ALBERT

Description: Abstract 3166 Background: In ENESTnd, nilotinib (NI) significantly reduced progression and demonstrated superior molecular response rates vs. imatinib (IM) in patients newly diagnosed with Philadelphia chromosome positive chronic phase chronic myelogenous leukemia (CML-CP). Additionally, fewer NI patients discontinued therapy vs. IM (Table 1). Previously reported 24-month analyses of PROs indicated similar health-related quality of life (HRQoL) and functioning mean scores between the treatment arms, and scores similar to general population norms. Aim: To evaluate the PROs for patients in ENESTnd with minimum follow-up of 36 cycles and to understand cohort-level (per treatment arm) HRQoL outcomes Methods: In ENESTnd, nNewly diagnosed CML-CP patients were randomized to NI 300 mg twice daily (BID), NI 400 mg BID, or IM 400 mg once daily (QD). HRQoL was assessed using the Functional Assessment of Cancer Therapy – Leukemia (FACT-Leu) and the Short Form 36 Health Survey (SF-36). The FACT-Leu has two components: a) the FACT-General (FACT-G) which measures physical, social/family, emotional, and functional well-being and b) a 17-item leukemia specific subscale. The SF-36 assesses eight domains (physical functioning, role-physical, bodily pain, general health, vitality, social functioning, role-emotional, and mental health) that enable a mental component score (MCS) and physical component score (PCS). Questionnaires were administered at enrollment and end of cycles 3, 12, 24, and 36. Mixed effects models for longitudinal data were used to compare trends over time between treatment arms. Study discontinuation prevented collection of PROs from patients with inadequate response or intolerance to treatment. Pattern-mixture models were fit to attempt to control for missing PRO data resulting from discontinuation or other reasons. Separately, missing data were imputed consistently across arms for cohort-level analysis of the FACT-Leu subscale so mean cohort scores could be reported (Table 1). Results: In both mixed effects and pattern-mixture models, FACT-Leu subscale, FACT-G, and SF-36 scores (PCS and MCS) were similar across treatment arms over time. Of the patients remaining on study and completing PRO questionnaires, SF-36 PCS and MCS scores at cycle 36 in all arms were comparable to the general US population; FACT-G scores were slightly better (Table 2). In the cohort-level analysis with imputation according to reason, IM FACT-Leu subscale scores began to trend increasingly lower vs. NI arms beginning at day 168 (Figure 1). By day 1008, the IM arm mean score is 10% and 13% lower than the NI 300 mg and NI 400 mg arms, respectively (Figure 1). Higher rates of discontinuation in the IM arm are the main factors that lead to the HRQoL deficit. Conclusions: In ENESTnd, patients who respond to and tolerate treatment have consistent HRQoL that is comparable to the general population. Cohort-level analysis indicates that discontinuation rates due to inadequate response and intolerance must be considered when determining the HRQoL across the entire cohort. These results suggest that in a population of newly diagnosed patients with CML-CP, NI results in higher HRQoL than IM. These findings may have particularly important implications for payers and policy makers when evaluating treatment options. Disclosures: Beaumont: Novartis: Research Funding. Magestro:Novartis: Employment. Coombs:Novartis: Employment. Fan:Novartis Pharmaceuticals Corp: Employment. Kemp:Novartis Pharmaceuticals Corp: Employment. Waltzman:Novartis: Employment.

