AMPK-induced novel phosphorylation of RUNX1 inhibits STAT3 activation and overcome imatinib resistance in chronic myelogenous leukemia (CML) subjects.

Imatinib resistance remains an unresolved problem in CML disease. Activation of JAK2/STAT3 pathway and increased expression of RUNX1 have become one reason for development of imatinib resistance in CML subjects. Metformin has gained attention as an antileukemic drug in recent times. However, the molecular mechanism remains elusive. The present study shows that RUNX1 is a novel substrate of AMP-activated kinase (AMPK), where AMPK phosphorylates RUNX1 at Ser 94 position. Activation of AMPK by metformin could lead to increased cytoplasmic retention of RUNX1 due to Ser 94 phosphorylation. RUNX1 Ser 94 phosphorylation resulted in increased interaction with STAT3, which was reflected in reduced transcriptional activity of both RUNX1 and STAT3 due to their cytoplasmic retention. The reduced transcriptional activity of STAT3 and RUNX1 resulted in the down-regulation of their signaling targets involved in proliferation and anti-apoptosis. Our cell proliferation assays using in vitro resistant cell line models and PBMCs isolated from CML clinical patients and normal subjects demonstrate that metformin treatment resulted in reduced growth and improved imatinib sensitivity of resistant subjects.

Robust Reference Intervals for Serum Kappa and Lambda Free Light Chains from a Multi Centre Study Population from Hyderabad, India: Myeloma Diagnostic Implications.

The International Myeloma Working Group considers the serum free light chain (SFLC) assay to be an adjunct to traditional tests. Apart from the FLC ratio, the absolute values of individual free light chains also are gaining importance as they appear to be more relevant in certain clinical settings. Automated assays are available for their determination. As laboratories put new test systems into use catering to different disease populations, they are required by accreditation and certification bodies to verify or establish performance specifications, including reference intervals (RIs) representative of their population. Our aim was to establish local RIs for SFLC in a multicentre representative healthy population using a robust method. There was no significant relationship between SFLC levels and age, gender and creatinine levels. The 95% RI for κSFLC was 4.81 to 33.86mg/L, for ? SFLC was 5.19 to 23.67mg/L and for κ/?SFLC was 0.36 to 2.33, significantly higher than the values given by the manufacturer. The κ/? SFLC ratio at 2.23, covering 100% of the data, showed 72% sensitivity (95% CI=39.0 - 94.0), 100% specificity (95% CI=71.5 - 100.0), 100% PPV (95% CI=21.5 - 100.0), 95% NPV (95% CI=75.4 - 99.9), and 79% accuracy (95% CI=56.0 - 93.0). In the patient group, kit RI for κ /? SFLC ratio classified 45.5% (n=5) as positive vs 9.1% (n=1) positive by the study RI, while the kit RI for kappa FLC classified 90.9% (n=10) as positive vs 54.5% (n=6) , indicating increased probability of false positive test results with the kit RI when applied to our patient population. Appropriate and specific reference intervals and criteria values result in fewer false-positive and false-negative results which means fewer wrong or missed diagnoses.

Correlation of C/EBPα expression with response and resistance to imatinib in chronic myeloid leukaemia.

OBJECTIVE: Altered differentiation is a common feature of haematopoietic malignancies with poor prognosis. CAAT/enhancer binding protein alpha (C/EBPα) is a key transcription factor that regulates myeloid differentiation. This study is aimed to know the prognostic value of CAAT/enhancer binding protein alpha expression and correlate its expression with response to imatinib therapy. METHODS: We quantified the expression of C/EBPα gene in 126 chronic myeloid leukaemia samples (82 from newly diagnosed and 44 from imatinib-resistant patients) and 20 control samples. C/EBPα mRNA level was measured by real-time quantitative polymerase chain reaction using the ΔΔCT method. RESULTS: C/EBPα expression level was significantly lower in the imatinib-resistant group than in the pretreatment and control group (P = 0.0398). Low CAAT/enhancer binding protein alpha levels in the imatinib-resistant group were significantly associated with advanced phase (P = 0.04), with more peripheral blasts (P = 0.0001), high BCR-ABL levels (P = 0.018) and T315I and P-loop mutations (P = 0.0002). In the pretreatment group, low expression showed association with high EUTOS risk score (P = 0.03) and possible partial cytogenetic response (P = 0.010). CONCLUSIONS: Our results suggest that low expression of CAAT/enhancer binding protein alpha might have a role in the response to imatinib and progression of disease in patients with chronic myeloid leukaemia.

Incidence of Bcr-Abl kinase domain mutations in imatinib refractory chronic myeloid leukemia patients from South India.

Mutations in the Bcr-Abl kinase domain (KD) are a major cause for acquired resistance to imatinib (IM) treatment and have been associated with progression and poor prognosis in chronic myeloid leukemia patients. The present study includes 63 patients resistant to standard imatinib dose of 400 mg according to ELN guidelines. Direct sequencing method is used for mutational analysis. The present study revealed 15 exonic mutations in 46.03 % of patients; among them, seven cases (24.13 %) had multiple mutations. Mutations were found to be higher in sokal high- (45.0 %) and intermediate- (68.42 %) compared to low-risk (29.16 %) group. Mutations were observed in 38.09 % of patients with EUTOS (European Treatment and Outcome Study) high risk and in 50.0 % with low risk. The frequency of mutations was 50.0 % in advanced phase, 47.36 % in late chronic-phase, and 43.33 % in chronic-phase patients. 42.10 % of patients with primary resistance and 52.0 % with secondary resistance had mutations. P-loop and T315I mutations were associated with poor survival in advanced phase patients (85.71 %) (P = 0.03). No significant variation was observed with Bcr-Abl transcript levels between the patients with the presence or absence of mutations (P = 0.73). Bcr-Abl levels were found to be significantly elevated in P-loop and T315I mutation carriers (P = 0.001) and also in T315I mutation-positive patients (P = 0.01). P-loop mutations and T315I are frequent in advanced phases and strongly associated with poor prognosis and survival. Hence, the identification of mutations in IM-resistant CML patients will help in treatment optimization with 2nd- or 3rd-generation tyrosine kinase inhibitors (TKIs).

Early molecular response in chronic myeloid leukemia patients predicts future response status.

Imatinib is the frontline therapy for chronic myeloid leukemia (CML) management. Most of the CML patients achieve major responses, but a proportion (nearly 25-35%) of them develop drug resistance. Molecular monitoring using quantitative real-time PCR at regular intervals according to European LeukemiaNet (ELN) helps in the assessment of long-term outcomes in imatinib-treated CML patients. Eighty-four CML patient samples (42 at diagnosis and 42 at 3-month intervals from the same patients) were analyzed for Bcr-Abl transcript levels. Quantification results revealed that the patients with <10% Bcr-Abl levels at 3 months had higher rates of complete cytogenetic response (CCyR) and optimal responses compared to patients with >10% Bcr-Abl levels (P < 0.0001). Patients with >10% Bcr-Abl levels were found to have 25.0% of suboptimal response and 3.57% of failure to imatinib at standard dose. Hence, the present study confirms that early molecular monitoring at 3 months after imatinib initiation helps in predicting the concurrent cytogenetic response and treatment optimization in CML patients.