Role of Extracellular Signal-Regulated Kinase-1/2 (ERK) on Complete Remission Achievement of Primary Adult Acute Lymphoblastic Leukemia Patients Enrolled in the GIMEMA Protocol LAL 2000: In Vitro Activity of the MEK Inhibithor PD98059.
Authors: Gregorj Chiara , Petrucci Maria T. , Scerpa Maria C. , Ricciardi Maria R. , De Cave Fabiana , Gervasoni Jacopo , Vignetti Marco , Gubbiotti Stefania , Ariola Cristina , Specchia Giorgina , Chiarenza Annalisa , Ferrara Felicetto , Fabbiano Francesco , Camera Andrea , Fioritoni Giuseppe , Miraglia Eustachio , Annino Luciana , Mosna Federico , Cantore Nicola , Luppi Mario , Nobile Francesco , Leone Giuseppe , Depaoli Lorella , Peta Antonio , Martelli Massimo A. , Majolino Ignazio , Montanaro Marco , Milella Michele , Meloni Giovanna , Foà Robert , Tafuri Agostino 
Published: April 2020

Abstract
The mitogen-activated protein kinase (MAPK) pathway contributes to the proliferative and differentiative control of hematopoietic cells. Extracellular signal-regulated kinase-1/2 (ERK) is one of the members of the MAPK pathway; its constitutive activation (p-ERK) has been found in a broad spectrum of hematological malignancies, particularly in acute myeloid leukemia (AML). A molecular therapeutic approach based on MAPK inhibitors has been therefore attempted. The aim of this study was to assess the expression and the clinical role of p-ERK in adult acute lymphoblastic leukemia (ALL) evaluating, in addition, the in vitro effects of the MEK inhibitor PD98059, used alone or in combination with chemotherapy, on the proliferation and apoptosis of ALL blast cells. Primary samples from 131 uniformly treated de novo patients enrolled in the GIMEMA LAL 2000 protocol were evaluated, using a p-ERK1/2-specific MoAb (clone E10), by Western blot analysis and flow cytometric assay which allowed single cell phospho-protein determination by the Kolmogorov-Smirnov statistic test (D-value). Lymphoid cell lines (Jurkat, CEM, RAJI and RPMI8866), as well as normal PMA-activated PBL expressed p-ERK, whereas resting PBL exhibited minimal levels (D-value &lt;0.10). In clinical ALL samples p-ERK ranged between 0 and 0.83, constitutive activation (D-value ≥ 0.10) was found in 45/131 samples (34.5%) and resulted significantly associated with higher WBC values (&gt;50x109/L; P=0.0128) and lower RNA-index of G1 phase (P=0.0296). Age, leukemia phenotype and molecular characteristics did not associate with p-ERK expression, while phospho-protein levels, measured as a continuous variable, correlated with failure to achieve CR (P=0.069). According to cell availability, we evaluated the effects of the MEK inhibitor PD98059 at 25μM on 30 fresh ALL samples, cultured in vitro for 24 hours. Eight of them were characterized by constitutive ERK activation, which was efficiently abrogated in 6; however, this effect did not induce cell cycle changes or apoptosis either alone or in combination with chemotherapeutic agents (Ara-C and/or Dexametasone). In summary, our study demonstrated that p-ERK is expressed in one third of adult ALL samples at presentation. Patients with p-ERK expression are characterized by higher leukemic mass, with a trend to induction treatment failure. MEK inhibition by PD98059 is capable of modulating p-ERK in primary ALL cells, though it does not result in cell cycle/apoptotic changes. In conclusion, ERK signaling appears only partially involved in the control of proliferative signals in adult ALL and modulation of additional pathways may result in more effective therapeutic strategies.