PROTEOMIC ANALYSIS OF RELAPSE AML IDENTIFIES OPPORTUNITIES FOR THERAPEUTIC INTERVENTION IN INDIVIDUAL PATIENTS
(Abstract release date: 05/19/16)
EHA Library. Britton D. 06/09/16; 135353; LB2242
Dr. David Britton
Contributions
Contributions
Abstract
Abstract: LB2242
Type: Eposter Presentation
Background
A poor outcome in patients with acute myeloid leukaemia (AML) is usually related to chemorefractory disease or relapse after an initial response. Standard therapy generally includes days 1-3 daunorubicin (D) and days 1-7 cytarabine (A) followed by several similar courses should complete remission be achieved. Resistance of AML blasts to such treatment can be attributed to the activity of pro-survival enzymes, some of which can be inhibited by pharmacological inhibitors, therefore could potentially be repurposed for relapsed AML in a case by case basis. However, finding the right inhibitor for the right patient presents a challenge due to the plethora of possible causes of resistance in each case. Liquid chromatography – tandem mass spectrometry (LC-MS/MS) proteomics is a powerful discovery technology enabling quantification of global protein expression and enzymatic activity in samples. Hence this approach may be an effective way to identify suitable drug targets in biopsies and better understand the biochemistry of cells following chemotherapy.
Aims
Our primary aim was to identify biochemical pathways modulated in AML blasts and cell lines as a result of treatment with standard chemotherapy. We also aimed to test pharmaceutical alternatives to personalise AML treatment following thorough investigation into the expression and activity of protein drug targets in AML blasts and cell lines before and after chemotherapy.
Methods
We used LC-MS/MS proteomics and phosphoproteomics to investigate global protein expression and kinase activity in 19 matched diagnosis and relapse AML biopsies, as well as in 3 AML cell lines before and after chemotherapy. Briefly, we collected frozen biopsy specimens from Barts and the London tissue bank. After thawing the AML blasts were incubated in FBS containing media for 2 hr at 37oC. Cell lines (HL60, MV411 and p31Fuj) were treated ± D and/or A (2, 6, or 24 hr). After incubation, cells were centrifuged and washed in PBS, then proteins extracted in urea lysis buffer. For proteomics, proteins were digested with trypsin, and resulting peptides analysed directly by LC-MS/MS. For phosphoproteomics, phosphorylated peptides were first enriched using TiO2 prior to analysis. Commercial (Mascot) and in-house (Pescal, KSEA) software were utilised to identify and quantify proteins, determine kinase activities and investigate intracellular signalling. Cell Viability of blasts ± treatments were recorded using the Guava ViaCount Reagent and Cytometer.
Results
On average we identified >3000 proteins and >9,000 phosphorylation sites per sample. We observed that AML blasts showed high expression and activity of enzymes involved in DNA repair (e.g. PARP1, ATR and PRKDC) at diagnosis and several increased significantly after relapse (e.g. PLK3 and APEX1). Pro-survival signalling pathways and those regulating apoptosis and metabolism were also modulated after relapse but these were patient specific. AML cell lines were more sensitive to D than A. HL60s were most sensitive while p31Fuj cells were least sensitive. Chemotherapy induced significant increase in activity of ATM, ATR, PRKDC and CHEK2. Simultaneous inhibition of ATM and ATR significantly reduced cell viability ± A.
Conclusion
We identified the most abundant and active protein drug targets in AML primary samples and cell lines. Treating AML cell lines with chemotherapy uncovered biochemical pathways involved in DNA repair and survival that determined sensitivity or resistance to these drugs. Proteins significantly modulated in expression and activity after relapse include those involved in DNA repair and pro-survival but these were patient specific, suggesting that targeted therapies will have to be personalized.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, DNA repair, Proteomics, Relapse
Type: Eposter Presentation
Background
A poor outcome in patients with acute myeloid leukaemia (AML) is usually related to chemorefractory disease or relapse after an initial response. Standard therapy generally includes days 1-3 daunorubicin (D) and days 1-7 cytarabine (A) followed by several similar courses should complete remission be achieved. Resistance of AML blasts to such treatment can be attributed to the activity of pro-survival enzymes, some of which can be inhibited by pharmacological inhibitors, therefore could potentially be repurposed for relapsed AML in a case by case basis. However, finding the right inhibitor for the right patient presents a challenge due to the plethora of possible causes of resistance in each case. Liquid chromatography – tandem mass spectrometry (LC-MS/MS) proteomics is a powerful discovery technology enabling quantification of global protein expression and enzymatic activity in samples. Hence this approach may be an effective way to identify suitable drug targets in biopsies and better understand the biochemistry of cells following chemotherapy.
