Bivalent promoter marks and a latent enhancer may prime the leukaemia oncogene LMO1 for ectopic expression in T-cell leukaemia

LMO1 is a transcriptional regulator and a T-acute lymphoblastic leukaemia (T-ALL) oncogene. Although first identified in association with a chromosomal translocation in T-ALL, the ectopic expression of LMO1 occurs far more frequently in the absence of any known mutation involving its locus. Given that LMO1 is barely expressed in any haematopoietic lineage, and activation of transcriptional drivers in leukaemic cells is not well described, we investigated the regulation of this gene in normal haematopoietic and leukaemic cells. We show that LMO1 has two promoters that drive reporter gene expression in transgenic mice to neural tissues known to express endogenous LMO1. The LMO1 promoters display bivalent histone marks in multiple blood lineages including T-cells, and a 3' flanking region at LMO1 +57 contains a transcriptional enhancer that is active in developing blood cells in transgenic mouse embryos. The LMO1 promoters become activated in T-ALL together with the 3' enhancer, which is bound in primary T-ALL cells by SCL/TAL1 and GATA3. Taken together, our results show that LMO1 is poised for expression in normal progenitors, where activation of SCL/TAL1 together with a breakdown of epigenetic repression of LMO1 regulatory elements induces ectopic LMO1 expression that contributes to the development and maintenance of T-ALL.

Supplementary Information accompanies this paper on this Leukemia website (http://www.nature.com/leu) OPEN Sequential combination of azacitidine and lenalidomide in del(5q) higher-risk myelodysplastic syndromes or acute myeloid leukemia: a phase I study Leukemia (2013) 27, 1403-1407; doi:10.1038/leu.2013.26 Clonal deletion of the chromosome 5 long arm (del(5q)) has important prognostic implications for patients with myelodysplastic syndromes (MDS) or acute myeloid leukemia (AML). 1 Even standard intensive therapy including induction chemotherapy and allogeneic hematopoietic stem cell transplantation is often associated with treatment failure. 2 Combination azacitidine plus lenalidomide represents a potential innovative approach in these patients, and is also supported by existing data from patients with higher-risk non-del(5q) MDS or AML. 3,4 The primary objective of the Azacitidine-Lenalidomide multicenter phase I study was to identify the dose-limiting toxicity and maximum tolerated dose of lenalidomide in combination with a fixed regimen of azacitidine during treatment cycle 1 in patients with higher-risk MDS or AML and del(5q). Secondary endpoints were clinical and cytogenetic response rates, safety and mutational analyses. A standard 3 þ 3 design was used to determine the maximum tolerated dose. Eligible patients were: aged X18 years with del(5q) karyotypic abnormalities; had an Eastern Accepted article preview online 28 January 2013; advance online publication, 19 February 2013 Letters to the Editor Cooperative Oncology Group performance status score of 0-3; had previously treated or untreated International Prognostic Scoring System Intermediate-2-or high-risk MDS or AML (430% bone marrow (BM) blasts according to French-American-British classification); and were ineligible for immediate allogeneic hematopoietic stem cell transplantation due to donor unavailability. The study was conducted in accordance with the Declaration of Helsinki, and received approval from appropriate institutional review boards or ethics committees. All patients provided written informed consent. ClinicalTrials.gov identifier NCT00923234.
