The p73 protein is a p53 homolog and acts on cell cycle and apoptosis regulation. Resistance to apoptosis is a common feature of Acute Myeloid Leukemia (AML), but mutations on the genes p53 and p73 are rare. It is translated in two distinct isoforms: TAp73 and ΔNp73. The later does not possess the N-terminal transactivation domain and exerts a dominant negative action over TAp73 and p53 functions. Theoretically an aberrant high expression of ΔNp73 may lead to a block of p53 and TAp73, thus conferring a proliferative advantage to the leukemic cells. In order to evaluate this issue, we proposed to: Compare the gene expression levels of TAp73 and ΔNp73 isoforms in the bone marrow from de novo AML patients and normal individuals; Correlate these expression patterns with the presence of the rearrangements PML-RARα, AML1-ETO and CBFβ-MHY11, (previously determined by RT-PCR according the BIOMED-1 protocol). From 137 AML patients whose samples were evaluated by Real Time PCR, 78 harbored the genetic rearrangements (referred to as RP group): PML-RARα (n = 30), AML1-ETO (n = 16) or CBFβ-MHY11 (n = 32), whereas in the 59 remaining samples these rearrangements were not detected (RN group). Additionally, CD34+ cell samples of 22 normal bone marrow donors were also evaluated. Sample input was normalized by GAPDH expression and the relative expression was calculated using the cell line k562 as reference sample. The mean expression of TAp73 and ΔNp73 was significantly lower on normal CD34+ cell compared to leukemic samples [(TAp73: mean (m) = 0.0162 ± standard deviation = 0.004 vs m = 0.623 ± 0.0845, p = 0,0047); (ΔNp73: m = 0.277 ± 0.09 vs m = 8.09 ± 1.34, p = 0,0215)]. A higher expression of TAp73 and ΔNp73 was observed on RN compared to RP samples [(TAp73: m = 0.992 ± 0.171 vs m = 0.344 ± 0.055, p < 0,0001); (ΔNp73: m = 12.44 ± 2.434 vs m = 4.80 ± 1.382, p = 0,0046)]. There was no difference in the expression of TAp73 between PML-RARα positive samples (m = 0.391 ± 0.095) and the remaining leukemic samples (m = 0.688 ± 0.104, p = 0,1476). However, the expression levels of ΔNp73 were significantly lower in the PML-RARα positive samples (m = 2.656 ± 0.370 vs m = 9.62 ± 1.69, p = 0,0317). No significant difference was observed in ΔNp73 and TAp73 expression between PML-RARα positive samples and the remaining samples with gene rearrengements (TAp73: m = 0.391 ± 0.095 vs m = 0.3144 ± 0.0671, p = 0,4990; ΔNp73: m = 2.656 ± 0.37 vs m = 6.153 ± 2.221, p = 0,2205). When compared to AML1-ETO and CBFβ-MHY11, the RN samples had a higher expression level of TAp73 (m = 0.3144 ± 0.0672 vs m = 0.992 ± 0.1717, p = 0.001), while there was no significant difference on the expression levels of ΔNp73 (m = 6.15 ± 2.22 vs m = 12.44 ± 2.43, p = 0.0642). These findings suggest that both p73 isoforms pathways are involved in the leukemogenic process. Moreover, the lower expression of ΔNp73 in the group with gene rearrangements may contribute to its better prognosis. The distinct pattern of ΔNp73 isoforms expression in AML with PML-RARα rearrangements suggests that it may be associated to a distinct response to apoptotic stimuli and to treatment outcome.
