The Role Of RAS Mutation And PHLPP1in Myeloid Leukemia

Part1. Development of a high-resolution melting method for thedetection of RAS mutationObjective: RAS mutation is one of the most common gene alterations in acutemyeloid leukemia(AML). We aimed to establish a method using high resolution meltinganalysis(HRMA) for screening mutations in NRAS and KRAS at those hot-spot codons,whose sensitivity, specificity and reliability were evaluated.Methods: Primers for HRMA and DNA sequencing were designed to amplify DNAfragments spanning those hot-spot codons of NRAS and KRAS. PCR products usingPrimers for HRMA were then screened for mutations by HRMA. Positive samples werevalidated by DNA sequencing. The sensitivity of HRMA was assessed by a dilution seriesof mutant in a background of wild-type DNA.Results: The heterogeneous mutations of NRAS and KRAS were easily distinguishedfrom the wild-type because their melting curves were significantly different, while themelting curves of the homozygosis mutations of NRAS and KRAS were similar with thewild-type, with melting peak obviously drifted. All positive samples detected by HRMAwere confirmed by DNA sequencing.Conclusions: We successfully established the HRMA method for screening NRASand KRAS mutations using LightScanner, which is rapid, sensitive, high-throughput andreliable and could be used in clinical detection in AML. Part2. RAS mutation analysis in a large cohort of Chinese AcuteMyeloid Leukemia patientsObjective: We examined RAS mutational status and correlated this with presentingmorphology, cytogenetics, clinical outcome and other gene aberrations in a large cohort ofChinese acute myeloid leukemia (AML) patients.Methods: N-RAS and K-RAS were screened for mutations at hot-spot codons12,13and61using high resolution melting analysis (HRMA) and direct DNA sequencing in504Chinese AML patients and their clinical relevance was analyzed.Results: The frequencies of mutations of N-RAS and K-RAS were9.7%(49/504) and2.9%(15/504), respectively. c.35G>A (rs121913237: G>A; p.Gly12Asp and rs121913529:G>A; p.Gly12Asp) and c.38G>A (rs121434596: G>A; p.Gly13Asp and rs112445441:G>A; p.Gly13Asp) were the most common base substitutions (46%in N-RAS and60%inK-RAS, respectively). AML patients with RAS mutations presented significantly higherwhite blood cell count (WBC) at diagnosis than those without mutations (p<0.001). RASmutations were underrepresented in patients with t(15;17)(2.9%, p=0.01), whileoverrepresented in cases with abn11q23(50%, p=0.002) and inv(16)(66.6%, p=0.04). Inthe FAB subtypes M4and M5, RAS mutations were more frequent (21.6%and20.6%,respectively) than they were in other subtypes (7.5%, p=0.006and0.005, respectively).FLT3-ITD and RAS mutation were rarely coexistent (p=0.03). RAS mutation didn’tinfluence overall survival (OS) either in the entire cohort or within some definedsubgroups.Conclusions: RAS mutations are associated with some biologically specific subtypesof AML but don’t impact clinical outcome in Chinese patients. Part3. The role of PHLPP1in Chronic Myelocytic LeukemiaObjective: To investigate in vitro the effects of PHLPP1on proliferation, apoptosis,and cell cycle of human chronic myelocytic leukemia(CML) cell line K562and theexpression of PHLPP1in CML primary cells. Methods: The PHLPP1mRNA expression and PHLPP1protein in cell lines andprimary cells were detected by real-time PCR and Western-blotting, respectively.Recombinant PCDNA3.0-PHLPP1plasmid or the empty vector was transfected into K562using Lipofectamine2000. To determine the rate of proliferation, the numbers of cells werecounted daily for3days using trypan blue. Cell cycle distribution and cell apoptosis wereassessed by flow cytometry. Real-time PCR was also used to detect the expression ofmiR190in CML primary cells.Results:1.Both of the levels of PHLPP1mRNA and protein in CML cell lines K562andMeg01were significant decreased compared to AML cell lines THP1and HL-60(P<0.01).2.PHLPP1was overexpressed in K562cells transfected with PCDNA3.0-PHLPP1plasmid according to the results of Real-time PCR and Western-blotting (P<0.01),indicating stable K562cell line overexpressing PHLPP1(K562-PHLPP1) was establishedsuccessfully.3.Overexpression of PHLPP1signifcantly slowed the rate of proliferation of K562cells. Specifically, the number of K562-PHLPP1cells was significantly lower than that ofK562cells transfected with empty vector (K562-control) at hour48(p<0.05).4.Overexpression of PHLPP1induced higher G1/S ratio of K562-PHLPP1cells(0.695±0.026) than that of K562-control cells (0.416±0.019) in cell cycle distribution(P<0.05), which was similar with the result under the serum-starve condition.5.Overexpression of PHLPP1alone was unable to induce a significant amount ofapoptosis, but enhanced apoptotic effect caused by Imatinib mesylate (STI571)significantly. Treating cells with STI571induced apoptosis of both K562-control andK562-PHLPP1cells in a time-dependent manner. Specifically, the proportion of apoptoticcells in the K562-PHLPP1group was significantly higher than that in the K562-controlgroup at hour48treated with1.0μM STI571(P<0.05).6.The PHLPP1level in K562cells treated with2.0μM STI571was detected byWestern-blotting at hour0,12,24, and36, respectively. The PHLPP1level in K562cellstreated with STI571increased significantly at hour12(P<0.05), reaching the highest levelat hour36(P<0.01).7.The average PHLPP1level in CML primary cells was comparable with K562cellline (P>0.05) and significantly lower than that in AML cell line THP1(P<0.01). 8.There was no correlation between the expression of miR190and the PHLPP1mRNA level in CML primary cells (r=－0.07, P=0.78).Conclusions:1.The PHLPP1levels in CML cell lines were decreased. Overexpression of PHLPP1in K562inhibited the proliferation of cells through G1arrest in cell cycle.2.Overexpression of PHLPP1in K562alone was unable to induce a significantamount of apoptosis, but enhanced apoptotic effect caused by Imatinib mesylate (STI571)significantly. STI571could up-regulate the levels of endogenous PHLPP1in K562cells.3.The average PHLPP1level in CML primary cells was significantly decreased andthere was no correlation between the PHLPP1mRNA leveland the expression of miR190.