Description: Abstract 3826 Background: Imatinib is the current standard of care for chronic myelogenous leukemia (CML). Nilotinib is a highly potent and the most selective inhibitor of BCR-ABL. A Phase III multi-center, open label, randomized study (ENESTnd) was conducted comparing these two therapies in adult patients with newly diagnosed Philadelphia Chromosome positive (Ph+) CML in chronic phase. The primary endpoint analysis at 12 months demonstrated that major molecular response (MMR) was significantly improved with nilotinib 300 mg BID (44%) and nilotinib 400 mg BID (43%) compared to imatinib 400 mg QD (22%; p 〈 0.001). The discontinuation rate due to adverse events was lowest among the nilotinib 300mg BID treatment arm (5%) compared to 7% in the imatinib arm, and nilotinib 400 mg BID (9%). Based on the results of this clinical trial, nilotinib 300 mg BID was approved for initial use for CML-CP in the US. Aim: To evaluate the occurrence and rate of hospitalizations and time away from usual activities in this phase III trial. Methods: A total of 846 patients were randomized to receive nilotinib 300 mg BID (n=282), nilotinib 400 mg BID (n=281) or imatinib 400mg QD (n=283). Hospitalizations, defined as any visit to the hospital requiring an overnight stay, excluding pre-planned or elective surgery, were assessed throughout the study period. Overdispersed Poisson regression models were used to compare the days hospitalized per 1,000 patient-days on study. Patients were asked to report time-off, defined as average number of hours per week taken away from all usual activities due to CML and side effects of CML treatment over the past 4 weeks, at Baseline and at the end of Months 3 and 12. The Wilcoxon rank-sum test was used to compare the time off from usual activities at each assessment; and t-tests were used to evaluate the within-group changes in time off. Results: There were a total of 57 hospitalizations in the imatinib arm versus 48 hospitalizations in the nilotinib 300 mg BID arm, and 74 hospitalizations in the nilotinib 400 mg BID arm (Table). Descriptive statistics for length of stay (LOS) are presented in the Table. The hospitalization rate, expressed as hospital days per 1,000 patient days, was 47% higher in the imatinib arm compared to the nilotinib 300 mg BID arm (p=0.057) and 8% higher compared to the nilotinib 400 mg BID arm (p=0.68). Patients in the nilotinib 300mg BID arm had fewer stays and shorter LOS than the imatinib arm, whereas patients in the nilotinib 400mg BID arm had more stays than the imatinib arm but shorter LOS on average resulting in fewer total hospital days. The majority of hospitalizations (56%) in all three arms occurred within the first 9 months. Time off from usual activities, which began at an average level of 8–10 hours per week at Baseline, decreased in each arm, but the decrease did not significantly differ between arms (Table). Similar results were observed when patients reporting zero hours of time off were excluded from the analysis. There was no association between time off and age. Summary/conclusions: In patients with newly diagnosed CML-CP, nilotinib resulted in less hospital time compared to imatinib, although this difference did not reach statistical significance. Additionally, patients in all three treatment groups reported significant improvements from baseline in time off from usual activities. Disclosure: Beaumont: Novartis: Research Funding. Coombs:Novartis: Employment, Equity Ownership. Bollu:Novartis: Employment, Equity Ownership. Woodman:Novartis Oncology: Employment. Cella:Novartis: Research Funding.

Description: Hemopoietic lineage switch (Hls) 5 and 7 were originally isolated as genes up-regulated during an erythroid-to-myeloid lineage switch. We have shown previously that Hls7/Mlf1 imposes a monoblastoid phenotype on erythroleukemic cells. Here we show that Hls5 impedes erythroid maturation by restricting proliferation and inhibiting hemoglobin synthesis; however, Hls5 does not influence the morphology of erythroid cells. Under the influence of GATA-1, Hls5 relocates from cytoplasmic granules to the nucleus where it associates with both FOG-1 and GATA-1. In the nucleus, Hls5 is able to suppress GATA-1–mediated transactivation and reduce GATA-1 binding to DNA. We conclude that Hls5 and Hls7/Mlf1 act cooperatively to induce biochemical and phenotypic changes associated with erythroid/myeloid lineage switching.

Description: Abstract 1025 Background: Nilotinib is a more potent and more selective inhibitor of BCR-ABL in-vitro than imatinib. A randomized Phase III study was conducted comparing these two therapies in adult patients with newly diagnosed Philadelphia Chromosome positive (Ph+) chronic myelogenous leukemia (CML) in chronic phase (CP). Aim: To evaluate whether baseline health-related quality of life (HRQL) or symptom scores were predictive of clinical outcome in this phase III trial. Methods: A total of 846 patients were randomized to receive nilotinib 300 mg BID (n=282), nilotinib 400 mg BID (n=281) or imatinib 400mg QD (n=283). For these analyses to evaluate the impact of baseline HRQL on clinical outcomes, the treatment arms were combined. HRQL was assessed using the Functional Assessment of Cancer Therapy–Leukemia (FACT-Leu). The FACT-Leu consists of four general subscales measuring physical, social/family, emotional, and functional well-being and a 17-item leukemia-specific subscale (LeuS). The FACT-G score is the sum of the four general well-being subscales. The questionnaire was administered at baseline, and at 3, 12, and 24 months. Baseline FACT-G and LeuS scores were divided into three equal-sized groups (i.e., tertiles). Patients with high, mid, and low baseline scores were compared on several clinical outcomes: best