AML1-ETO9a Research Articles

Loss of the Cell Cycle Regulator p26INCA1 Induces Exhaustion of Leukemic Stem Cells

AbstractAbstract 96Acute myeloid leukemia is a clonal disease characterized by a malignant proliferation and accumulation of myeloid progenitor cells. Current therapeutic strategies are often not able to eradicate the leukemic cells. Malignancy is associated with deregulation of cell cycle check- points and the deregulation of checkpoints is associated with altered stem cell properties. A better understanding of malignant stem cells and their cell cycle regulation might help to develop new therapies. Recently, we identified p26INCA1 as a novel cell cycle regulator. GST pulldown assays revealed binding of INCA1 predominantly to CDK2- specific Cyclins and we demonstrated an inhibitory effect of INCA1 on CDK2/ CyclinA complexes in kinase activity assays. The loss of INCA1 and its inhibitory effect on the cell cycle regulation led to an increased cell cycling and consequently to an enlarged stem cell pool in vivo. Upon cytotoxic stress, the loss of Inca1 enhanced cell cycling and bone marrow exhaustion. To analyze a potential role of INCA1 in leukemogenesis we retrovirally transduced wildtype and Inca1−/− bone marrow cells with AML1-ETO9a (A1E9a) and transplanted these cells into wildtype recipients. Most of the wildtype cell- transplanted recipients died due to AML. In contrast, only one of the Inca1−/− cell- transplanted mice developed AML. Engraftment was higher upon transplantion of Inca1−/− cells but engraftment was not sustained. To consider the repopulation capacity of the leukemic cells we performed transplantation of primary leukemic cells into secondary recipients. A1E9a induced leukemia in Inca1 wildtype cells was transplantable and lethal. However Inca1−/− leukemic cells were severely impaired in leukemia development in secondary recipients. Colony forming units and replating capacity were significantly reduced in A1E9a Inca1−/− bone marrow cells although these cells showed increased CDK2 activity. Exhaustion of leukemic cells in the absence of Inca1 was confirmed by cloning efficiency assays. Further analyses were performed with c-myc induced leukemias. Interestingly, Inca1 deletion precluded the development of leukemias in secondary recipients. Taken together, these findings identify an important role for p26INCA1 in the maintenance of leukemia and potentially the self-renewal capacity of leukemic stem cells. Disclosures:No relevant conflicts of interest to declare.

The Acetylation of AML1-ETO Is Required for Leukemogenesis.

AbstractAbstract 1588Transcription factors and histones are similarly modified through acetylation, phosphorylation, ubiquitination and methylation, which impact on the transcriptional regulation of gene expression and various biological processes in normal and malignant hematopoiesis. The t(8;21) associated AML1-ETO fusion protein is found in 40% of the FAB M2 subtype of acute myeloid leukemia, but how the post-translational modification of AML1-ETO affects its leukemogenicity is largely unknown. Here we show that AML1-ETO directly interacts with the lysine acetyltransferase, p300, via the region containing NHR1 domain and that p300 can acetylate two lysine residues in AML1-ETO and AML1-ETO (exon 9a) in human and mouse leukemia cells. To understand the biological effects of AML1-ETO acetylation, we used human CD34+ cord blood cells as a preleukemia model. The maintenance of CD34+ cells by the acetylation defective form of AML1-ETO was 5 fold less than with AML1-ETO (p<0.01) in the liquid culture assay, and unlike the effect of AML1-ETO, the number of the cobble stone area forming cells (CAFC) was not increased by the mutant AML1-ETO in CAFC assay. However, the block in erythroid and myeloid differentiation conferred by AML1-ETO was still seen in the AML1-ETO acetylation mutant transduced human CD34+ cells. We then approved the impact of acetylation on leukemogenicity using the AML1-ETO9a (AE9a) mouse leukemia model. Mice receiving AE9a acetylation mutant transduced fetal liver cells have not developed leukemia by Day 250, whereas all the mice receiving AE9a transduced cells died due to leukemia before Day 160, with a mean survival time of 109 days (p<0.001). These results suggest that the acetylation of AML1-ETO is required not only for its self-renewal promoting effects and but also for the development of acute leukemia. To gain insight into the mechanisms of AML1-ETO acetylation, we performed luciferase assays and found that the AML1-ETO acetylation mutant lost the ability to activate an M-CSFR promoter driven reporter construct. Furthermore, the expression levels of AML1-ETO activated target genes related to self-renewal were not upregulated in AML1-ETO acetylation mutant transduced human CD34+ cells. These results indicated that the acetylation is crucial to AML1-ETO induced transcription activation. We have also been studying the role of the region containing NHR1 domain (245 to 430 aa) in AML1-ETO: deletion of this region abrogated the binding of p300 to AML1-ETO and led to loss of AML1-ETO lysine acetylation. Furthermore, loss of the region containing NHR1 domain abrogated the self-renewal properties of AML1-ETO and the activation of AML1-ETO target genes in human CD34+ cord blood cells, without affecting its differentiation-blocking activity or its ability to repress gene expression. Given the importance of the acetylation of AML1-ETO in its biological effects, we inhibited p300 function, chemically and using RNA interference; this blocked the transcriptional activation of AML1-ETO target genes, and inhibited the growth of AML1-ETO expressing AML cells in both pre-leukemic and leukemia models. All together, we have found that the acetylation of AML1-ETO via p300 is indispensable for its leukemia-promoting activity and for its ability to activate gene expression. Our work suggests that inhibition of p300 function may represent an important new anti-leukemia strategy that targets self-renewing, leukemia-initiating cells. Disclosures:No relevant conflicts of interest to declare.

