ALBERT

Description: The t(15;17) translocation in acute promyelocytic leukemia (APL) yields a PML/RAR-α fusion messenger RNA species that can be detected by reverse transcription–polymerase chain reaction (RT-PCR) amplification. Breakpoints within intron 3 of PML produce a short PML/RAR-α isoform, whereas breakpoints within intron 6 result in a longer form. Using RT-PCR, serial evaluations were performed on the bone marrow of 82 patients with APL (median follow-up, 〉 63 months) who received retinoic acid (RA) induction followed by postremission treatment with chemotherapy, RA, and biologic agents. Sixty-four patients attained a clinical complete remission and had at least 2 RT-PCR assays performed after completing therapy. Forty of 47 patients (85%) with newly diagnosed APL who were induced using RA had residual disease detectable by RT-PCR before additional therapy. After 3 cycles of consolidation therapy, residual disease was found in only 4 of 40 evaluable patients (10%). Among newly diagnosed patients who had 2 or more negative RT-PCR assays, only 3 of 41 (7%) had a relapse, whereas all 4 patients (100%) who had 2 or more positive results had a relapse. Among 63 newly diagnosed patients, those who expressed the short isoform appeared to have shorter disease-free and overall survival durations than patients who expressed the long isoform. These data indicate that 2 or more negative RT-PCR assays on bone marrow, performed at least 1 month apart after completing therapy, are strongly associated with long-term remissions. Conversely, a confirmed positive test is highly predictive of relapse.

Description: The nucleoporin gene NUP98 is fused to several genes including HOXD13 in patients with myelodysplastic syndromes (MDS), acute myeloid leukemia, and chronic myeloid leukemia, blast crisis. Genetically engineered mice that express a NUP98-HOXD13 (NHD13) transgene (Tg) display the phenotypic features of MDS, including cytopenias, bone marrow dysplasia, and transformation to acute leukemia. Here we show that short-term treatment with the p53 inhibitor Pifithrin-α partially and transiently rescued the myeloid and lymphoid abnormalities found in NHD13+ Tg mice, with no improvement in the anemia, while the genetic deletion of 2 alleles of p53 rescued both the myeloid progenitor cell and long-term hematopoietic stem cell compartments. Nonetheless, loss of one or both alleles of p53 did not rescue the MDS phenotype, but instead exacerbated the MDS phenotype and accelerated the development of acute myeloid leukemia. Our studies suggest that while targeting p53 may transiently improve hematopoiesis in MDS, over the long-term, it has detrimental effects, raising caution about abrogating its function to treat the cytopenias that accompany this disease.

Description: Abstract 1588 Transcription 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