Aims
Our primary aim was to identify biochemical pathways modulated in AML blasts and cell lines as a result of treatment with standard chemotherapy. We also aimed to test pharmaceutical alternatives to personalise AML treatment following thorough investigation into the expression and activity of protein drug targets in AML blasts and cell lines before and after chemotherapy.
Methods
We used LC-MS/MS proteomics and phosphoproteomics to investigate global protein expression and kinase activity in 19 matched diagnosis and relapse AML biopsies, as well as in 3 AML cell lines before and after chemotherapy. Briefly, we collected frozen biopsy specimens from Barts and the London tissue bank. After thawing the AML blasts were incubated in FBS containing media for 2 hr at 37oC. Cell lines (HL60, MV411 and p31Fuj) were treated ± D and/or A (2, 6, or 24 hr). After incubation, cells were centrifuged and washed in PBS, then proteins extracted in urea lysis buffer. For proteomics, proteins were digested with trypsin, and resulting peptides analysed directly by LC-MS/MS. For phosphoproteomics, phosphorylated peptides were first enriched using TiO2 prior to analysis. Commercial (Mascot) and in-house (Pescal, KSEA) software were utilised to identify and quantify proteins, determine kinase activities and investigate intracellular signalling. Cell Viability of blasts ± treatments were recorded using the Guava ViaCount Reagent and Cytometer.
Results
On average we identified >3000 proteins and >9,000 phosphorylation sites per sample. We observed that AML blasts showed high expression and activity of enzymes involved in DNA repair (e.g. PARP1, ATR and PRKDC) at diagnosis and several increased significantly after relapse (e.g. PLK3 and APEX1). Pro-survival signalling pathways and those regulating apoptosis and metabolism were also modulated after relapse but these were patient specific. AML cell lines were more sensitive to D than A. HL60s were most sensitive while p31Fuj cells were least sensitive. Chemotherapy induced significant increase in activity of ATM, ATR, PRKDC and CHEK2. Simultaneous inhibition of ATM and ATR significantly reduced cell viability ± A.
Conclusion
We identified the most abundant and active protein drug targets in AML primary samples and cell lines. Treating AML cell lines with chemotherapy uncovered biochemical pathways involved in DNA repair and survival that determined sensitivity or resistance to these drugs. Proteins significantly modulated in expression and activity after relapse include those involved in DNA repair and pro-survival but these were patient specific, suggesting that targeted therapies will have to be personalized.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, DNA repair, Proteomics, Relapse
Abstract: LB2242
Type: Eposter Presentation
Background
A poor outcome in patients with acute myeloid leukaemia (AML) is usually related to chemorefractory disease or relapse after an initial response. Standard therapy generally includes days 1-3 daunorubicin (D) and days 1-7 cytarabine (A) followed by several similar courses should complete remission be achieved. Resistance of AML blasts to such treatment can be attributed to the activity of pro-survival enzymes, some of which can be inhibited by pharmacological inhibitors, therefore could potentially be repurposed for relapsed AML in a case by case basis. However, finding the right inhibitor for the right patient presents a challenge due to the plethora of possible causes of resistance in each case. Liquid chromatography – tandem mass spectrometry (LC-MS/MS) proteomics is a powerful discovery technology enabling quantification of global protein expression and enzymatic activity in samples. Hence this approach may be an effective way to identify suitable drug targets in biopsies and better understand the biochemistry of cells following chemotherapy.
Aims
Our primary aim was to identify biochemical pathways modulated in AML blasts and cell lines as a result of treatment with standard chemotherapy. We also aimed to test pharmaceutical alternatives to personalise AML treatment following thorough investigation into the expression and activity of protein drug targets in AML blasts and cell lines before and after chemotherapy.
Methods
We used LC-MS/MS proteomics and phosphoproteomics to investigate global protein expression and kinase activity in 19 matched diagnosis and relapse AML biopsies, as well as in 3 AML cell lines before and after chemotherapy. Briefly, we collected frozen biopsy specimens from Barts and the London tissue bank. After thawing the AML blasts were incubated in FBS containing media for 2 hr at 37oC. Cell lines (HL60, MV411 and p31Fuj) were treated ± D and/or A (2, 6, or 24 hr). After incubation, cells were centrifuged and washed in PBS, then proteins extracted in urea lysis buffer. For proteomics, proteins were digested with trypsin, and resulting peptides analysed directly by LC-MS/MS. For phosphoproteomics, phosphorylated peptides were first enriched using TiO2 prior to analysis. Commercial (Mascot) and in-house (Pescal, KSEA) software were utilised to identify and quantify proteins, determine kinase activities and investigate intracellular signalling. Cell Viability of blasts ± treatments were recorded using the Guava ViaCount Reagent and Cytometer.
