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Functional protein network activation mapping reveals new potential molecular drug targets for poor prognosis pediatric BCP-ALL.

Accordi B, Espina V, Giordan M, VanMeter A, Milani G, Galla L, Ruzzene M, Sciro M, Trentin L, De Maria R, te Kronnie G, Petricoin E, Liotta L, Basso G - PLoS ONE (2010)

Bottom Line: Further we also found an association between high levels of CYCLIN E and relapse incidence.Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis.We conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.

View Article: PubMed Central - PubMed

Affiliation: Oncohematology Laboratory, Department of Pediatrics, University of Padova, Padova, Italy. benedetta.accordi@unipd.it

ABSTRACT

Background: In spite of leukemia therapy improvements obtained over the last decades, therapy is not yet effective in all cases. Current approaches in Acute Lymphoblastic Leukemia (ALL) research focus on identifying new molecular targets to improve outcome for patients with a dismal prognosis. In this light phosphoproteomics seems to hold great promise for the identification of proteins suitable for targeted therapy.

Methodology/principal findings: We employed Reverse Phase Protein Microarrays to identify aberrantly activated proteins in 118 pediatric B-cell precursor (BCP)-ALL patients. Signal transduction pathways were assayed for activation/expression status of 92 key signalling proteins. We observed an increased activation/expression of several pathways involved in cell proliferation in poor clinical prognosis patients. MLL-rearranged tumours revealed BCL-2 hyperphosphorylation through AMPK activation, which indicates that AMPK could provide a functional role in inhibiting apoptosis in MLL-rearranged patients, and could be considered as a new potential therapeutic target. Second, in patients with poor clinical response to prednisone we observed the up-modulation of LCK activity with respect to patients with good response. This tyrosine-kinase can be down-modulated with clinically used inhibitors, thus modulating LCK activity could be considered for further studies as a new additional therapy for prednisone-resistant patients. Further we also found an association between high levels of CYCLIN E and relapse incidence. Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis.

Conclusions/significance: We conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.

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Related in: MedlinePlus

Validation of RPMA results through Western Blot.(A) Hyperactivation of the AMPK pathway in MLL-rearranged patients vs non-translocated ones (independent sets of pediatric BCP-ALL at diagnosis: patients 11–15 are MLL-rearranged -all MLL-AF4-, patients 16–20 are non-translocated). RS4;11 cell lysate was used as positive control for antibody staining. (B) Total forms of the AMPK pathway proteins in previously described patients: 11–15 are MLL-rearranged and 16–20 are non-rearranged. RS4;11 cell lysate was used as positive control for antibody staining. There are no substantial differences on total protein form levels between MLL-rearranged and non-translocated patients. (C) AMPK pathway inhibition after Compound C treatment. RS4;11 and SEM cells (both MLL-rearranged) were treated with the AMPK inhibitor Compound C 8 µM for 48 hours. Phosphorylation of AMPK pathway proteins was evaluated through WB in control, DMSO treated and Compound C treated cells.
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pone-0013552-g002: Validation of RPMA results through Western Blot.(A) Hyperactivation of the AMPK pathway in MLL-rearranged patients vs non-translocated ones (independent sets of pediatric BCP-ALL at diagnosis: patients 11–15 are MLL-rearranged -all MLL-AF4-, patients 16–20 are non-translocated). RS4;11 cell lysate was used as positive control for antibody staining. (B) Total forms of the AMPK pathway proteins in previously described patients: 11–15 are MLL-rearranged and 16–20 are non-rearranged. RS4;11 cell lysate was used as positive control for antibody staining. There are no substantial differences on total protein form levels between MLL-rearranged and non-translocated patients. (C) AMPK pathway inhibition after Compound C treatment. RS4;11 and SEM cells (both MLL-rearranged) were treated with the AMPK inhibitor Compound C 8 µM for 48 hours. Phosphorylation of AMPK pathway proteins was evaluated through WB in control, DMSO treated and Compound C treated cells.