Description: Abstract 2804 The myelodysplastic syndromes (MDS) are clonal stem cell disorders characterized by ineffective hematopoiesis leading to blood cytopenias and a high rate of progression to acute myelogenous leukemia (AML) (Nimer, 2008). Recent studies implicated an important role for p53 in 5q−MDS, a subtype where haploinsufficiency of the RPS14 ribosomal protein drives the anemia that accompanies this disease (Ebert et al., 2008; Barlow et al., 2010 and Dutt et al., 2011). The elevated level of p53 activity apparently triggers the excessive apoptosis and the dysplastic morphology seen in the erythroid cells of the “5q-mice” that lack one copy of the chromosomal region syntenic to the human 5q region that contains the RPS14 gene (Barlow et al., 2010). Thus, the cytopenias in some patients with MDS may be the result of defective ribosomal biosynthesis, leading to activation of p53 and excessive apoptosis. The nucleoporin gene NUP98 is fused to a number of different genes including HOXD13 by chromosomal translocations that are found in patients with MDS, AML and CML, blast crisis. Genetically engineered mice that express a NUP98-HOXD13 (NHD13) transgene (Tg) display the phenotypic features of MDS, including cytopenias, bone marrow dysplasia, and transformation to acute leukemia (Lin et al., 2005). We obtained these mice and analyzed the hematopoietic stem cells (HSC) and the erythroid compartment and found significantly decreased rps14 mRNA expression in the NHD13+ CD71+Ter119+ cells compared to the WT controls. Furthermore, flow cytometry analysis revealed increased intracellular p53 levels in the NHD13+ Lin−Sca-1+ c-Kit+ (LSK) and CD71+Ter119+ cells. These data suggest that defective rps14 ribosomal protein production and an increased p53 level may contribute to the anemic phenotype in NHD13+ Tg mice. To examine whether inhibition of p53 function can improve the cytopenias of NHD13+ Tg mice, mice were injected with Pifithrin-α (a reversible inhibitor of p53-mediated apoptosis and p53-dependent gene transcription, 2 mg/kg body weight) daily for five weeks. We observed partial rescue of the myeloid and lymphoid lineage differentiation defects, with no improvement in the hemoglobin level. To further investigate whether the presence or absence of p53 affects the MDS or AML phase of NHD13 driven disease, we generated NHD13+p53+/− and NHD13+p53−/− mice. Deletion of one allele of p53 rescues the myeloid progenitor cell compartment, while deletion of both alleles reversed the deficit in both the LSK and the MPP populations that was observed in the NHD13+ Tg mice. The clinically healthy NHD13+p53−/− mice (aged 3 to 5 months) and the NHD13+p53+/− mice (aged from 3 to 7 months) both had more severe leukopenia and anemia than the NHD13+ Tg mice or the WT controls (aged 4 to 7 months). 60% of the NHD13+p53−/− mice and the NHD13+p53+/− mice developed MDS with a median survival of 138 d and 190 d respectively; in contrast 30% of the NHD13+ Tg mice had MDS with a median survival of 236 d. These data indicate that the relative or absolute lack of p53 hastens the development of MDS, and shortens the median survival. Lack of one or two p53 alleles significantly accelerated the development of AML, which in NHD13+p53+/− mice resulting in a median survival of 278 d. However, AML in the NHD13+p53−/− mice is more undifferentiated with a median survival of 133 d, and the NHD13+ Tg mice showed AML with a median survival of 324 d. Taken together, these data demonstrated that the chronic loss one allele or two alleles of p53 does not rescue the MDS phenotype induced by NHD13 fusion gene. Rather it accelerates the development of NHD13 driven MDS and leukemia. Our studies suggest that targeting p53 transiently may temporally improve hematopoiesis in MDS, but over the long term has detrimental effects on hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 942 Background: Early death in APL, most often due to bleeding, has emerged as the major cause of treatment failure now that curative strategies exist. Despite the routine use of ATRA, EDR remains high (Lehmann et al Leukemia 2011, Park et al Blood 2011). Although the optimal strategy to prevent early death is not clear, the recommendation is to initiate ATRA immediately at first suspicion of the disease without waiting for genetic confirmation. Therefore, we examined the timing of ATRA administration. Methods: To determine time interval from initial presentation to ATRA administration, we retrospectively collected data on all newly diagnosed APL pts presenting between 1992–2009 to 4 institutions: Northwestern University, Chicago, IL (university medical center); Memorial Sloan-Kettering Cancer Center, New York, NY (free standing cancer center); John J. Stroger Hospital of Cook County, Chicago, IL (public hospital); and Rambam Medical Center, Haifa, Israel, (Northern Israel's largest hospital; a tertiary referral center). We also examined 3 other time intervals: presentation to suspicion of APL, suspicion of APL to ordering ATRA, and ordering ATRA to its administration. Results: We identified 205 newly diagnosed APL pts: 46% men, median 48 years (range 1.5–85). Median white blood cell (WBC) count at presentation was 2,100/uL (range 300/uL-222,500/uL); 25% had high risk (HR) disease (WBC 〉10,000/uL). Median time interval from initial presentation to suspicion of APL was 1 day and median time from suspicion of APL to ordering ATRA was an additional day (table 1). ATRA was ordered on the day APL was suspected in 32% pts, the next day in 31%; 2 days after suspicion in 17%; and after 3 or more days in 16%. 89% received ATRA on the day ordered. At least 1 bleeding episode was identified in 34% of 185 pts with bleeding data available. Bleeding was associated with higher WBC count (p=0.0003) and lower hemoglobin (p=0.027) at presentation. 46 of 186 pts with complete information on time from presentation to administration of ATRA died. 23 (12%) died within 30 days of presentation; comprising half of all fatalities. Causes of death were: hemorrhage −15, sepsis −4, suspected MI −2, pneumonia −1, and sepsis plus differentiation syndrome -1. Among deaths within 30 days, 48%, 22%, 26% and 7% were in 1st through 4th weeks, respectively. 4 (18%) of these 23 pts died before ATRA was administered, all day 1 or 2 after presentation and all from bleeding. Only 15/182 pts received ATRA on day of presentation. Two of these 15 (13%) died within 30 days (none from bleeding). In comparison, 7/40 (18%) who received ATRA on day after presentation died within 30 days (71% from bleeding). 10/127 (8%) who received ATRA on day 2 or after died within 30 days (6 from bleeding). 20% in each group who received ATRA on either day of presentation or day 1 after presentation had HR disease. For this subgroup, if ATRA was administered on the day APL was suspected or one of the following 2 days, EDR was 19% (7/37). However, if ATRA was initiated on day 3 or 4, EDR was 80% (4/5); (p=0.013). 59% received ATRA prior to confirmation and 41% received ATRA on the day APL was confirmed or later. Conclusions: (1) APL was suspected rapidly upon presentation, usually within 1 day and ATRA was almost always administered on the day ordered. (2) However, ATRA was not given to most pts the day APL was suspected and for some 2 or more days elapsed. This time interval contributed to the delay in ATRA administration, suggesting physicians waited for marrow morphology or genetic confirmation before ordering ATRA. (3) Although at these centers a lower EDR (12%) than reported from the SEER database (17.3%) was observed, the current recommendation to give ATRA at first suspicion of APL was often not practiced. (4) There appears to be an association between EDR and timing of ATRA administration; other factors contribute to the EDR including variability in blood product support. (5) Our study argues that educating health care professionals who are the first to encounter APL pts as to the urgency of ATRA administration will reduce early deaths that occur even in the ATRA era. Disclosures: No relevant conflicts of interest to declare.

