinib in the Treatment of Imatinib-Sensitive and Imatinib-Resistant Chronic Myeloid Leukemia.

Chronic myelogenous leukemia (CML) and Philadelphia chromosome positive (Ph+) acute lymphoblastic leukemia (ALL) are caused by the Bcr-Abl tyrosine kinase oncogene. The Abl inhibitor imatinib is an effective, frontline therapy for early, chronic phase CML. However, accelerated or blast crisis phase CML and Ph+ ALL patients often relapse because of drug resistance that results from the emergence of imatinib-resistant point mutations within the Bcr-Abl kinase domain. The aminopyrimidine ATP-competitive inhibitor, AMN107, was designed to fit into the ATP-binding site of the Bcr-Abl protein in such a way as to exhibit higher efficacy against imatinib-resistant Bcr-Abl point mutants. AMN107 is active against many imatinib-resistant Bcr-Abl mutants in vitro and in vivo, and is significantly more potent than imatinib against wild-type Bcr-Abl. AMN107 is currently showing promise in phase I/II clinical trials involving CML patients who are unresponsive to imatinib, and thus could potentially be used as a single agent in selected patients resistant or intolerant to imatinib. Alternatively, the use of more than one inhibitor of Abl should effectively lower the number of residual Bcr-Abl-expressing cells having the potential to undergo mutation, and therefore could potentially suppress the emergence of drug-resistant Bcr-Abl mutations. Thus, AMN107 and imatinib could be administered together to achieve higher responsiveness in CML patients. In the current study, we investigated the combination of imatinib and AMN107 in a panel of wild-type and imatinib-resistant Bcr-Abl-expressing cell lines, including 32D.p210, K562, F486S-Ba/F3, F317L-Ba/F3, M351T-Ba/F3, and T315I-Ba/F3. We found evidence of additive to synergistic effects in several of the cell lines examined. In addition, the combination of AMN107 and imatinib was studied in vivo using a bioluminescent Bcr-Abl model of CML. Mice harboring murine 32D.p210 cells engineered to stably express firefly luciferase were treated with vehicle, AMN107 alone (15mg/kg), imatinib alone (75mg/kg), or both AMN107 and imatinib at their respective doses. Mice treated with both agents were observed to carry an overall lower tumor burden (as measured by levels of total body bioluminescence and percent spleen weights) than vehicle-treated mice and mice treated with each agent alone. These results suggest that the combination of imatinib and AMN107 may be a more effective treatment for CML than either agent alone.

BCR-ABL Activates STAT3 Via MEK Kinase 1 in Embryonic Stem Cells.

BCR-ABL oncogene, the molecular hallmark of chronic myelogenous leukemia, arises in a primitive hematopoietic stem cell that has the capacity for both differentiation and self-renewal. Its product, Bcr-Abl protein, has been shown to activate STAT3 and to promote self-renewal in ES cells, even in the absence of leukemia inhibitory factor (LIF). MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase involved in Bcr-Abl signal transduction (Oncogene 22:7774, 2003). To investigate the role of MEKK1 in Bcr-Abl-induced transformation of ES cells, p210 Bcr-Abl was stably transfected into wild type (WT+p210) and MEKK1−/− (MEKK1−/−+p210) ES cells. Bcr-Abl enhanced both MEKK1 expression and activation in ES cells, as it does in other Bcr-Abl-transformed cells. In the absence of LIF, WT+p210 cells showed constitutive STAT3 activation and formed compact colonies having strong alkaline phosphatase activity, a characteristic phenotype of undifferentiated ES cells. MEKK1−/−+p210 cells, by contrast, showed less STAT3 activity than WT+p210 cells and formed large, flattened colonies having weak alkaline phosphatase activity, a phenotype of differentiated ES cells. These results indicate that MEKK1 plays an essential role in Bcr-Abl-induced STAT3 activation and in the capacity for LIF-independent self-renewal, and may thus be involved in Bcr-Abl-mediated leukemogenesis in stem cells.

AMD107: Efficacy as a Selective Inhibitor of the Tyrosine Kinase Activity of BCR-ABL in Murine Leukemia Models.

