Cancerous inhibitor of protein phosphatase 2A (CIP2A) modifies energy metabolism via 5' AMP-activated protein kinase signalling in malignant cells.

Cancerous inhibitor of protein phosphatase 2A (CIP2A) is an adverse biomarker across many malignancies. Using K562 cells engineered to have high or low CIP2A expression, we show that high CIP2A levels significantly bias cellular energy production towards oxidative phosphorylation (OXPHOS) rather than glycolysis. Mass spectrometric analysis of CIP2A interactors and isobaric tagging for relative and absolute protein quantitation (ITRAQ) experiments identified many associated proteins, several of which co-vary with CIP2A level. Many of these CIP2A associating and co-varying proteins are involved in energy metabolism including OXPHOS, or in 5' AMP-activated protein kinase (AMPK) signalling, and manipulating AMPK activity mimics the effects of low/high CIP2A on OXPHOS. These effects are dependent on the availability of nutrients, driven by metabolic changes caused by CIP2A. CIP2A level did not affect starvation-induced AMPK phosphorylation of Unc-51 autophagy activating kinase 1 (ULK-1) at Ser555, but autophagy activity correlated with an increase in AMPK activity, to suggest that some AMPK processes are uncoupled by CIP2A, likely via its inhibition of protein phosphatase 2A (PP2A). The data demonstrate that AMPK mediates this novel CIP2A effect on energy generation in malignant cells.

The hOCT1 SNPs M420del and M408V alter imatinib uptake and M420del modifies clinical outcome in imatinib-treated chronic myeloid leukemia.

Although the prognosis of chronic myeloid leukemia (CML) patients treated with imatinib is good, many fail to develop an optimal response or lose one. This heterogeneity could be attributed to the presence of human organic cation transporter-1 (hOCT1) single nucleotide polymorphisms (SNPs). In the present study, we analyzed the effect of 23 hOCT1 SNPs on imatinib treatment outcome in newly diagnosed CML patients using MassARRAY sequencing and pyrosequencing. The only SNP associated with outcome was M420del (rs35191146), with patients with the M420del demonstrating an increased probability of imatinib treatment failure. In CML cell lines transfected with M420del and/or M408V, M420del significantly decreased imatinib uptake, but this effect was countered if the M408V (rs628031) SNP was also present. A similar effect was seen for the uptake of the hOCT1 substrates TEA(+) and ASP(+). Finally, apparent hOCT1 mRNA levels were studied using both our earlier primers covering the M420del and another set that did not. Different mRNA expression was observed, explaining the disparity in published data on the prognostic importance of hOCT1 mRNA and highlighting the importance of avoiding common SNP sites in primer design. These data demonstrate that the common M420del SNP can modulate the outcome of imatinib treatment.

Evidence that the pregnane X and retinoid receptors PXR, RAR and RXR may regulate transcription of the transporter hOCT1 in chronic myeloid leukaemia cells.

The expression and activity of the uptake transporter human organic cation transporter 1 (hOCT1; SLC22A1) is an independent predictor of response to imatinib treatment in patients with chronic myeloid leukaemia (CML). We have recently shown that peroxisome proliferator-activated receptor (PPAR) activation can increase the killing effect of imatinib in CML cells, due to upregulated hOCT1 gene expression and increased imatinib uptake. To investigate the role of activation of nuclear receptors other than PPAR in the transcriptional regulation of hOCT1, CML cells were treated with agonists for 13 adopted orphan receptors and endocrine receptors. It was found that hOCT1 expression was upregulated by the agonists for pregnane X receptor (PXR), retinoid acid receptor (RAR) and retinoid X receptor (RXR) in CML cell line and primary CML cells (P = 0.04; Wilcoxon rank test). Hence, agonists for PXR, RAR and RXR may be potentially used to improve the efficacy of imatinib in patients with CML.

Validation of CIP2A as a Biomarker of Subsequent Disease Progression and Treatment Failure in Chronic Myeloid Leukaemia.