molecular response by Month 24, best cytogenetic response by Month 24, treatment discontinuation, hospitalization, dose modification of any kind (interruption, increase, or reduction), grade 3 or 4 adverse events, and missed cytogenetic tests. Chi-square tests were used to compare these dichotomous and categorical outcomes between baseline HRQL groups. The relationship between baseline HRQL scores and Sokal Risk groups was evaluated using analysis of variance. Higher scores on the FACT-G and LeuS indicate better HRQL and less symptom burden. Results: Mean baseline FACT-G scores were 97.8, 85.0, and 66.3, and mean baseline LeuS scores were 61.6, 53.8, and 42.2, for the high/mid/low tertile groups, respectively. There was no significant association between best molecular response by Month 24 or cytogenetic response by Month 24 and baseline FACT-G scores (p=0.149 and p=0.094, respectively). There was an association between best molecular response by Month 24 and baseline LeuS scores (p=0.043) but no significant association with best cytogenetic response by Month 24 (p=0.316). There were no significant associations with either dose modifications (p=0.252 for FACT-G, p=0.643 for LeuS), grade 3 or 4 adverse events (p=0.531 for FACT-G, p=0.831 for LeuS), or missed cytogenetic tests (p=0.722 for FACT-G, p=0.374 for LeuS). There was a significant association between treatment discontinuation and baseline FACT-G scores (p=0.007). Only 18% of patients with the highest baseline FACT-G scores discontinued treatment compared to 26% in the middle group and 31% in the group with the lowest baseline FACT-G scores. This relationship was not statistically significant for baseline LeuS scores (p=0.070). Fourteen percent of patients with high baseline FACT-G scores were hospitalized at some point during the study, compared to 15% of patients with mid FACT-G scores, and 22% with low baseline FACT-G (p=0.099). However, 11% of patients with high LeuS scores were hospitalized compared to 20% of patients with mid-range LeuS scores, and 21% of patients with low LeuS scores (p=0.018). The mean baseline FACT-G scores were 81.4, 83.5, and 83.4 (p=0.288), and the mean baseline LeuS scores were 51.1, 53.4, and 52.5 (p=0.042), for patients with high, intermediate, and low Sokal scores, respectively. Conclusions: In patients with newly diagnosed CML-CP, worse general HRQL at baseline was predictive for treatment discontinuation, but not predictive for best molecular response. Leukemia related symptoms at baseline were associated with a greater likelihood of subsequent hospitalization and moderately associated with molecular response. Baseline HRQL was not clearly associated with Sokal scores. These findings suggest that among patients with newly diagnosed CML-CP, examination of baseline HRQL and symptoms may allow patients and clinicians to better anticipate outcomes such as hospitalizations and continuation of therapy. Disclosures: Beaumont: Novartis: Research Funding. Nowinski:Novartis: Research Funding. Coombs:Novartis: Employment, Equity Ownership. Szczudlo:Novartis: Employment, Equity Ownership. Blakesley:Novartis: Employment, Equity Ownership. Gallagher:Novartis: Employment, Equity Ownership. Burns:Novartis: Research Funding. Cella:Novartis: Research Funding.

Description: The J2E erythroblastoid cell line responds to erythropoietin (Epo) by morphological maturation and hemoglobin synthesis. However, on rare occasions, these cells have undergone a spontaneous lineage switch and display features of monoblastoid cells which do not respond to Epo. Amongst the genes up-regulated in the monoblastoid variants were Hemopoietic lineage switch (Hls) 5 and 7. Hls5 is a recently identified member of the RING finger, B Box, Coiled coil (RBCC) or tripartite motif (TRIM) family, which includes PML, a gene involved in acute promyelocytic leukaemia. Hls7 is the murine orthologue of Myeloid leukaemia factor 1 (Mlf1), a gene involved in a t(3;5), associated with acute myeloid leukaemia. We have shown previously that Hls7/Mlf1 imposes a dramatic phenotypic change upon the erythroid cells, rendering them monoblastoid (Williams, J. et al EMBO 1999). We have studied the role of Hls5 and Mlf1 in erythroid commitment and differentiation. Ectopic expression of Hls5 inhibits globin production in erythroid cells and suppresses development of B-FUE and C-FUE. A yeast-two-hybrid screen identified FOG-1 as a binding partner of Hls5. Significantly, FOG-1 is a transcriptional co-regulator of GATA-1, which controls globin gene expression. While Hls5 is able to enhance the repression of GATA-1 activity imposed by FOG-1, it is also able to repress GATA-1 transcriptional activity in the absence of FOG-1. Using electrophoretic mobility shift assay we have shown that Hls5 is able to reduce GATA-1 binding to DNA in a dose dependant manner. This observation that Hls5 reduces GATA-1 binding to promoter elements is mirrored by chromatin immunoprecipitation assays. Expression of MLF1 is highest in CD34+ cells, but is markedly down regulated during erythroid differentiation. Microarray analysis identified a number of known transcriptional regulators differentially expressed in the presence of Mlf1 including ets1, Myc intron binding protein and Tbr2. Mlf1 is able to bind DNA and luciferase reporter assays demonstrated that Mlf1 is able to affect transcription. In addition, Mlf1 interacts with a novel member of the hnRNP family viz Mlf1 associated nuclear protein (Manp). Manp binds to DNA, is able to influence the subcellular localisation of Mlf1 by translocating Mlf1 from the cytoplasm to the nucleus. Importantly, Manp also has an affect on transcription. These data demonstrate that both Hls5 and Mlf1 affect transcription of genes associated with erythroid differentiation.