Patient relapse due to the emergence of imatinib-resistance in advanced chronic myelogenous and Ph+ acute lymphocytic leukemias has prompted the search for improved Bcr-Abl kinase inhibitors. Up to 40% imatinib-resistant patients express mutant forms of the Bcr-Abl kinase that reduce binding of imatinib without significantly decreasing kinase activity. However, some mutants are only partially resistant, since higher concentrations of imatinib may still block kinase activity and have a beneficial clinical effect. More potent Bcr-Abl inhibitors or inhibitors with different contact points with the kinase domain are predicted to be useful for the treatment of imatinib-resistant disease. Rational drug design based upon the crystal structure of an Abl-imatinib complex, together with medicinal chemistry paradigms, resulted in the discovery of AMN107. AMN107 is a low molecular weight compound that selectively inhibits the c-Abl/Bcr-Abl, imatinib-resistant mutants of Bcr-Abl, PDGFR, and c-Kit tyrosine kinases, with IC50 values for the inibition of autophosphorylation (cell capture ELISA) of 23 nM (32D cells transfected with p210 Bcr-Abl), 83 nM (A31 cells) and 192 nM (GIST cells), respectively. Following oral administration to mice (20 mg/kg in NMP-PEG300 10:90 v/v), AMN107 was well absorbed, with a mean plasma level after 2 h in the range of 6.0-12.1 M, which corresponds to >100-fold that required to inhibit Bcr-Abl autophosphorylation in 32D.p210 cells. Following injection of 32D.p210-luciferase cells into Balb/c mice, serial imaging of the leukemic clone in live mice indicated that AMN107 could substantially reduce the accumulation of leukemic cells in the marrow, nodes, liver, and spleen, compared to a vehicle control. Further, administration of AMN107 (75 mg/kg/day p.o.) to BALB/c mice over an 18 day period, commencing 3 days after the injection of 32 D.p210 cells, resulted in the protection of 15/20 animals over 100 days observation, whereas 19/20 vehicle treated animals developed a lethal leukemic disease(all deaths occurred within 36 days of cell injection). The spleen sizes of the surviving animals in the treated group were in the normal range (0.142±0.043 g; n = 17), whereas those of the vehicle treated animals were 0.50±0.045 (n = 20). These results were confirmed in a bone marrow transplant assay, where AMN107 was found to prolong survival of mice transplanted with marrow cells infected with a p210Bcr/Abl retrovirus. AMN107 also prolonged survival of mice transplanted with a Bcr/Abl mutant (E255V) associated with imatinib resistance in patients, while imatinib treatment was unsuccessful. These in vivo studies, along with in vitro studies reported separately, indicate that AMN107 is a highly active inhibitor of Bcr/Abl that may have clinical utility in patients with Bcr/Abl+ leukemias.

Histone deacetylase inhibitor NVP-LAQ824 has significant activity against myeloid leukemia cells in vitro and in vivo

NVP-LAQ824 is a novel potent hydroxamic acid-derived histone deacetylase inhibitor that induces apoptosis in nanomolar concentrations in myeloid leukemia cell lines and patient samples. Here we show the activity of NVP-LAQ824 in acute myeloid leukemia cells and BCR/ABL-expressing cells of mouse and human origin, both sensitive and resistant to imatinib mesylate (Gleevec, STI-571). Whereas imatinib inhibited overall cellular tyrosine phosphorylation in Ba/F3.p210 cells, NVP-LAQ824 did not inhibit tyrosine phosphorylation, and did not affect BCR/ABL or ABL protein expression. Neither compound was able to inhibit cellular tyrosine phosphorylation in the imatinib-resistant Ba/F3.p210-T315I cell line. These data taken together suggest that BCR/ABL kinase activity is not a direct target of NVP-LAQ824. Synergy between NVP-LAQ824 and imatinib was demonstrated against BCR/ABL-expressing K562 myeloid leukemia cell lines. In addition, we show that NVP-LAQ824 was well tolerated in vivo in a pre-clinical murine leukemia model, with antileukemia activity resulting in significant prolongation of the survival of mice when treated with NVP-LAQ824 compared to control mice. Taken together, these findings provide the framework for NVP-LAQ824 as a novel therapeutic in myeloid malignancies.