Identification of the JAK/STAT Signaling Pathway as a Valid Therapeutic Target of T(8;21) Acute Myeloid Leukemia Using Combined Gene Expression and Promoter Occupancy Profiling.

t(8;21) is one of the most common chromosomal abnormalities associated with acute myeloid leukemia (AML). The resulting AML1-ETO fusion protein is directly involved in the pathogenesis of AML but is not able to cause leukemia on its own. We have shown previously that a C-terminal splice variant of t(8;21), AML1-ETO9a (AE9a), is sufficient to promote AML in mice. Here we show that the leukemia-initiating cells reside in the Lin-/Sca1-/cKit+ population. To identify molecular targets directly modulated by AE9a, we compared the gene expression profile to the promoter occupancy (ChIP-on-chip) profile of this population. We identified 1485 genes deregulated by AE9a, among which 544 genes are also ChIP-on-chip targets. CD45, a negative regulator of cytokine/growth factor receptor and JAK/STAT signaling, was greatly down-regulated in AE9a leukemia cells. We further show that t(8;21) AML-M2 patient samples have lower CD45 levels compared to non-t(8;21) AML-M2 samples. Interestingly, JAK1 and JAK2 were upregulated by AE9a and both CD45 and JAK1 are ChIP-on-chip targets. In addition we show that JAK/STAT signaling was enhanced in the leukemia cells. Consequently, the leukemia cells are more susceptible to JAK2 inhibitors than wild type cells. Our results indicate that AE9a enhances JAK/STAT signaling by directly modulating regulators of this signaling pathway, which provides a potential novel approach to treating t(8;21) AML.

Identification of Gfi1 as a Direct Target of the Leukemogenic Fusion Protein AML1-ETO9a

AML1-ETO9a (AE9a), a C-terminal splice variant of t(8;21), is highly leukemogenic in mice. Using combined gene expression and promoter occupancy (ChIP-on-chip) profiling of an AE9a induced murine AML model, we identified Gfi1, a transcriptional repressor important for neutrophil differentiation, as a direct target of AE9a. The level of Gfi1 mRNA was down-regulated following leukemia development. We further show that t(8;21) AML-M2 patient samples have lower Gfi1 mRNA levels compared to non-t(8;21) AML-M2 samples. In addition, Gfi1 protein was down-regulated in the human hematopoietic cell line U937 upon AE or AE9a expression. Two consensus AML1 sites (TGTGGT) located at positions −1750 and +405 from the Gfi1 transcriptional initiation site, respectively, are found in the proximity of the peaks of ChIP-on-chip profiling. We confirmed direct DNA binding of AE9a to these regions by ChIP assays. Furthermore, we showed that the activity of the Gfi1 promoter region −1880 to +481 is repressed by AE and AE9a in luciferase reporter assays. To address the effects of down-regulation of Gfi1 on proliferation, we overexpressed it in a primary AML1-ETO C-terminal mutant (AML1-ETOtr) leukemia cell line. Its overexpression slowed down growth and promoted apoptosis and differentiation of this cell line. In this report, we provide evidence that AE9a directly down-regulates Gfi1, thereby inhibiting hematopoietic differentiation, which may contribute to leukemia development.

The Coexpression of AML1-ETO9a Isoform and AML1-ETO in Acute Myeloid Leukemia M2 Subtype and Its Clinical Significance.

