Page 78 - Haematologica-April 2018

A. Thivakaran et al.
Gfi1b/GFI1B-deficient/low and Gfi1b/GFI1B high- expressing leukemic cells (Figure 7A). As ROS plays an important role in the pathogenesis of AML,38 we exam- ined the level of ROS. For non-malignant HSCs, it was shown that HSCs with low ROS had a high self-renewal capacity.39 In contrast, HSCs with elevated ROS were mostly located in the vascular niche, had a reduced self- renewal capacity, and were more restricted with regard to their differentiation potential.39 Based on this and our pre- vious report that loss of Gfi1b leads to higher level of ROS in HSCs,6 we examined whether ROS level might differ between Gfi1b-deficient and Gfi1b expressing LSCs. Due to the difficulty of defining a distinct LSC population in each set of AML samples, we used CD117 (c-Kit) expres- sion as a surrogate marker to define a population which is enriched for LSCs. CD117 expression has been used to identify a fraction that is enriched for LSCs.40 We identi- fied two distinct populations in the CD117+ blast cells that differ with regard to their ROS expression (a popula- tion with low ROS expression and a population with high ROS expression). Loss of Gfi1b led to an increased level of ROS (defined as the mean fluorescent intensity, MFI) in both ROS-low and ROS-high populations (Figure 7B-D and Online Supplementary Figure S8B).
Altered activity of the AKT pathway in Gfi1b-deficient AML
Elevated levels of ROS promote AML development, but ROS also activates various redox-sensitive signaling transduction cascades,41 including the MAPK pathway, which limits the stemness of the affected cells, at least in a non-malignant setting.42 In the presence of ROS, the MAPK pathway component p38 is activated, which sub- sequently results in an exhaustion of the HSC population.43 It has also been shown that activation of p38 limits oncogenic transformation.44 Despite a higher level of ROS, in our models, Gfi1b-deficient leukemic cells have escaped p38 activation, indicated by a decreased level of phosphorylated p38 compared to Gfi1bfl/flMxCrewtNUP98/HOXD13tg (Figure 7E). The fact that Gfi1b might directly or indirectly regulate p38 is also supported by the analysis of differentially acetylated genes in Gfi1b-deficient and Gfi1b-expressing leukemic cells. Because decreased p38 levels are associated with higher oncogenic potential,44 this could partially explain the higher number of functional LSCs we observed in the Gfi1b-deficient leukemic cells. Activation of p38 leads to an increased level of AktSer473.45 AktSer473 activity inversely correlates with the number of LSCs in AML.46 We thus examined the connection between loss of Gfi1b and AktSer473 and found that the level of phosphorylated AktSer473 is reduced in Gfi1b-deficient leukemia (Figure 7F). Akt represses the function of FoXO3, and since FoXO3 acts as an oncogene in AML,46 we determined the FoXO3 protein level. Gfi1b-deficient leukemic cells showed an increased expression of FoXO3 protein in the nucleotide (NER) and cytoplasmatic (CER) cell fraction compared to the expression level of FoXO3 in Gfi1b-expressing leukemic cells (Figure 7G). To obtain an insight into whether this increased level of FoXO3 also has any func- tional consequences, we re-examined the whole genome expression datasets in Gfi1b-expressing and Gfi1b-defi- cient leukemic cells and found an enrichment of FoXO3 binding sites among the promoter areas of those genes, which were differentially expressed between cells with
absence of Gfi1b expression and cells with intact expres- sion of Gfi1b (Online Supplementary Figure S9), showing that altered level of FoXO3 might be one additional explanation for our observations (Figure 7H).
Discussion
In the datasets analyzed by us, GFI1B was expressed at a lower level in LSCs compared to the control. Low GFI1B was also indicative of an inferior prognosis for MDS and AML patients, with the caveat that these state- ments are based on retrospective studies. Larger prospec- tive studies would be required to make such a claim on a solid basis. We previously reported that low GFI1 expres- sion level in AML blasts was associated with poor out- come and here we report that low GFI1B expression lev- els were associated with poor survival. This might appear surprising since GFI1 and GFI1B repress each other, but in our cohorts we observed that low GFI1B expression level can also be associated with low GFI1 expression (data not shown), therefore, the reciprocal regulation between GFI1 and GFI1B might be different in leukemic cells. We pos- tulate that GFI1B plays a dose-dependent role in human/murine AML pathogenesis. Anguita et al. showed that a mutated dominant-negative form of GFI1B con- tributes to AML development. These reports show how altering the function of GFI1B can influence normal and malignant development. Recent studies have highlighted the role of different isoforms of GFI1B in the course of erythroid and megakaryocytic development.6,47-49 It remains to be elucidated whether altering the expression of these isoforms might also contribute to AML develop- ment.
Loss of Gfi1b in our murine models increased the num- ber of LSCs on a functional level. These data are in line with our previous reports that loss of Gfi1b leads to an expansion of functional HSCs.6 On a molecular level, loss of Gfi1b resulted in an increased level of H3K9ac among its target genes, which is in line with other reports regard- ing the epigenetic function of Gfi1b.11,16 Among these tar- get genes are a number of genes involved in the regulation of leukemogenesis and stem cell regulation, indicating that the absence of Gfi1b leads to a gene expression sig- nature that directly or indirectly contributes to an increased number of LSCs.
Both murine and human data also indicated a possible connection between Gfi1b and ROS/p38/Akt signaling. P38 and AktSer473 activation limit oncogenic and stemness potential.43,44,46 Conceivably, lower expression of these proteins would increase the oncogenic potential. P38 and Akt were down-regulated in Gfi1b-deficient leukemic cells in vivo. In line with this, Saleque et al. demonstrated that Gfi1b is involved in the regulation of p38 and that reduced Gfi1b levels are associated with lower p38 signal- ing.50 In addition to the ROS/p38/Akt/FoXO3 signaling cascade, other pathways were altered. It remains to be elucidated which role these pathways might play in the pathogenesis of human and murine AML. In addition, how Gfi1b/GFI1B regulates ROS, p38, Akt and FoXO3 levels remains to be analyzed.
In summary, epigenetic changes and alteration of the ROS/p38/Akt/FoXO signaling cascade might facilitate the progression of normal hematopoietic cells to LSCs. In the future, testing will be needed to determine whether alter-
622
haematologica | 2018; 103(4)