Regulation of hTERT by BCR-ABL at multiple levels in K562 cells

<p>Abstract</p> <p>Background</p> <p>The cytogenetic characteristic of Chronic Myeloid Leukemia (CML) is the formation of the Philadelphia chromosome gene product, BCR-ABL. Given that BCR-ABL is the specific target of Gleevec in CML treatment, we investigated the regulation of the catalytic component of telomerase, hTERT, by BCR-ABL at multiple levels in K562 cells.</p> <p>Methods</p> <p>Molecular techniques such as over expression, knockdown, real-time PCR, immunoprecipitation, western blotting, reporter assay, confocal microscopy, telomerase assays and microarray were used to suggest that hTERT expression and activity is modulated by BCR-ABL at multiple levels.</p> <p>Results</p> <p>Our results suggest that BCR-ABL plays an important role in regulating hTERT in K562 (BCR-ABL positive human leukemia) cells. When Gleevec inhibited the tyrosine kinase activity of BCR-ABL, phosphorylation of hTERT was downregulated, therefore suggesting a positive correlation between BCR-ABL and hTERT. Gleevec treatment inhibited <it>hTERT </it>at mRNA level and significantly reduced telomerase activity (TA) in K562 cells, but not in HL60 or Jurkat cells (BCR-ABL negative cells). We also demonstrated that the transcription factor STAT5a plays a critical role in <it>hTERT </it>gene regulation in K562 cells. Knockdown of STAT5a, but not STAT5b, resulted in a marked downregulation of <it>hTERT </it>mRNA level, TA and hTERT protein level in K562 cells. Furthermore, translocation of hTERT from nucleoli to nucleoplasm was observed in K562 cells induced by Gleevec.</p> <p>Conclusions</p> <p>Our data reveal that BCR-ABL can regulate TA at multiple levels, including transcription, post-translational level, and proper localization. Thus, suppression of cell growth and induction of apoptosis by Gleevec treatment may be partially due to TA inhibition. Additionally, we have identified STAT5a as critical mediator of the <it>hTERT </it>gene expression in BCR-ABL positive CML cells, suggesting that targeting STAT5a may be a promising therapeutic strategy for BCR-ABL positive CML patients.</p

A Novel murine model identifies cooperating mutations and therapeutic targets critical for chronic myeloid leukemia progression

The introduction of highly selective ABL-tyrosine kinase inhibitors (TKIs) has revolutionized therapy for chronic myeloid leukemia (CML). However, TKIs are only efficacious in the chronic phase of the disease and effective therapies for TKI-refractory CML, or after progression to blast crisis (BC), are lacking. Whereas the chronic phase of CML is dependent on BCR-ABL, additional mutations are required for progression to BC. However, the identity of these mutations and the pathways they affect are poorly understood, hampering our ability to identify therapeutic targets and improve outcomes. Here, we describe a novel mouse model that allows identification of mechanisms of BC progression in an unbiased and tractable manner, using transposon-based insertional mutagenesis on the background of chronic phase CML. Our BC model is the first to faithfully recapitulate the phenotype, cellular and molecular biology of human CML progression. We report a heterogeneous and unique pattern of insertions identifying known and novel candidate genes and demonstrate that these pathways drive disease progression and provide potential targets for novel therapeutic strategies. Our model greatly informs the biology of CML progression and provides a potent resource for the development of candidate therapies to improve the dismal outcomes in this highly aggressive disease.Work in the Huntly laboratory is funded by CRUK, The European Research Council (ERC), Leukaemia Lymphoma Research, the Kay Kendall Leukaemia Fund, Wellcome Trust, the Medical Research Council (UK), the Leukemia Lymphoma Society America and the Cambridge NIHR Biomedical Research centre. David Adams is funded by Cancer Research UK and Wellcome Trust. Steffen Koschmieder has received funding from Deutsche José Carreras Leukämie-Stiftung (DJCLS; grant 10/23).This is the final published version. It first appeared at http://dx.doi.org/10.1084/jem.2014166

