Effect of the unfolded protein response and oxidative stress on mutagenesis in CSF3R: a model for evolution of severe congenital neutropenia to myelodysplastic syndrome/acute myeloid leukemia.

Severe congenital neutropenia (SCN) is a rare blood disorder characterised by abnormally low levels of circulating neutrophils. The most common recurrent mutations that cause SCN involve neutrophil elastase (ELANE). The treatment of choice for SCN is the administration of granulocyte-colony stimulating factor (G-CSF), which increases the neutrophil number and improves the survival and quality of life. Long-term survival is however linked to the development of myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). About 70% of MDS/AML patients acquire nonsense mutations affecting the cytoplasmic domain of CSF3R (the G-CSF receptor). About 70% of SCN patients with AML harbour additional mutations in RUNX1. We hypothesised that this coding region of CSF3R constitutes a hotspot vulnerable to mutations resulting from excessive oxidative stress or endoplasmic reticulum (ER) stress. We used the murine Ba/F3 cell line to measure the effect of induced oxidative or ER stress on the mutation rate in our hypothesised hotspot of the exogenous human CSF3R, the corresponding region in the endogenous Csf3r, and Runx1. Ba/F3 cells transduced with the cDNA for partial C-terminal of CSF3R fused in-frame with a green fluorescent protein (GFP) tag were subjected to stress-inducing treatment for 30 days (~51 doubling times). The amplicon-based targeted deep sequencing data for days 15 and 30 samples show that although there was increased mutagenesis observed in all the three genes of interest (partial CSF3R, Csf3r and Runx1), there were more mutations in the GFP region compared with the partial CSF3R region. Our findings also indicate that there is no correlation between the stress-inducing chemical treatments and mutagenesis in Ba/F3 cells. Our data suggest that oxidative or ER stress induction does not promote genomic instability, affecting partial C-terminal of the transduced CSF3R, the endogenous Csf3R and the endogenous Runx1 in Ba/F3 cells that could account for these targets to being mutational hotspots. We conclude that other mechanisms to acquire mutations of CSF3R that help drive the evolution of SCN to MDS/AML.

The ubiquitin ligase RNF38 promotes RUNX1 ubiquitination and enhances RUNX1-mediated suppression of erythroid transcription program.

RUNX1 is a member of RUNX transcription factors and plays important roles in hematopoiesis. RUNX1 function is under the tight control through posttranslational modifications, including phosphorylation and ubiquitination. We previously developed a luminescence-based binding assay (AlphaScreen) to systematically detect RUNX1-interacting E3 ubiquitin ligases. In this study, we showed that a nuclear ubiquitin ligase RNF38 induced ubiquitination of RUNX1. RNF38-induced RUNX1 ubiquitination did not promote RUNX1 degradation, but rather stabilized RUNX1 protein. We also found that RNF38 enhanced RUNX1-mediated transcriptional repression of the erythroid master regulator KLF1 in K562 cells. Consequently, RNF38 cooperated with RUNX1 to inhibit erythroid differentiation of K562 cells. Thus, our study identified RNF38 as a novel E3 ligase that modifies RUNX1 function without inducing its degradation.

Acute myeloid leukemia in an 86-year-old man with AML1/ETO treated with Homoharringtonine and Arsenic Trioxide: A case report.

RATIONALE: Acute myeloid leukemia (AML) is a malignantly clonal and highly heterogeneous disease. Although the treatment of AML has brought promising outcomes for younger patients, prognosis of the elderly remains dismal. Innovative regimens are increasingly necessary to be investigated. PATIENT CONCERNS: We present an 86-year-old AML patient with fever, cough, and sputum production. DIAGNOSES: A diagnosis of AML with maturation (AML-M2) and AML1/ETO was made. INTERVENTIONS: The patient was treated with a regimen of Homoharringtonine coupled with arsenic trioxide. OUTCOMES: The AML-M2 patient with AML1/ETO achieved incomplete remission, but showed few toxic side effects and improved survival. Besides, we analyzed the dynamic counts of complete blood cells during the treatment. The count of white blood cell had a positive correlation with the percentage of blast cells (r = 0.65), both of which had a negative correlation with the percentage of segmented neutrophils (r = -0.63, -0.89). LESSONS: Homoharringtonine and arsenic trioxide may induce both the apoptosis and differentiation of leukemic cells in AML-M2 with AML1/ETO.

