Several preclinical and clinical studies suggest the importance of naturally occurring polymorphisms of drug transporters in the individual difference of drug response. To functionally validate the nonsynonymous polymorphisms of ABCB1 (P-glycoprotein/MDR1) in vitro, we generated SNP variant forms (i.e., S400N, R492C, R669C, I849M, A893P, A893S, A893T, M986V, A999T, P1051A, and G1063A) and expressed them in Sf9 cells. The kinetic properties (Km and Vmax) of those variants were analyzed by measuring the ATPase activity to obtain the ATPase profile for each variant toward structurally unrelated substrates. On the basis of the experimental data, we determined the substrate specificity of ABCB1 WT and its variants by the quantitative structure-activity relationship (QSAR) analysis method. While several SNP variants appeared to influence the substrate specificity of ABCB1, the nonsynonymous polymorphisms of 2677G > T, A, or C at amino acid position 893 (Ala > Ser, Thr, or Pro) have great impacts on both the activity and the substrate specificity of ABCB1. The A893P variant (2677G > C), a rare mutation, exhibited markedly high activity of ATPase toward different test compounds. Molecular dynamics (MD) simulation based on a three-dimensional structural model of human ABCB1 revealed that multiple kinks are formed in the intracellular loop between transmembrane domains 10 and 11 of the A893P variant (2677G > C) protein. The polymorphisms of 2677G, 2677T, and 2677A exhibit wide ethnic differences in the allele frequency, and these nonsynonymous polymorphisms are suggested to be clinically important because of their altered ATPase activity and substrate specificity toward different drugs.

Sirtuins, NAD-dependent protein deacetylases, play important roles in cellular functions such as metabolism and differentiation. Whether sirtuins function in tumorigenesis is still controversial, but sirtuins are aberrantly expressed in tumors, which may keep cancerous cells undifferentiated. Therefore, we investigated whether the inhibition of sirtuin family proteins induces cellular differentiation in leukemic cells. The sirtuin inhibitors tenovin-6 and BML-266 induce granulocytic differentiation in the acute promyelocytic leukemia (APL) cell line NB4. This differentiation is likely caused by an inhibition of SIRT2 deacetylase activity, judging from the accumulation of acetylated α-tubulin, a major SIRT2 substrate. Unlike the clinically used differentiation inducer all-trans retinoic acid, tenovin-6 shows limited effects on promyelocytic leukemia–retinoic acid receptor α (PML-RAR-α) stability and promyelocytic leukemia nuclear body formation in NB4 cells, suggesting that tenovin-6 does not directly target PML-RAR-α activity. In agreement with this, tenovin-6 induces cellular differentiation in the non-APL cell line HL-60, where PML-RAR-α does not exist. Knocking down SIRT2 by shRNA induces granulocytic differentiation in NB4 cells, which demonstrates that the inhibition of SIRT2 activity is sufficient to induce cell differentiation in NB4 cells. The overexpression of SIRT2 in NB4 cells decreases the level of granulocytic differentiation induced by tenovin-6, which indicates that tenovin-6 induces granulocytic differentiation by inhibiting SIRT2 activity. Taken together, our data suggest that targeting SIRT2 is a viable strategy to induce leukemic cell differentiation.

BackgroundThe gain-of-function mutation JAK2V617F is frequently found in Philadelphia-chromosome-negative myeloproliferative neoplasm (MPN) patients. However, the tumorigenic properties of JAK2V617F have mostly been characterized in in vivo and in vitro murine models due to the lack of appropriate human cell lines.MethodsUsing the multipotent hematologic cell line UT-7/GM, we established D9, a novel human cell line that expresses JAK2V617F upon tetracycline addition. We assessed cellular differentiation in UT-7/GM cells when JAK2V617F was induced, and we used microarrays to analyze changes in mRNA expression caused by JAK2V617F.ResultsUsing the human D9 cell line, we demonstrated that the induction of JAK2V617F leads to cytokine-independent cell growth with increased STAT activation and erythroid differentiation, mimicking the characteristics observed in polycythemia vera, making it a suitable in vitro model for studying this disorder. Interestingly, JAK2V617F-dependent erythroid cell differentiation was blocked when GM-CSF was added to the culture, suggesting that the GM-CSF pathway antagonizes JAK2V617F-induced erythroid cell differentiation. Our microarray analysis identified several genes involved in inflammasome activation, such as AIM2, IL1B, and CASP1, which were significantly up-regulated in JAK2V617F-induced cells.ConclusionsThe observed inflammasome activation following JAK2V617F induction is consistent with a recent report demonstrating the involvement of IL1B in myelofibrosis development in a JAK2V617F model mouse. These results indicate that the D9 cell line should be useful for characterizing the signaling pathways downstream of JAK2V617F, allowing for the identification of effector molecules that contribute to the development of MPN.Electronic supplementary materialThe online version of this article (doi:10.1186/s40164-016-0032-7) contains supplementary material, which is available to authorized users.