Rapid generation of a tetracycline-inducible BCR-ABL defective retrovirus using a single autoregulatory retroviral cassette

The development of chronic myelogenous leukemia (CML) models in mice using an inducible BCR-ABL gene has been hampered by the requirement of sequential expression of tTA (Tet repressor-VP16 fusion protein) and Tet-OP sequences in the same cells after separate transfection. This double transfection strategy is time consuming as it requires screening of many hundreds of individual clones and cannot be applied to primary hematopoietic cells. To generate a tetracycline-inducible BCR-ABL retrovirus, we have subcloned BCR-ABL p210 cDNA in the SIN-Retro-TET vector, which allows regulated expression of a gene of interest in a single autoregulatory cassette, containing both tTA and Tet OP sequences. Retroviral particles were obtained by transfecting the SIN-BCR-ABL p210 construct into the 293 cells and by VSVG pseudotyping. To determine the functionality of the retrovirus, the IL-3-dependent murine Ba/F3 cell line was retrovirally transduced and clones were grown in the absence of both IL-3 (to select for transformed cells) and a tetracycline analog, doxycycline (to induce BCR-ABL expression). Using this technique, polyclonal Ba/F3 cells and several growth factor-independent Ba/F3 clones expressing BCR-ABL were obtained within 2-3 weeks. A single dose of doxycycline added to the medium (1 microg/ml), induced in different clones, a reduction of BCR-ABL protein levels by 60-90% at 24 h, leading to cell death in the absence of IL-3. In several individual clones, BCR-ABL expression was further reduced to become almost undetectable at 48 h. The doxycycline-regulated BCR-ABL expression was stable, as many clones maintained in culture for >8 months showed a persistent inhibitory response to doxycycline addition in the medium. In in vivo experiments, subcutaneous injection of 2 x 10(6) Ba/F3-SIN p210 cells in nude mice induced visible tumors in 2 weeks and all established tumors completely regressed upon addition of doxycycline in the drinking water (200 microg/ml). To determine the functionality of the inducible BCR-ABL retrovirus in vivo, primary Lin- bone marrow cells were transduced with SIN-p210 and transplanted in lethally irradiated mice. All transplanted mice had successful hematopoietic reconstitution and BCR-ABL integration was found in the peripheral blood of seven out of 14 mice available for long-term analysis (>6 months). However, despite evidence of retrovirus-mediated gene transfer, there was no evidence of leukemia, due either to low viral titers or to the relative inefficiency of the minimal CMV promoter in primary hematopoietic cells. Thus, these results demonstrate for the first time, to our knowledge, the feasibility to generate an inducible BCR-ABL retrovirus in a single step, in the context of an immortalized cell line. Our data suggest that with further improvements of the retrovirus-mediated gene transfer technology, it might be possible to generate inducible leukemia models in mice by the use of single retroviral constructs.

Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL–transformed hematopoietic cell lines

The tyrosine kinase activity of the Bcr/Abl oncogene is required for transformation of hematopoietic cells. The tyrosine kinase inhibitor STI571 (formerly called CGP57148B, Novartis Pharmaceuticals) inhibits BCR/ABL, TEL/ABL, and v-ABL kinase activity and inhibits growth and viability of cells transformed by any of these ABL oncogenes. Here we report the generation of 2 BCR/ABL–positive cell lines that have developed partial resistance to STI571. BCR/ABL–transformed Ba/F3 hematopoietic cells and Philadelphia-positive human K562 cells were cultured in gradually increasing concentrations of STI571 over a period of several months to generate resistant lines. Resistant Ba/F3.p210 cells were found to have an increase in Bcr/Abl messenger RNA, amplification of the Bcr/Abl transgene, and a greater than tenfold increase in the level of BCR/ABL protein. In contrast to Ba/F3.p210 cells, drug-resistant K562 cells did not undergo detectable amplification of the BCR/ABL gene, although they displayed a 2-fold to 3-fold increase in p210BCR/ABL protein. The addition of STI571 to both resistant Ba/F3.p210 and K562 cells resulted in a rapid reduction of tyrosine phosphorylation of cellular proteins, similar to that observed for nonresistant cells. However, the inhibition of kinase activity was transient and partial and was not accompanied by apoptosis. The results suggest that resistance to STI571 may be multifactorial. Increased expression of the target protein BCR/ABL was observed in both lines, and resulted from oncogene amplification in one line. However, altered drug metabolism, transport, or other related mechanisms may also contribute to drug resistance.