Description: The ability of hematopoietic stem cells to tightly regulate the transition from relative quiescence and self-renewal to the transiently amplifying, differentiating progenitor fate is critical for HSC homeostasis as well as their regenerative capacity. We have recently described the diminished frequency and rapid exhaustion of HSC self-renewal capacity in the absence of the dominant negative helix-loop-helix molecule Id1. Furthermore, Id1 null HSCs have an increased rate of cycling, coupled with accelerated myeloid commitment both in vivo and in vitro. This is reflected in the elevated expression of myelo-erythroid transcription factors (c/EBPalpha and GATA1) within the Lin−c-kit+Sca-1+ population - “myeloid priming”. The major targets of Id1 mediated transcriptional repression are the ubiquitous E protein E2A as well as Ets transcription factors (Ets1 and Ets2). We hypothesized that the unrestrained activity of these and/or other targets of Id1 transcriptional repression leads to premature HSC commitment in Id1 null animals. Indeed, we show that HSC differentiation in culture can be delayed by transduction of E2A directed shRNA specifically in Id1 null, but not in wild-type Id1 expressing cells. This indicates an abnormal E2A activity in Id1 null HSCs that could be responsible for their increased differentiation status. To further define the transcriptional deregulation in Id1 null HSCs, we have used the Affymetrix microarray technology. We observed ~3 fold increased expression of the CDK inhibitor p21 in freshly isolated Id1 null HSCs and have confirmed this result by multiple independent qPCR measurements. The transcriptional induction of p21 by E2A as well as its repression by Id1 have been well established. Therefore, the observed p21 induction could be explained by the elevated level of E2A activity in HSCs in the absence of Id1 expression. To explore the functional significance of Id1 mediated p21 regulation in HSCs, we have generated p21/Id1 double knockout animals. Surprisingly, despite its reported function in restricting the cell cycle entry of normal HSCs, we show that in the context of Id1 loss, p21 expression is required for the accelerated HSC cycling, and unlike Id1 single null HSCs, p21/Id1 double knockout HSCs do not show accelerated myeloid differentiation in culture. Therefore, we propose that Id1 actively represses E2A activity in HSCs, as well as the induction of p21, which could be an important component of the HSC commitment program. Further studies will be presented defining the in vivo relevance of the Id1/p21 genetic interaction for HSC growth and differentiation.