P-glikoprotein i aktivirane molekule signalnih putova PI3K/Akt i MAPK u blastima akutne mijeloične leukemije

Prijić, Sanja (2014) P-glikoprotein i aktivirane molekule signalnih putova PI3K/Akt i MAPK u blastima akutne mijeloične leukemije. PhD thesis, Sveučilište u Zagrebu.

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Abstract

Acute myeloid leukemia (AML) is a heterogeneous group of malignant clonal disorders of the hematopoietic system originating from malignant transformation of the myeloid precursor cell due to chromosome alterations or gene mutations. Cytogenetic rearrangements and specific mutations are the most important prognostic factors and form the basis for latest AML classification system. Nevertheless, since genetic alterations are not present in a significant number of AML patients, it provides space for scientists to investigate other biological characteristics of leukemic cells with the ultimate goal of improvement of patients' risk stratification and treatment regimens. Accordingly, this study investigated the expression and activity level of chemotherapy resistance mediator P-glycoprotein (P-gp), as well as the phosphorylation level of intracytoplasmic signalling molecules Akt, ERK1/2 and p38 in leukemic blasts of patients with de novo AML by flow cytometry. Specific objectives of the study were to analyze those leukemic cells’ features in relation to the known prognostic clinical and laboratory parameters of patients with AML, their interconnection as well as their potential prognostic impact on the survival of patients with AML. This research showed P-gp expression in 34% and its activity in 25% of AML patients. It was confirmed that the expression and activity of P-gp correlated with older age, higher number of leukocytes, immature cell markers expression (CD34 and TdT) and cytomorphologically immature AMLs, and that they represent unfavourable prognostic factors for overall survival. Constitutive phosphorylation of Akt molecule of the PI3K signalling pathway was found in 48%, while MAPK signalling pathway was activated in 39% (ERK1/2) and 44% (p38) of all tested AML samples, respectively. Significant correlation was found between Akt and p38 phosphorylation levels, but not with the ERK1/2 molecule. Serum lactat-dehydrogenase (LDH) level, an indicator of tumor burden, correlated with the phosphorylation levels of all three signalling molecules. In addition, Akt phosphorylation correlated with overall survival and relapse-free survival, ERK1/2 with younger patient age, while p38 correlated with the number of red blood cells at diagnosis of AML. Moreover, negative correlation was found between levels of p38 phosphorylation and P-gp activity in AML, indicating a possible role of p38 in P-gp regulation. Multivariate Cox regression analysis showed that P-gp activity and Akt phosphorylation are independent negative predictors of overall survival, while phosphorylation of ERK1/2 is an independent positive predictor of overall survival and disease free survival of AML patients. This P-gp activity and level of constitutive phosphorylation of the signalling pathways molecules in combination with other previously established clinical and laboratory features could represent additional parameters for the prognostic stratification of patients with AML.

Abstract in Croatian

Akutna mijeloična leukemija (AML) jest heterogena skupina malignih klonskih bolesti krvotvornog sustava koje nastaju malignom transformacijom mijeloidnih progenitorskih stanica zbog oštećenja kromosoma ili mutacije genâ. Specifične citogenetičke promjene i nalaz specifičnih mutacija danas predstavljaju glavne prognostičke čimbenike i temelj suvremene klasifikacije AML. Međutim, budući da se u značajnog broja bolesnika s AML navedene promjene ne nalaze, to znanstvenicima pruža prostora za istraživanje drugih još uvijek nepoznatih gena i biološki relevantnih obilježja leukemijskih stanica u cilju što bolje terapijske i prognostičke stratifikacije bolesnika. Sukladno tome, u ovom su radu ispitani izražaj i aktivnost medijatora kemorezistenicje P-glikoproteina (P-gp) te razina fosforilacije unutarcitoplazmatskih signalnih molekula Akt, ERK1/2 i p38 u blastima bolesnika s novootkrivenom AML metodom protočne citometrije. Specifični ciljevi rada bili su analizirati navedena obilježja leukemijskih stanica u odnosu na poznate prognostičke kliničko-laboratorijske parametre bolesnika s AML, njihovu međusobnu povezanost kao i njihov potencijalni prognostički utjecaj na preživljavanje bolesnika s AML. Istraživanjem je pokazan izražaj P-gp na blastima 34%, a aktivnost u blastima 25% bolesnika s AML. Potvrđeno je da izražaj i aktivnost P-gp koreliraju sa starijom dobi, većim brojem leukocita pri dijagnozi, izražajem nezrelih biljega (CD34 i TdT) i citomofološki nezrelijim oblicima AML te da predstavljaju prognostičke čimbenike za kraće ukupno preživljavanje. Konstitutivna fosforilacija Akt signalnog puta PI3K/Akt nađena je u blastima 48% bolesnika, a signalnog puta MAPK u 39% (ERK1/2), odnosno 44% (p38) bolesnika, pri čemu nalaz fosforilacije Akt korelira s nalazom fosforilacije molekule p38, ali ne i molekule ERK1/2. Konstitutivna fosforilacija Akt, ERK1/2 i p38 u blastima korelira s pokazateljem tumorske mase – serumskim LDH pri dijagnozi, fosforilacija Akt s ukupnim preživljavanjem i preživljavanjem bez znakova bolesti, fosforilacija ERK1/2 s mlađom dobi bolesnika, a fosforilacija p38 s brojem eritrocita pri dijagnozi. Štoviše, nađena je i negativna korelaciju između razine fosforilacije p38 i aktivnosti P-gp u blastima AML što bi indirektno moglo govoriti u prilog potencijalne uloge p38 u regulaciji P-gp. U multivarijatnoj analizi s pomoću Coxovog regresijskog modela pokazano je da je aktivnost P-gp nezavisni negativni prediktor za ukupno preživljavanje bolesnika, fosforilacija ERK1/2 nezavisni pozitivni prediktor za ukupno preživljavanje i preživljavanje bez pojave bolesti, a fosforilacija Akt nezavisni negativni prediktor za ukupno preživljavanje bolesnika. To govori u prilog da aktivnost P-gp i razina konstitutivne fosforilacije signalnih putova Akt i ERK1/2 u kombinaciji s poznatim kliničkolaboratorijskim čimbenicima bi mogli predstavljati dodatne parametre za prognostičku stratifikaciju bolesnika s AML.

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