Quantitative single cell determination of ERK phosphorylation and regulation in relapsed and refractory primary acute myeloid leukemia

Abstract

We investigated the constitutive activation of the MEK/ERK pathway in acute myelogenous leukemia (AML) via a flow cytometric technique to quantitate expression of phosphorylated ERK (p-ERK). A total of 42 AML samples (16 newly diagnosed, 26 relapsed/refractory) were analyzed. Normal bone marrow CD34(+) cells (n = 10) had little or no expression of p-ERK, while G-CSF-mobilized CD34(+) cells exhibited enhanced p-ERK levels. Markedly elevated p- ERK levels were found in 83.3% of the AML samples, with no differences observed between the newly diagnosed and relapsed/refractory samples. Treatment with a MEK inhibitor resulted in significantly decreased p- ERK levels in both the newly diagnosed and relapsed/refractory samples, which was associated with growth arrest, but not apoptosis induction. In summary, we defined conditions for the analysis of MAPK signaling in primary AML samples. Normal CD34(+) cells expressed very low levels of p- ERK, and increased p- ERK levels were found in normal G-CSF-stimulated circulating CD34(+) cells. Constitutively high p-ERK levels observed in the majority of AML samples suggest deregulation of this pathway that appears to be independent of disease status. The ability of ERK inhibition to promote growth arrest rather than apoptosis suggests that clinical trials of MEK/ERK inhibitors may be more effective when combined with chemotherapy

Contributions of the Raf/MEK/ERK, PI3K/PTEN/Akt/mTOR and Jak/STAT pathways to leukemia

“…139 The Raf/MEK/ERK pathway is frequently activated in AML. 140 Consistently, in some AML cases, treatment of blast cells with pharmacological inhibitors of ERK 1/2 signaling (U0126) suppressed p70 S6K phosphorylation. 137 GSK-3b can negatively regulate mTOR by phosphorylating and activating TSC-2.…”

“…220 It has been reported that a high frequency of AML and ALL (450%) displays constitutive activation of the Raf/MEK/ERK pathway in absence of any obvious genetic mutation. 140,231 While there may be some unidentified mutation at one component of the pathway or a chromosomal translocation that feeds into the pathway or a phosphatase that regulates the activity of the pathway, the genetic nature of constitutive activation of the Raf/MEK/ERK pathway is unknown. Elevated expression of ERK in AMLs and ALLs is associated with a poor prognosis.…”

Antitumor activity of sorafenib in FLT3-driven leukemic cells

“…18,19 In a recent study looking at AML samples from 42 patients, markedly elevated phospho-ERK levels were found in 83.3% of the AML samples and treatment with a MEK inhibitor resulted in significantly decreased phospho-ERK levels, which was associated with growth arrest but not apoptosis induction. 20 On that last point, other studies on pharmacological inhibition of the Ras/Raf/Mek pathway in AML cells in vitro have reported conflicting data, with some studies showing mostly cell cycle arrest without immediate significant induction of apoptosis in treated cells 21,22 whereas other studies have demonstrated induction of apoptosis. 23,24 Sorafenib also has potent activity against kinases reported to potentially be contributing to AML physiopathology such as PDGFR, KIT and VEGFR.…”

Proapoptotic activity and chemosensitizing effect of the novel Akt inhibitor perifosine in acute myelogenous leukemia cells

“…Therefore, this drug was capable of downmodulating two signaling pathways which are of fundamental importance for AML cell survival and drug resistance. 24,44,45 However, even at higher concentrations, perifosine was unable to completely dephosphorylate ERK 1/2 in THP-1 cells, even though the upstream kinase MEK 1/2 was almost completely dephosphorylated. In THP-1 cells, upregulation of the PI3K-Akt pathway is due to overexpression of p110d PI3K which leads to higher levels of PIP 3 , 46 whereas the reason for constitutive activation of ERK 1/2 is unknown.…”