The effects of azacitidine are cell-cycle S-phase-dependent, whereas lenalidomide inhibits cell-cycle progression; therefore, we considered sequential administration of azacitidine followed by lenalidomide to maximize their additive effects. 5 In accordance with a previous study, 4 and to avoid potentially higher hematotoxicity associated with azacitidine in direct combination with lenalidomide, patients received azacitidine 75 mg/m 2 /day subcutaneously (days 1-5) then oral lenalidomide (days 6-19) starting at 10 mg/day, escalating in 5 mg increments to 25 mg/day. Patients who experienced dose-limiting toxicity could continue at the next lower dose level. In patients with partial response or stable disease, induction therapy was continued up to eight cycles, or until progressive disease, unacceptable toxicity, or complete response (CR). To prevent continued hematotoxicity, patients who achieved complete BM blast clearance (o5% BM blasts or CR) after X2 cycles of induction therapy received maintenance therapy, which comprised azacitidine 30 mg/m 2 /day subcutaneously (days 1-5) followed by lenalidomide at maximum tolerated dose (days 6-19) every 8 weeks, up to six cycles or until progressive disease. Response was evaluated using International Working Group 2003 criteria for AML and 2006 criteria for MDS. 6,7 Conventional chromosome banding analyses from BM cultures and fluorescence in situ hybridization analyses of peripheral blood CD34 þ cells were performed. Evaluation of molecular mutations in candidate genes including TP53 was performed using nextgeneration deep-sequencing of whole-BM cells before the study. 8 Screening for ASXL1 mutations was performed using Sanger capillary sequencing. Changes in TP53 mutation levels were evaluated in patients who achieved both complete hematologic and cytogenetic responses.
A median of two induction cycles (range, 1-6) were administered; two patients received maintenance cycles. Three of six patients treated with lenalidomide 25 mg experienced a doselimiting toxicity, including: one patient with absence of hematologic recovery despite achieving a BM CR (suggesting response, but no recovery of counts due to drug-associated toxicity); one patient with pneumonia considered probably related to the study drugs; and one patient with grade 3 deep-vein thrombosis considered possibly related to lenalidomide, which required temporary treatment interruption then dose reduction. Of an additional three patients to receive lenalidomide 20 mg, one patient had a treatment delay of 44 weeks, resulting from a grade 4 sepsis considered probably related to study drugs. Although the lenalidomide maximum tolerated dose of 20 mg in combination with azacitidine was lower than in previous reports, 3,9 the heavily pretreated patient population together with the sequential approach may have potentiated the hematologic toxicity profile of both compounds. In addition, del(5q) progenitors are more sensitive to the antiproliferative effects of lenalidomide compared with other cytogenetic aberrations. 5 The adverse event profile of sequential azacitidine and lenalidomide was consistent with that of the individual drugs. 10,11 The most common non-hematologic grade X3 AEs were pneumonia (40%) and sepsis (15%). The most common treatment-related grade 3-4 hematologic AEs were thrombocytopenia (45%) and neutropenia (35%). Overall, 13 of 29 serious AEs were considered to be possibly, probably, or definitely related to the study drugs, including: three events of grade 3 febrile neutropenia; one of grade 4 pancytopenia; two of pneumonia; and one event each of grade 4 sepsis and grade 3 herpes zoster (in one patient), grade 3 deep-vein thrombosis, grade 3 nausea and vomiting, grade 4 thrombocytopenia, grade 3 anemia and grade 4 cerebral ischemia. Seven patients with serious AEs died (median one cycle), including two patients with Aspergillus pneumonia, one patient with atypical pneumonia, one patient with infectious sepsis and three patients with pneumonia. However, only one serious AE resulting in death (pneumonia in a patient who received one cycle of lenalidomide 25 mg) was considered related to study drugs. Overall, 10 patients survived X6 months after treatment initiation.