Description: Abstract 253 Hemopoietic lineage commitment is controlled, in part, by transcription factors that regulate specific genes required for the formation of mature blood cells. Differentiation along particular hemopoietic lineages is dependant not only on the presence of particular transcription factors, but also on appropriate concentrations - altering transcription factor levels can force cells into different hemopoietic pathways. Transcription factors undergo numerous post-translational modifications and are controlled spatially via sub-cellular localisation. De-regulation of transcription factors can result in leukemias, or other blood disorders. GATA-1 is an example of a key lineage-determining gene, essential for erythropoiesis. Increasing GATA-1 levels promotes maturation along the erythroid pathway, whereas reducing GATA-1 concentrations favours myelopoiesis. GATA-1 regulation occurs at multiple levels including transcription, translation and post-translational modifications such as phosphorylation, acetylation, ubiquitination and sumoylation. Although GATA-1 ubiquitination modifies the protein for proteasomal degradation, the effect of adding small ubiquitin-like modier (Sumo) to GATA-1 is unclear. Several examples of hemopoietic differentiation plasticity have been observed. We reported a lineage switch by erythroleukemic J2E cells which spontaneously developed a monoblastoid phenotype. Two genes (Hls5 and Hls7/Mlf1) were isolated from this lineage switch with potential lineage-determining features. Hls5 is a member of the RBCC (Ring finger, B-box, Coiled-coil) family of proteins, which includes PML. Ectopic expression of Hls5 impedes erythroid differentiation by reducing GATA-1 levels, and suppressing hemoglobin synthesis. Significantly, Hls5 relocates from the cytoplasm to associate with GATA-1 in the nucleus, where it interferes with DNA binding and transactivation of GATA-1. Several members of the RBCC family are ubiquitin E3 ligases, catalysing the final step in the ubiquitination process - these molecules play a vital role in regulating the levels of target proteins. Here we show that Hls5 is a bona fide ubiquitin E3 ligase, in partnership with several ubiquitin E2 enzymes. The Ring finger is critical for Hls5 ligase activity as mutation of key residues within the Ring finger ablates catalytic activity. Interestingly, a yeast 2 hybrid screen for Hls5 interactors identified Ubc9 and Pias1, which act as E2 and E3 enzymes in the sumoylation cascade. Co-immunoprecipitation, BRET and co-localization experiments confirmed the Hls5 association with Ubc9 and Pias1. Moreover, Hls5 binds Sumo-1 (but not Sumo-2 or 3), and co-localizes with Sumo-1 in discrete nuclear bodies. Thus, Hls5 interacts with several components of the intracellular sumoylation machinery. Hls5 can also reduce sumoylated proteins globally, indicating it may target these modified proteins for degradation. Recently, a new family of ubiquitin E3 ligases has been described which specifically mark sumoylated proteins for degradation. These Sumo-targeted ubiquitin ligases (STUbL) are found primarily in yeast, and only one mammalian STUbL has been identified. We postulated that Hls5 may be a STUbL, capable of regulating sumoylated GATA-1. Our data demonstrate that while Hls5 is able to bind GATA-1 via the B-box and Coiled-coil domains, it preferentially associates with sumoylated GATA-1 through a canonical Sumo interacting motif (SIM). This results in increased GATA-1 ubiquitination and, as a consequence, levels of sumoylated GATA-1 are reduced substantially. Since mutation of the lysine necessary for Sumo attachment does not affect GATA-1 transactivation, sumoylation may act as a prelude to ubiquitination and protein turn-over. We propose, therefore, that GATA-1 mediates transcription of target genes, and is subsequently sumoylated by Pias1 and Ubc9 – addition of Sumo moieties to GATA-1 enhance binding to Hls5, which in turn impedes GATA-1 DNA binding, and promotes ubiquitination for proteasomal degradation. This model is consistent with decreased levels of GATA-1 in erythroid cells ectopically expressing Hls5, and with the original isolation of Hls5 as a potential lineage-determining gene involved with the erythroid to monoblastoid lineage switch. Thus, Hls5 is a novel STUbL which plays a role in hemopoietic lineage commitment by modulating GATA-1 activity and content. Disclosures: No relevant conflicts of interest to declare.