The p73, a homologue to the tumor suppressor gene p53, is involved in oncogenesis, although its specific role remains unclear. This gene has two distinct promoters which encode for two protein isoforms with opposite transcriptional activities: full-length TAp73 and the ΔNp73, the latter differing by the lack of the N-terminal transactivating domain and, has a dominant-negative function towards p53 and TAp73 activity. In the present study, we have investigated, by Real Time PCR, the gene expression pattern of TAp73 and ΔNp73 in 147 samples from patients with de novo AML and 22 samples of CD34+ hematopoietic progenitor cells isolated from bone marrow of healthy donors. We detected a significantly higher expression of TAp73 and ΔNp73 genes in AML samples compared to normal hematopoietic progenitors. AML samples harboring the chromosomal translocations: PML-RARa, AML1-ETO and CBFβ-MYH11 presented significantly higher levels of the TAp73 than those negative for these rearrangements. In contrast, samples without these gene rearrangements expressed higher levels of the truncated form ΔNp73. No difference was observed among patients with PML-RARa, AML1-ETO and CBFβ-MYH11. In order to determine if the ratio between the relative expression of TAp73 and ΔNp73 genes was associated with differential susceptibility to pro-apoptotic stimuli, we performed an in vitro cytotoxicity assay using Ara-C. Blasts from 20 AML patients were incubated for 24 hours in a medium containing 100μg-ml of Ara-C and evaluated for apoptosis status using Anexin V and Propidium Iodite. The leukemic cells with a higher ΔNp73 / TAp73 ratio were significantly more apoptosis-resistant (Pearson’s R2: −0.607). We also assessed the protein expression by flow cytometry using saturating amounts of specific antibodies for each isoform and analyzing the mean fluorescence channel. Estimated protein expression corroborated the gene expression results. Our results suggest an association between the presence of PML-RARα, AML1-ETO or CBFβ-MYH11 chromosomal translocation and a higher TAp73 gene expression compared to ΔNp73 isoforms expression in AML samples. Moreover, the higher resistence to apoptosis detected in blasts with higher ΔNp73 / TAp73 values suggest that this pathway may contribute to prognosis.
1845 Acute Promyelocytic Leukemia (APL) patients present increased bone marrow microvascular density (MVD) compared to normal bone marrow, which has been associated with the aberrant secretion of the proangiogenic factor VEGF by leukemic cells. The APL associated fusion protein PML-RARα is thought to deregulate the TGF-β pathway, through its dominant negative action on cytoplasmic PML, thus down regulating SMAD2/3 signaling, and VEGF transcription. However, PML-RARα expression was associated with increased TGF-β gene transcription and secretion. We used the low molecular weight quinazolinone alkaloid Halofuginone (HF), which has been shown to be a potent TGF-β inhibitor, to test the association between TGF-β/VEGF/angiogenesis. HF inhibited the VEGF secretion by NB4 (an APL cell lineage) and cell proliferation. To determine the effects of HF in vivo, irradiated NOD/SCID mice were transplanted with leukemic cell from hCG-PML-RARα transgenic mice. Twenty-four hours after transplantation, mice were treated with 150μg/kg of HF by intraperitoneal injections for 21 days. All recipients developed leukemia, however the leukemic infiltration of bone marrow and WBC were significantly lower in animals treated HF (4.2 ± 3.89 vs. 20.6 ± 21.9, p <0.0001) and hemoglobin and platelet counts higher in this treated group (Hb: 12.0 ± 1.40 vs. 9.6 ± 1.67, p <0.0001; and Platelets: 932.0 ± 122.5 vs. 552.0 ± 83.2, p <0.001). Accordingly, a lower leukemic infiltration of spleen was detected (ratio spleen/body of 0.006 vs. 0.012 in treated and untreated groups respectively, p=0.0415). Furthermore, the differential count and immunophenotyping of bone marrow showed a lower percentage of immature myeloid cells (27 ± 9.3 vs. 66.3 ± 17.9; p=0.0037 and 16.88 ± 6.27 vs. 44.06 ± 27.06, respectively). HF treatment did not induce molecular remission nor increased the survival of leukemic animals. However, the lack of difference in survival could be atributted to HF hepatoxicity, as a significant rise in AST and ALT serum levels was observed (661.2 ± 262.6/177.9 ± 107.4 vs. 89.88 ± 11.65/27.86 ± 4.47 U/L, prespectively). Phosphorilated SMAD2 levels were determined by ELISA in NB4 cells and a significant dose dependent decrease was observed in samples treated with HF at the doses of 50, 100 and 200 ng/mL. Gene expression analysis showed that the HF treatment inhibited the expression of TgfB, Smad3, Smad4, Myc, Vegf and Egf and the immunohistochemistry analysis of BM sections revealed a significant decrease of VEGF staining (30 vs. 80%, p =0.0227), but there was no decrease in the microvascular density. Taken together, these results showed that angiogenesis is an important therapeutic target in APL, and despite the toxicity, HF has antileukemic potential due to its antiproliferative and proangiogenic factors inhibitory capabilities. Disclosures: Assis: FAPESP: Research Funding; CNPq: Research Funding.