Previous studies on either animal model or clinical settings have demonstrated that the expression of AML1/ETO9a (AE9a), a truncated version of AML1-ETO (AE) generated by alternative splicing during transcription, in patients with acute myeloid leukemia M2 subtype may have clinical significance. Moreover, the simultaneously expression of both AE and AE9a may even be more meaningful to the prognosis of the patients. To validate this hypothesis, we have measured the mRNA level of both AE and AE9a in each individual M2 patient by quantitative real-time RT-PCR, and correlated the ratio of AE/AE9a mRNA levels with the clinical outcome and prognosis in these patients. In 35 newly-diagnosed AML-M2 patients with t(8;21), expression of AE9a was detected in 30 cases (85.71%). The median value of the percentages of AE9a and AE within overall transcripts of the fusion gene in these patients were 18.16% and 81.84%, respectively (p<0.05). A positive correlation between the mRNA level of AE9a and AE was shown in 45 bone marrow samples from 19 patients during the follow-up, and the correlation coefficient was 0.900(P<0.01). The expression level of both AE9a and AE decreased in 16 patients after one course of standard conventional chemotherapy. Among them, a reduced percentage of AE9a mRNA level was prominent in 11 (68.75%) patients, while the percentage of mRNA level of AE decreased in 5 (31.25%) cases. The complete remission rate after first course of chemotherapy was 81.82%(9/11)in 11 patients, and 20.00%(1/5)in 5 patients, suggesting a higher CR rate in those patients who exhibited more conspicuous decrease in mRNA level of AE9a (P<0.05). The percentage of AE9a mRNA in the total fusion transcripts rose in all of the 5 relapsing cases. In a retrospective study on 92 patients of AML-M2 including 50 cases with t(8;21) and 42 case with either normal karyotype or other chromosome abnormalities, expression of AE9a has been found in these 50 cases by conventional RT-PCR. Between the AE9a positive and negative groups, no statistically significant difference had been found in complete remission rate, relapsing rate and the first CR period over a follow-up with medium time of 13 months (4–76 mths). However, 22 months of medium survival in AE9a positive group (38 cases) was remarkably shorter than 70 months in AE9a negative group (25 cases). Our results indicated that the mRNA of AE9a at a relatively lower level co-expressed with AE in most AML-M2 patients with t(8;21) translocation. The expression level of AE9a rather than AE may play more crucial role in development and progress of leukemia. AE9a positive patients have distinctly shorter survival time than the negative cases, predicting a worse prognosis.

The Expression of AML1-ETO9a Isoform in Acute Myeloid Leukemia M2 Subtype and Its Clinical Significance.

AML-ETO (AE) fusion protein caused by t(8;21) is believed to be involved in the development of AML. Most recent studies using a mouse models indicate that expression of a truncated version of AE by alternative splicing, namely AML1/ETO9a (AE9a), may be more closely related to development of leukemia. To validate the hypothesis, we have studied the expression of AE9a in AML-M2 patients and its prognostic value. The Expression of AE fusion gene and AE9a was detected by using RT-PCR in 40 cases of newly diagnosed patients and 30 cases in complete remission. The expression pattern of these fusion genes in M2 patients was compared with that in other subtypes of AML, MDS and healthy controls. In addition, we have also examined the expression level of AE and AE9a in 40 cases of M2 patients during follow-up and analyzed the correlation with clinical manifestations. Our Results demonstrated that 19 of 70 cases of AML-M2 patients simultaneously expressed AE and AE9a while 10 cases expressed only AE; 2 cases expressed AE9a alone; the rest expressed neither. 19 of 40 cases of newly diagnosed patients expressed AE9a (47.5%). Neither 30 AML-M2 cases in complete remission nor the other subtypes of AML, Myelodysplastic syndrome (MDS) and healthy controls expressed AE9a. After treatment by 1–3 course of conventional chemotherapy (MA, DA, DA or AAG regimen), 4 of 19 AE9a positive cases attained complete remission (21%), 7 cases relapsed after relieved (36.8%), 3 cases died after transplantation, 3 cases failed to attained remission and gave up(15.8%), 2 cases lost connection after transplant. While after same conventional chemotherapy, 17 of 21 AE9a negative cases (81%) attained remission, 3 cases(14.3%)relapsed after relieved, 1 case (4.8%) died of giving up treatment after attaining remission. These results confirmed that AE9a was closely related to AML-M2, and it may promote the development of leukemia in combination with AE fusion gene. The remission rate in AML1/ETO9a positive patients is remarkable lower than that in negative's. Thus AE9a expression may indicate an unfavorable prognosis of AML-M2 patients.