BACKGROUND: It would be clinically useful to prospectively identify the risk of disease progression in chronic myeloid leukaemia (CML). Overexpression of cancerous inhibitor of protein phosphatase 2A (PP2A) (CIP2A) protein is an adverse prognostic indicator in many cancers. METHODS: We examined CIP2A protein levels in diagnostic samples from the SPIRIT2 trial in 172 unselected patients, of whom 90 received imatinib and 82 dasatinib as first-line treatment. RESULTS: High CIP2A levels correlated with inferior progression-free survival (p = 0.04) and with worse freedom from progression (p = 0.03), and these effects were confined to dasatinib recipients. High CIP2A levels were associated with a six-fold higher five-year treatment failure rate than low CIP2A levels (41% vs. 7.5%; p = 0.0002), in both imatinib (45% vs. 11%; p = 0.02) and dasatinib recipients (36% vs. 4%; p = 0.007). Imatinib recipients with low CIP2A levels had a greater risk of treatment failure (p = 0.0008). CIP2A levels were independent of Sokal, Hasford, EUTOS (EUropean Treatment and Outcome Study), or EUTOS long-term survival scores (ELTS) or the presence of major route cytogenetic abnormalities. No association was seen between CIP2A levels and time to molecular response or the levels of the CIP2A-related proteins PP2A, SET, SET binding protein 1 (SETBP1), or AKT. CONCLUSIONS: These data confirm that high diagnostic CIP2A levels correlate with subsequent disease progression and treatment failure. CIP2A is a simple diagnostic biomarker that may be useful in planning treatment strategies.

Serotonin re-uptake transporter gene polymorphisms are associated with imatinib-induced diarrhoea in chronic myeloid leukaemia patients.

Tyrosine kinase inhibitors (TKIs), the treatment of choice for chronic myeloid leukaemia (CML), can cause lower gastrointestinal (GI) toxicity which is manifested as diarrhoea. The mechanisms are not fully understood. The enteroendocrine signalling compound, serotonin (5-HT), is important for regulating peristaltic motion, fluid secretion and visceral hypersensitivity in the GI tract, and has been implicated in diseases such as irritable bowel syndrome. In this study, we have evaluated whether TKI-induced diarrhoea may be related to variation in the serotonin re-uptake transporter (SERT) gene. CML patients with and without diarrhoea on the SPIRIT2 trial (imatinib, n = 319; and dasatinib, n = 297) were genotyped for the promoter 5-HTTLPR, intron 2 VNTR and rs25531 polymorphisms by PCR-based methods. Diarrhoea was more prevalent in imatinib, than in dasatinib treated patients (P = 0.015), which when stratified by gender was seen to be driven by female patients (P = 0.036). Logistic regression analysis revealed that age, and the dominant HTTLPR with the rs25531 single nucleotide polymorphism (SNP) model, explained the occurrence of diarrhoea in ~10% of imatinib-treated female CML patients. These data suggest SERT polymorphisms influence imatinib-induced diarrhoea but not that of dasatinib.

Peroxisome proliferator-activated receptor activation increases imatinib uptake and killing of chronic myeloid leukemia cells.

Low pretreatment expression of the imatinib uptake transporter human organic cation transporter 1 (hOCT1) is associated with inferior complete cytogenetic response rates, progression-free survival, and overall survival in imatinib-treated chronic myeloid leukemia (CML). Upregulation of hOCT1 can therefore increase the uptake of imatinib. The hOCT1 gene is transactivated by hepatocyte nuclear factor 4α in human liver, and peroxisome proliferator-activated receptors (PPAR) α and γ activation increases OCT1 expression in mouse hepatocytes. Here we report that no isoform of hepatocyte nuclear factor 4α is expressed in CML lines or in CML primary cells. In contrast, both PPARα and γ were expressed in all CML cell lines and primary cells studied. PPARα agonist treatment increased imatinib killing of CML KCL22 cells and primitive CD34(+) cells, and also upregulates hOCT1 gene expression and increases imatinib uptake into KCL22 cells and primary cells. PPARα agonists might potentially be of clinical use in CML patients failing imatinib.