Mentions: We compared primary leukemia samples isolated from 8 MLL-rearranged patients (5 with t(4;11), 2 with t(9;11), and one with t(11;19)) with 36 patients without known genomic aberrancies. Statistical analysis (Wilcoxon test with Benjamini-Hochberg multiplicity corrections) revealed different expression or activation of 9 proteins between the MLL-rearranged patients and the non-translocated ones. Our results show that 4 proteins were statistically significantly elevated in the MLL-rearranged patients group: CYCLIN E (p = 0.02425), ANNEXIN 2 (p = 0.02910), AMPKβ (S108) (p = 0.02910) and AMPKα (S485) (p = 0.03686). Furthermore a set of 3 more proteins, eNOS/NOS III (S116 – corresponding to S114 in human), LKB1 (S428) and BCL-2 (S70), was found to be differentially activated in the MLL-rearranged cohort using a Global Test analysis (p = 0.003) (Figure 1A). These 3 proteins are all known members of the AMPK pathway (see Discussion) and, along with the findings that AMPK itself was activated in the MLL-rearranged cohort, form the basis of a comprehensive pathway derangement in MLL-rearranged patients (presented schematically in Figure 1B). We thus identified a singular MLL-specific hyperactivated pathway that through AMPK phosphorylation leads to the activation of BCL-2. A heatmap was generated to highlight the relationships between clustering and protein expression levels (Figure 1C). RPMA results were validated by Western Blot in an independent set of patients (Figure 2A). Of note, total forms of the AMPK pathway proteins do not show substantial differences between MLL-rearranged and non-translocated patients (Figure 2B), corroborating the observation that the higher phosphorylation levels of the proteins in the AMPK pathway are the peculiar molecular derangement characteristic of MLL-rearranged BCP-ALL.


Functional protein network activation mapping reveals new potential molecular drug targets for poor prognosis pediatric BCP-ALL.

Accordi B, Espina V, Giordan M, VanMeter A, Milani G, Galla L, Ruzzene M, Sciro M, Trentin L, De Maria R, te Kronnie G, Petricoin E, Liotta L, Basso G - PLoS ONE (2010)

Validation of RPMA results through Western Blot.(A) Hyperactivation of the AMPK pathway in MLL-rearranged patients vs non-translocated ones (independent sets of pediatric BCP-ALL at diagnosis: patients 11–15 are MLL-rearranged -all MLL-AF4-, patients 16–20 are non-translocated). RS4;11 cell lysate was used as positive control for antibody staining. (B) Total forms of the AMPK pathway proteins in previously described patients: 11–15 are MLL-rearranged and 16–20 are non-rearranged. RS4;11 cell lysate was used as positive control for antibody staining. There are no substantial differences on total protein form levels between MLL-rearranged and non-translocated patients. (C) AMPK pathway inhibition after Compound C treatment. RS4;11 and SEM cells (both MLL-rearranged) were treated with the AMPK inhibitor Compound C 8 µM for 48 hours. Phosphorylation of AMPK pathway proteins was evaluated through WB in control, DMSO treated and Compound C treated cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2958847&req=5