Mechanism of resistance to the ABL tyrosine kinase inhibitor STI571 in BCR/ABL–transformed hematopoietic cell lines

AbstractThe tyrosine kinase activity of the Bcr/Abl oncogene is required for transformation of hematopoietic cells. The tyrosine kinase inhibitor STI571 (formerly called CGP57148B, Novartis Pharmaceuticals) inhibits BCR/ABL, TEL/ABL, and v-ABL kinase activity and inhibits growth and viability of cells transformed by any of these ABL oncogenes. Here we report the generation of 2 BCR/ABL–positive cell lines that have developed partial resistance to STI571. BCR/ABL–transformed Ba/F3 hematopoietic cells and Philadelphia-positive human K562 cells were cultured in gradually increasing concentrations of STI571 over a period of several months to generate resistant lines. Resistant Ba/F3.p210 cells were found to have an increase in Bcr/Abl messenger RNA, amplification of the Bcr/Abl transgene, and a greater than tenfold increase in the level of BCR/ABL protein. In contrast to Ba/F3.p210 cells, drug-resistant K562 cells did not undergo detectable amplification of the BCR/ABL gene, although they displayed a 2-fold to 3-fold increase in p210BCR/ABL protein. The addition of STI571 to both resistant Ba/F3.p210 and K562 cells resulted in a rapid reduction of tyrosine phosphorylation of cellular proteins, similar to that observed for nonresistant cells. However, the inhibition of kinase activity was transient and partial and was not accompanied by apoptosis. The results suggest that resistance to STI571 may be multifactorial. Increased expression of the target protein BCR/ABL was observed in both lines, and resulted from oncogene amplification in one line. However, altered drug metabolism, transport, or other related mechanisms may also contribute to drug resistance.

Characterization of Two SHP-2-associated Binding Proteins and Potential Substrates in Hematopoietic Cells

Multiple studies have demonstrated an important role for the Src homology 2-containing tyrosine phosphatase 2 (SHP-2) in receptor tyrosine kinase-regulated cell proliferation and differentiation. Recent studies have identified potential SHP-2 substrates which mediate these effects. SHP-2 also is implicated in several cytokine receptor signaling pathways and in Bcr-Abl transformation. However, its precise role and targets in normal and abnormal hematopoietic cells remain to be determined. We identified two novel tyrosyl-phosphorylated proteins associated with SHP-2 in hematopoietic cells. The first, a 97-kDa cytosolic protein (p97), associates inducibly with SHP-2 upon cytokine stimulation and constitutively in Bcr-Abl-transformed cells. In contrast, p135, a 135-kDa transmembrane glycoprotein, forms a distinct complex with SHP-2, independent of cytokine stimulation or Bcr-Abl transformation. Far Western analysis reveals that SHP-2, via its Src homology 2 domains, can interact directly with either protein. In vitro dephosphorylation experiments, as well as transient transfection studies using wild type and mutant SHP-2 constructs, suggest that p97 and p135 also are SHP-2 substrates. Our results indicate that SHP-2 forms at least two separate complexes in hematopoietic cells and point to new potential SHP-2 targets.

Retroviral Vector Design for Gene Therapy of Cancer: Specific Inhibition and Tagging of BCR-ABLp190 Cells

The main difficulty in providing effective treatment of patients with cancer is distinguishing between tumor and normal cells. The chimeric molecules created by cancer-associated chromosomal abnormalities (such as the BCR-ABL fusion protein) represent ideal therapeutic targets since they are unique to the disease state. A major challenge, however, is how to deliver the specific anti-tumor agent into every tumor cell. In this report we describe the use of a novel strategy to introduce specific anti-tumor reagents into every tumor cell. It uses retroviral vectors encoding both antisense transcripts specific for the BCR-ABLp190 fusion junction (the specific anti-tumor drug) and a truncated human CD5 cDNA, which allows selection of the infected cells. In order to coexpress the antisense molecule with the truncated human CD5 gene, the picornavirus internal ribosome-entry site was incorporated in the constructs. When the antisense transcripts in the CD5-retroviral vector were introduced into Ba/F3+p190 cells rendered interleukin 3 (IL-3) independent by expression of the BCR-ABL sequences, the cells died upon IL-3 withdrawal, as measured by the absence of CD5-positive cells. Control Ba/F3+p210 cells infected with the same virus did not die in the absence of IL-3. These data suggest a novel strategy for cancer treatment which incorporates the use of a retrovirus coexpressing both a selectable surface marker and a tumor-specific agent.