Methotrexate Is a JAK/STAT Pathway Inhibitor

Background: The JAK/STAT pathway transduces signals from multiple cytokines and controls haematopoiesis, immunity and inflammation. In addition, pathological activation is seen in multiple malignancies including the myeloproliferative neoplasms (MPNs). Given this, drug development efforts have targeted the pathway with JAK inhibitors such as ruxolitinib. Although effective, high costs and side effects have limited its adoption. Thus, a need for effective low cost treatments remains. Methods & Findings: We used the low-complexity Drosophila melanogaster pathway to screen for small molecules that modulate JAK/STAT signalling. This screen identified methotrexate and the closely related aminopterin as potent suppressors of STAT activation. We show that methotrexate suppresses human JAK/STAT signalling without affecting other phosphorylation-dependent pathways. Furthermore, methotrexate significantly reduces STAT5 phosphorylation in cells expressing JAK2 V617F, a mutation associated with most human MPNs. Methotrexate acts independently of dihydrofolate reductase (DHFR) and is comparable to the JAK1/2 inhibitor ruxolitinib. However, cells treated with methotrexate still retain their ability to respond to physiological levels of the ligand erythropoietin. Conclusions: Aminopterin and methotrexate represent the first chemotherapy agents developed and act as competitive inhibitors of DHFR. Methotrexate is also widely used at low doses to treat inflammatory and immune-mediated conditions including rheumatoid arthritis. In this low-dose regime, folate supplements are given to mitigate side effects by bypassing the biochemical requirement for DHFR. Although independent of DHFR, the mechanism-of-action underlying the low-dose effects of methotrexate is unknown. Given that multiple pro-inflammatory cytokines signal through the pathway, we suggest that suppression of the JAK/STAT pathway is likely to be the principal anti-inflammatory and immunosuppressive mechanism-of-action of low-dose methotrexate. In addition, we suggest that patients with JAK/STAT-associated haematological malignancies may benefit from low-dose methotrexate treatments. While the JAK1/2 inhibitor ruxolitinib is effective, a £43,200 annual cost precludes widespread adoption. With an annual methotrexate cost of around £32, our findings represent an important development with significant future potential

A Multicenter Study Evaluating the Efficacy and Safety of Bendamustine in the Treatment of Mantle Cell Lymphoma

Abstract Abstract 4940 Background: Bendamustine has evolved as an increasingly used chemotherapeutic agent for diverse lymphomas in the United States (US) and Europe. It is an alkylating agent that was approved in the US for use as a first line therapy for chronic lymphocytic leukemia and relapsed indolent B-cell non-Hodgkin's Lymphoma (NHL) in 2008. However, the two pivotal studies that established bendamustine for this indication included only few patients with mantle cell lymphoma (MCL). The goal of this retrospective study was to evaluate efficacy of bendamustine in MCL. Patients and Methods: We retrospectively reviewed the records of all MCL patients who were treated with bendamustine at Jackson Memorial Hospital and the Sylvester Comprehensive Cancer Center (both in Miami, FL) and the Stanford Cancer Center (in Stanford, CA). The primary endpoint was overall response rate (ORR) and secondary endpoints were overall survival (OS), time to treatment failure (TTF), and safety. Results: Thirty MCL patients received bendamustine, 28 in combination with rituximab and 2 as monotherapy. Nineteen of these patients were treated at the University of Miami and 11 at Stanford. The median age at diagnosis was 58 years and 77% of the patients were male. The most common extranodal site of involvement at diagnosis was the bone marrow (57%), followed by the GI tract (33%). Most patients presented with advanced disease, with 69% and 28% of the patients having stage IV and III disease at diagnosis, respectively. Seven patients (23%) were diagnosed with the blastic variant of MCL. Five patients received bendamustine as initial therapy and 25 for relapsed disease. Five of these patients had relapsed MCL after an autologous HSCT The most common schedule was bendamustine 100mg/m2 on days one and two and rituximab 375mg/m2 on day one of a 28 day cycle, for a median of six cycles. Of the 30 patients, there were fifteen complete responses (CR) and ten partial responses (PR), for an ORR of 83% (95% confidence interval [CI] 70–97%). With a median follow-up of 8 months (range 0.3–29.5 months), the median overall survival (OS) was not reached; the one-year OS rate was 73%; the two year OS rate was 67%. When OS and time to treatment failure (TTF) were stratified by response (using a 2-month landmark as the starting point for analysis), patients achieving a CR had a significantly longer OS and TTF compared to patients who did not achieve a CR (log rank p < 0.001). The TTF and OS were significantly worse for patients with the blastic variant versus the diffuse variant. In 5 patients with relapse post autologous HSCT, bendamustine induced a CR in three and a PR in one other patient. The majority of serious adverse events associated with bendamustine were hematological. Grade 3 or 4 neutropenia, anemia, and thrombocytopenia occurred in 23%, 3%, and 20%, respectively. Common non-hematologic grade 1 or 2 toxicities were fatigue (27%), edema (20%), erythematous rash (20%), and nausea (13%). Two patients developed neurological sequelae during or after treatment with bendamustine. The first patient developed left sided weakness and dysmetria ten months after completing therapy. A brain biopsy revealed progressive multifocal leukoencephalopathy. A second patient developed ataxia and incontinence while on rituximab plus bendamustine therapy and expired before a definitive diagnosis could be established. Conclusions: Bendamustine appears to have a favorable profile as a second-line agent for patients with relapsed MCL. The response rate is at least comparable to other therapies, especially when examined in light of the toxicity profile seen with other regimens. This study supports the need for continued investigation of bendamustine as a therapy for MCL patients both in the frontline and relapsed settings in prospective clinical trials, some of which are presently ongoing. Disclosures: No relevant conflicts of interest to declare