Telomeres are hexameric nucleotide repeats in tandem at the ends of linear chromosomes that function to protect chromosomes and prevent genomic DNA erosion. Telomeric DNA can be elongated by the telomerase complex, which is composed of a reverse transcriptase catalytic subunit (TERT), an RNA template (TERC), and associated proteins. Loss-of-function mutations in genes encoding telomerase complex components are leading causes for telomeres shortening and have been associated with several human pathologies, including aplastic anemia and dyskeratosis congenita, both with a proclivity for progression to acute myeloid leukemia. We have demonstrated that telomere dysfunction and telomerase mutations are genetic risk factors for AML (Calado et al. PNAS 2009; Calado et al. Leukemia 2012). However, the contribution of telomere integrity to acute promyelocytic leukemia (APL) and its clinical significance have not been explored. Here, we retrospectively determined the telomere content of leukemic cells in samples collected from 74 APL patients (age, 15-65y) treated according to combination of all-trans retinoic acid (ATRA) and antracycline (Rego et al. Blood 2013) from Ribeirão Preto,São Paulo, Brazil. The data were analyzed in order to correlate the relationship with clinical and laboratory features, hematologic recovery, relapse, and survival. As controls, peripheral blood cells from 338 healthy subjects (age, 0-87y) were collected. All participants gave written informed consent approved the local Ethics Committee in Human Research (CEP). Telomere content measurement was determined by real-time quantitative PCR (qPCR) using the Rotor-Gene SYBR Green PCR Master Mix (Calado et al. Leukemia 2012). Briefly, this method compares the abundance of telomere content in comparison to the abundance of a invariable standard single gene, providing relative measurement, as determined by a telomere:single gene (T/S) ratio. Telomere content was measured and patients divided into quartiles, according to their telomeres, and dichotomized into short (lower quartile) and long (upper three quartiles) telomeres. Telomeres were significantly and consistently short in APL blasts as compared to peripheral blood leukocytes of age-matched healthy donors (Figure 1; P<0,0001)). There was no relevant difference between APL patients with short (n=21) and long (n=53) telomeres with respect to clinical and laboratory features. Overall, 63 subjects (85%) achieved complete remission (CR). Patients with short telomere APL blasts tended to have poorer CR rate as compared to patients with longer telomere blasts, although the difference was statistically marginally significant (71% vs 90%; P=0.06). Eight patients (11%) experienced early mortality (i.e., death during induction therapy) and telomere length was predictive of early mortality (23% for those with short telomeres in comparison to 5% for those with longer telomeres; P=0.037). With a median follow-up of 33 months (range, 1-72 months), patients with long telomeres also appeared to have better 2-year overall survival (OS) (90%) compared with those with short telomeres (76%) by trend (P = 0.07). Telomere length had no impact on disease-free survival (DFS) rate (P =0.66). Our findings demonstrate that APL blast cells consistently have very short telomeres at presentation, suggesting that telomere dysfunction may contribute to genomic instability in leukemogenesis. Our results suggest that telomere length of APL blasts may predict the achievement of complete remission, early mortality, and overall survival. However, the number of patients enrolled in this study was relatively small and the follow-up was short. These results need to be confirmed in a larger number of patients followed for a longer period of time and validated by an independent cohort. If confirmed, as telomere length measurement is a simple and inexpensive test, it may be applied in the future in the risk assessment of patients with APL. Disclosures: No relevant conflicts of interest to declare.
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