We compared global gene expression profiles, proteomic and transcriptomic profiles, and the difference in susceptibility to antileukemic agents among patients.
Results. Patients were separated into one subset showing high constitutive activation and one subset showing low constitutive activation based on their constitutive pathway activation profiles. The high activation subset was characterized by reduced frequency of cells showing monocytic differentiation, increased frequency of adverse karyotypes, decreased constitutive cytokine release, and increased expression of certain integrins.
Conclusion. The two subsets differed in their expression of genes encoding regulators of protein phosphorylation, whereas phosphoproteomic analyses showed differences related to transcriptional regulation. These variations are a part of complex phenotypic differences.
3.2
Article II
Insulin-initiated activation of the PI3K-Akt-mTOR pathway in acute myeloid leukemia cells; a study of patient heterogeneity and pathway inhibitors
Authors: Ina Nepstad, Kimberley Joanne Hatfield, Elise Aasebø, Maria Hernandez-Valladares, Karen Marie Hagen, Kristin Paulsen Rye, Frode Berven, Frode Selheim, Håkon Reikvam and Øystein Bruserud
Background. Constitutive signaling through the PI3K-Akt-mTOR pathway is present in AML cells, and this pathway is considered a possible therapeutic target. Insulin can be a growth factor for AML cells, and we characterized the effect of insulin on PI3K-Akt-mTOR activation.
Methods. We investigated the phosphorylation level of 10 proteins involved in PI3K-Akt-mTOR signaling by flow cytometry for AML cells derived from 76 unselected patients. Patient subsets were compared by global gene expression, and proteomic and phosphoproteomic profiling.
Results. Patients were classified in two main subsets based on the constitutive activation of their AML cells; the overall results indicated insulin significantly increased the phosphorylation of all investigated mediators.
Strong insulin responders were characterized by a specific gene expression profile as well as proteomic and as phosphoproteomic differences with regard to regulators of transcription, RNA metabolism, and protein modification. Even though the overall effects of pathway inhibitors differed between patients, PI3K and Akt inhibition was characterized by a generally strong inhibition of AktpT308 and 4EBP1pT36pT45 whereas mTOR inhibition caused a strong inhibition of mTORpS2448 and S6pS244.
Conclusion. Insulin modulates PI3K-Akt-mTOR signaling in primary human AML cells, but the insulin effect differs between patient subsets, which can be identified through their mRNA or proteomic profiles. The effects of pathway inhibitors on activation differs between patients and depends on the molecular target of the inhibitor.
3.3
Article III
Resistance to the antiproliferative in vitro effect of PI3K-Akt-mTOR Inhibition in primary human acute myeloid leukemia cells is associated with altered cell metabolism
Authors: Ina Nepstad, Håkon Reikvam, Annette K. Brenner, Øystein Bruserud, Kimberley J. Hatfield
Background. The PI3K-Akt-mTOR pathway plays a central role in the regulation of proliferation, differentiation, and survival of hematopoietic cells, and constitutive signaling through this pathway has been observed in AML cells. This pathway is a possible therapeutic target in human AML and we therefore investigated possible associations between cellular metabolism and sensitivity to PI3K-Akt-mTOR pathway inhibitors, in relation to heterogeneity of the disease.
Methods. A non-targeted metabolite profiling was performed to compare the metabolome variances of primary human AML cells derived from patients susceptible or resistant to the in vitro antiproliferative effects of inhibitors to the pathway.
Additionally, using flow cytometry, we investigated the phosphorylation profile of 18 proteins involved in PI3K-Akt-mTOR signaling, together with the effect of the cyclooxygenase inhibitor indomethacin on their phosphorylation status.
Results. Inhibitors of the PI3K-Akt-mTOR pathway have antiproliferative effects on leukemia cells for only a subset of patients. We compared the metabolite profiles of AML cells defined as either susceptible to or resistant to in vitro treatment with pathway inhibitors and found 627 metabolites could be detected. Non-responders showed increased levels of metabolites reflecting energy metabolism (citric acid, isocitric acid), amino acid metabolism (proline, aspartic acid, glutamine, taurine), and arachidonic acid metabolism (4,7,10,13-eicosateraenoic acid, 4,7,10,13,16-doocosapentaenoic acid).
Decision tree analysis showed that the two patient groups could be identified based on the levels of cysteinyl-cysteine and threonic acid. Furthermore, the cyclooxygenase inhibitor indomethacin altered the phosphorylation of mTOR and its downstream mediators.
Conclusion. Differences were found in leukemia cells that are susceptible or resistant to PI3K-Akt-mTOR inhibitors in energy, amino acid, and arachidonic acid metabolism.
Results shows that modulation of arachidonic acid metabolism alters the activation of mTOR and its downstream mediators.
3.4
Article IV
Clonal heterogeneity reflected by PI3K-Akt-mTOR signaling in human acute myeloid leukemia cells and its association with adverse prognosis
Authors: Ina Nepstad, Kimberley Joanne Hatfield, Tor Henrik Anderson Tvedt, Håkon Reikvam and Øystein Bruserud
Background. Clonal heterogeneity is seen for a subset of AML patients, and detection of separate subclones based on karyotyping is associated with adverse prognoses.
However, the use of another methodological strategy is required for a more general evaluation of the prognostic impact of clonal heterogeneity in human AML. Constitutive activation of the PI3K-Akt-mTOR pathway is present in AML cells, and this pathway is considered a possible therapeutic target in human AML. However, the degree of pathway activation varies between patients. In this study, we suggest that this pathway reflects biologically important clonal heterogeneity.
Methods and Results. We investigated constitutive PI3K-Akt-mTOR pathway activation in primary human AML cells derived from 114 patients, together with 18 mediators central to the signaling in this pathway. The cohort included patients with normal karyotype or single karyotype abnormalities, with an expected heterogeneity regarding molecular genetic abnormalities. Clonal heterogeneity reflected as pathway mediator activation was detected for 39 patients, and the study shows that primary AML cells derived from patients with and without dual PI3K-Akt-mTOR cell copulations have differences in their global gene expression profiles. Finally, detection of AML subclones based on PI3K-Akt-mTOR signaling was associated with adverse prognosis for patients receiving intensive antileukemic treatment.
Conclusion. Clonal heterogeneity, as shown in the activation status of selected mediators in the PI3K-Akt-mTOR pathway, was related to differences in gene expression profiles and had an independent prognostic impact. This biological heterogeneity reflected in the intracellular signaling status should be further investigated as a potential prognostic biomarker in human AML.