pone-0013552-g002: Validation of RPMA results through Western Blot.(A) Hyperactivation of the AMPK pathway in MLL-rearranged patients vs non-translocated ones (independent sets of pediatric BCP-ALL at diagnosis: patients 11–15 are MLL-rearranged -all MLL-AF4-, patients 16–20 are non-translocated). RS4;11 cell lysate was used as positive control for antibody staining. (B) Total forms of the AMPK pathway proteins in previously described patients: 11–15 are MLL-rearranged and 16–20 are non-rearranged. RS4;11 cell lysate was used as positive control for antibody staining. There are no substantial differences on total protein form levels between MLL-rearranged and non-translocated patients. (C) AMPK pathway inhibition after Compound C treatment. RS4;11 and SEM cells (both MLL-rearranged) were treated with the AMPK inhibitor Compound C 8 µM for 48 hours. Phosphorylation of AMPK pathway proteins was evaluated through WB in control, DMSO treated and Compound C treated cells.
Mentions: We compared primary leukemia samples isolated from 8 MLL-rearranged patients (5 with t(4;11), 2 with t(9;11), and one with t(11;19)) with 36 patients without known genomic aberrancies. Statistical analysis (Wilcoxon test with Benjamini-Hochberg multiplicity corrections) revealed different expression or activation of 9 proteins between the MLL-rearranged patients and the non-translocated ones. Our results show that 4 proteins were statistically significantly elevated in the MLL-rearranged patients group: CYCLIN E (p = 0.02425), ANNEXIN 2 (p = 0.02910), AMPKβ (S108) (p = 0.02910) and AMPKα (S485) (p = 0.03686). Furthermore a set of 3 more proteins, eNOS/NOS III (S116 – corresponding to S114 in human), LKB1 (S428) and BCL-2 (S70), was found to be differentially activated in the MLL-rearranged cohort using a Global Test analysis (p = 0.003) (Figure 1A). These 3 proteins are all known members of the AMPK pathway (see Discussion) and, along with the findings that AMPK itself was activated in the MLL-rearranged cohort, form the basis of a comprehensive pathway derangement in MLL-rearranged patients (presented schematically in Figure 1B). We thus identified a singular MLL-specific hyperactivated pathway that through AMPK phosphorylation leads to the activation of BCL-2. A heatmap was generated to highlight the relationships between clustering and protein expression levels (Figure 1C). RPMA results were validated by Western Blot in an independent set of patients (Figure 2A). Of note, total forms of the AMPK pathway proteins do not show substantial differences between MLL-rearranged and non-translocated patients (Figure 2B), corroborating the observation that the higher phosphorylation levels of the proteins in the AMPK pathway are the peculiar molecular derangement characteristic of MLL-rearranged BCP-ALL.

Bottom Line: Further we also found an association between high levels of CYCLIN E and relapse incidence.Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis.We conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.

View Article: PubMed Central - PubMed

Affiliation: Oncohematology Laboratory, Department of Pediatrics, University of Padova, Padova, Italy. benedetta.accordi@unipd.it

ABSTRACT

Background: In spite of leukemia therapy improvements obtained over the last decades, therapy is not yet effective in all cases. Current approaches in Acute Lymphoblastic Leukemia (ALL) research focus on identifying new molecular targets to improve outcome for patients with a dismal prognosis. In this light phosphoproteomics seems to hold great promise for the identification of proteins suitable for targeted therapy.

Methodology/principal findings: We employed Reverse Phase Protein Microarrays to identify aberrantly activated proteins in 118 pediatric B-cell precursor (BCP)-ALL patients. Signal transduction pathways were assayed for activation/expression status of 92 key signalling proteins. We observed an increased activation/expression of several pathways involved in cell proliferation in poor clinical prognosis patients. MLL-rearranged tumours revealed BCL-2 hyperphosphorylation through AMPK activation, which indicates that AMPK could provide a functional role in inhibiting apoptosis in MLL-rearranged patients, and could be considered as a new potential therapeutic target. Second, in patients with poor clinical response to prednisone we observed the up-modulation of LCK activity with respect to patients with good response. This tyrosine-kinase can be down-modulated with clinically used inhibitors, thus modulating LCK activity could be considered for further studies as a new additional therapy for prednisone-resistant patients. Further we also found an association between high levels of CYCLIN E and relapse incidence. Moreover, CYCLIN E is more expressed in early relapsed patients, who usually show an unfavourable prognosis.

Conclusions/significance: We conclude that functional protein pathway activation mapping revealed specific deranged signalling networks in BCP-ALL that could be potentially modulated to produce a better clinical outcome for patients resistant to standard-of-care therapies.

Show MeSH
Related in: MedlinePlus