Inducible expression of AML1-ETO fusion protein endows leukemic cells with susceptibility to extrinsic and intrinsic apoptosis.

AML1-ETO, a leukemia-associated fusion protein generated by the frequently occurred chromosome translocation t(8;21) in acute myeloid leukemia, was shown to exert dichotomous functions in leukemic cells, that is, growth arrest versus differentiation block. By the analysis of oligonucleotide microarray, AML1-ETO was shown to modulate the expressions of an impressive array of pro- and anti-apoptotic genes. Here, we investigate potential effects of the ecdysone inducible AML1-ETO expression on apoptosis of leukemic U937 cell line. We show that AML1-ETO significantly stabilizes death receptor Fas protein and increases proapoptotic Bak in addition to reducing Bcl-2 expression. Accordingly, inducible AML1-ETO expression is followed by apoptosis to a lower degree. Especially, AML1-ETO endows leukemic cells with the susceptibility to anti-Fas agonist antibody, ultraviolet light and camptothecin analog NSC606985-induced apoptosis with increased activation of caspase-3/8. Considering that apoptosis-enhancing effect of AML1-ETO would not be favorable to the leukemogenesis harboring the t(8;21) translocation, it must be overcome to fulfill their leukemogenic potential. Complementary to this prediction is that two AML1-ETO-carrying leukemic cells, Kasumi-1 and SKNO-1, present similar sensitivity to apoptosis induction with AML1-ETO-negative leukemic cells. Therefore, genetic and/or epigenetic screenings of apoptosis-related genes modulated by AML1-ETO deserve to be explored for understanding the mechanisms of AML1-ETO-induced leukemogenesis.

Genotoxicity of etoposide: greater susceptibility of MLL than other target genes.

The ability of topoisomerase 2 inhibitors to induce DNA breakage is well recognized. Previous studies, however, have concentrated on the effects on individual genes. The effects of etoposide on the MLL, RUNX1, and MLLT3 genes were simultaneously studied in the same hemopoietic cell population. We found MLL to be more susceptible to etoposide-induced cleavage than RUNX1 and MLLT3, with maximum cleavage at a lower drug concentration. A higher level of MLL than other gene cleavage was also detected after cellular exposure to all drug concentrations. Greater susceptibility to topoisomerase 2 inhibitor-induced cleavage may explain the more frequent involvement of MLL in treatment-related leukemogenesis.

Biologic activity of triptolide in t(8;21) acute myeloid leukemia cells.

Triptolide is a compound isolated from the traditional Chinese medicinal herb Tripterygium wilfordii that shows potent anti-tumor activities, but its effects on acute myeloid leukemia with t(8;21) remain unclear. Here we report that triptolide inhibits cell proliferation and induces apoptosis in a dose- and time-dependent manner of t(8;21)-bearing Kasumi-1, SKNO-1 and CD34+ cells harvested from bone marrow samples of patients with t(8;21) leukemia. We show that triptolide triggers cleavage of the resultant AML1-ETO fusion protein of t(8;21), and causes downregulation of C-KIT followed by inhibition of JAK-STAT signaling. Triptolide downregulates p65 and inhibits the DNA-binding activity of NF-κB. Our data indicate that triptolide might be an effective agent for t(8;21) leukemia.

[Reversed effect of valproic acid on transcription inhibition of AML1-ETO fusion protein of kasumi-1 leukemic cell line].

This study was aimed to investigate the mechanism of histone deacetylase (HDAC) inhibitor, valproic acid (VPA), reversing transcription inhibition of AML1-ETO fusion protein in Kasumi-1 cell line. The mRNA expressions of AML1-ETO, AML1 and cyclin D2 were detected by semi-quantitation RT-PCR after treating kasumi-1 cells with VPA at different doses/and different time points. The results indicated that the mRNA expression of AML1-ETO showed no obvious change, when kasumi-1 cells were treated with VPA. Compared with control group, the expression level of AML1 mRNA significantly increased in a dose-dependent manner. Compared with control group, the expression level of cyclin D2 mRNA significantly decreased when kasumi-1 cells had been treated with 3 mmol/L VPA as well as kasumi-1 cells were treated with different concentrations of VPA for 3 days. In conclusion, VPA could remove transcription inhibition of AML1-ETO fusion protein, increase transcription of AML1 and down-regulate mRNA expression of AML1 target gene cyclin D2 through HDAC inhibiting activity.