Results
On average we identified >3000 proteins and >9,000 phosphorylation sites per sample. We observed that AML blasts showed high expression and activity of enzymes involved in DNA repair (e.g. PARP1, ATR and PRKDC) at diagnosis and several increased significantly after relapse (e.g. PLK3 and APEX1). Pro-survival signalling pathways and those regulating apoptosis and metabolism were also modulated after relapse but these were patient specific. AML cell lines were more sensitive to D than A. HL60s were most sensitive while p31Fuj cells were least sensitive. Chemotherapy induced significant increase in activity of ATM, ATR, PRKDC and CHEK2. Simultaneous inhibition of ATM and ATR significantly reduced cell viability ± A.
Conclusion
We identified the most abundant and active protein drug targets in AML primary samples and cell lines. Treating AML cell lines with chemotherapy uncovered biochemical pathways involved in DNA repair and survival that determined sensitivity or resistance to these drugs. Proteins significantly modulated in expression and activity after relapse include those involved in DNA repair and pro-survival but these were patient specific, suggesting that targeted therapies will have to be personalized.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, DNA repair, Proteomics, Relapse
Type: Eposter Presentation
Background
A poor outcome in patients with acute myeloid leukaemia (AML) is usually related to chemorefractory disease or relapse after an initial response. Standard therapy generally includes days 1-3 daunorubicin (D) and days 1-7 cytarabine (A) followed by several similar courses should complete remission be achieved. Resistance of AML blasts to such treatment can be attributed to the activity of pro-survival enzymes, some of which can be inhibited by pharmacological inhibitors, therefore could potentially be repurposed for relapsed AML in a case by case basis. However, finding the right inhibitor for the right patient presents a challenge due to the plethora of possible causes of resistance in each case. Liquid chromatography – tandem mass spectrometry (LC-MS/MS) proteomics is a powerful discovery technology enabling quantification of global protein expression and enzymatic activity in samples. Hence this approach may be an effective way to identify suitable drug targets in biopsies and better understand the biochemistry of cells following chemotherapy.
Aims
Our primary aim was to identify biochemical pathways modulated in AML blasts and cell lines as a result of treatment with standard chemotherapy. We also aimed to test pharmaceutical alternatives to personalise AML treatment following thorough investigation into the expression and activity of protein drug targets in AML blasts and cell lines before and after chemotherapy.
Methods
We used LC-MS/MS proteomics and phosphoproteomics to investigate global protein expression and kinase activity in 19 matched diagnosis and relapse AML biopsies, as well as in 3 AML cell lines before and after chemotherapy. Briefly, we collected frozen biopsy specimens from Barts and the London tissue bank. After thawing the AML blasts were incubated in FBS containing media for 2 hr at 37oC. Cell lines (HL60, MV411 and p31Fuj) were treated ± D and/or A (2, 6, or 24 hr). After incubation, cells were centrifuged and washed in PBS, then proteins extracted in urea lysis buffer. For proteomics, proteins were digested with trypsin, and resulting peptides analysed directly by LC-MS/MS. For phosphoproteomics, phosphorylated peptides were first enriched using TiO2 prior to analysis. Commercial (Mascot) and in-house (Pescal, KSEA) software were utilised to identify and quantify proteins, determine kinase activities and investigate intracellular signalling. Cell Viability of blasts ± treatments were recorded using the Guava ViaCount Reagent and Cytometer.
Results
On average we identified >3000 proteins and >9,000 phosphorylation sites per sample. We observed that AML blasts showed high expression and activity of enzymes involved in DNA repair (e.g. PARP1, ATR and PRKDC) at diagnosis and several increased significantly after relapse (e.g. PLK3 and APEX1). Pro-survival signalling pathways and those regulating apoptosis and metabolism were also modulated after relapse but these were patient specific. AML cell lines were more sensitive to D than A. HL60s were most sensitive while p31Fuj cells were least sensitive. Chemotherapy induced significant increase in activity of ATM, ATR, PRKDC and CHEK2. Simultaneous inhibition of ATM and ATR significantly reduced cell viability ± A.
Conclusion
We identified the most abundant and active protein drug targets in AML primary samples and cell lines. Treating AML cell lines with chemotherapy uncovered biochemical pathways involved in DNA repair and survival that determined sensitivity or resistance to these drugs. Proteins significantly modulated in expression and activity after relapse include those involved in DNA repair and pro-survival but these were patient specific, suggesting that targeted therapies will have to be personalized.
Session topic: E-poster
Keyword(s): Acute myeloid leukemia, DNA repair, Proteomics, Relapse
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