Despite most patients having a complex aberrant karyotype and heavily pretreated, response rates were encouraging (Table 1). Of 19 patients evaluable for response, 26 and 42% achieved hematologic (CR, CR with incomplete recovery of peripheral blood counts or partial response) and cytogenetic responses, respectively. Among previously untreated patients, hematologic and cytogenetic response rates were 44 and 56%, respectively. All three patients who achieved a cytogenetic response without hematologic response discontinued treatment early due to AEs. Median duration of hematologic and cytogenetic responses was 2.3 months (range, 0.9-8.1) and 3.2 months (range, 1.9-6.4), respectively. All responders achieved an initial response after cycle one, in contrast to data with azacitidine alone. 12 Although other studies of sequential azacitidine 75 mg/m 2 and lenalidomide (up to 75 mg) have reported higher response rates (up to 60%), among untreated patients with higher-risk MDS or AML, they enrolled a comparatively low percentage of patients with adverse baseline cytogenetics. 3,9 Furthermore, the majority of our patients displayed a TP53 mutation. This is consistent with the high-rate of TP53 mutations observed in advanced del(5q) MDS, which appears to underlie MDS pathophysiology in some patients with del(5q). 13 An interesting preliminary finding suggested that monitoring of peripheral blood del(5q)-positive CD34 þ cells may be a surrogate marker of response. Compared with baseline, the percentage of peripheral blood CD34 þ cells with the del(5q) clone was reduced in the five hematologic responders (including three with TP53 and one with DNMT3A mutations) after one cycle of therapy (P ¼ 0.001) (Figure 1a), but remained unchanged in nonresponders (Figure 1b). Furthermore, for the first time we performed minimal residual disease monitoring in consecutive BM samples of patients with complete cytogenetic and hematologic responses using next-generation deep-sequencing. Sequential treatment resulted in a rapid decline and disappearance of the TP53-mutant clone in one patient (Figure 1c), and decline followed by steady reemergence in the other (Figure 1d). Re-emergence of the TP53 clone might have occurred owing to treatment continuation by consolidation (lower doses, cycles every 8 instead of 4 weeks), and preceded hematologic and cytogenetic relapse by several months. In addition, del(5q) and del(17p) levels in peripheral blood CD34 þ cells increased concomitantly with the re-emergence of the TP53mutated clones in the BM (Figure 1d). Intensification of combination treatment especially for the first six cycles under molecular minimal residual disease monitoring might require further investigation. Effects on TP53 mutated clonal cells have not been reported with lenalidomide alone, reinforcing the potential benefit of its combination with azacitidine. 14,15 Both lower-and higher-risk del(5q) MDS patients harboring TP53 mutations have lower response rates to lenalidomide and a high-risk of AML evolution compared with unmutated patients. 14,15 Whether the Letters to the Editor addition of azacitidine improves treatment outcomes needs to be evaluated in future studies. In summary, in a population of del(5q) higher-risk MDS and AML patients, including a majority with complex karyotypes, the sequential combination of azacitidine and lenalidomide was shown to be a feasible and potentially effective treatment strategy, even in patients with TP53-mutated clones. We observed a correlation between the percentage of peripheral CD34 þ cells with del(5q) and response, suggesting that monitoring of this cell population may be a surrogate marker of response. Our results encourage application of sequential azacitidine and lenalidomide as first-line therapy for MDS patients with del(5q) in future trials, using a maximum lenalidomide dose of 20 mg, and with close surveillance of hematologic side-effects during the first two induction cycles.  Immunomodulatory derivatives of thalidomide (IMiD compounds) like lenalidomide (LEN) have impressive clinical activity in patients with both relapsed or refractory and newly diagnosed multiple myeloma. 1 Hematopoietic stem cell (HSC) transplantation is still the 'backbone' in the treatment of newly diagnosed multiple myeloma patients and very often combined with LEN in order to achieve high response rates. 2 Unfortunately, clinical data have shown that in up to 43% of patients, standard mobilization with granulocyte colony-stimulating factor (G-CSF) alone failed to mobilize hematopoietic progenitors, raising concerns about potential stem cell toxicity of LEN. 3,4 We have shown that neither LEN nor pomalidomide (POM) are toxic to HSC, 5,6 and it is known that mobilization of CD34 þ cells with Plerixafor (AMD3100) overcomes mobilization failures after LEN treatment. 7 The fact that AMD3100 antagonizes the binding of the chemokine stromal-cell-derived factor-1a (SDF-1a) to its receptor CXC chemokine receptor 4 (CXCR4) suggests a potential role of the CXCR4/SDF-1a axis in mediating mobilization failure after LEN treatment. To further analyze why blocking CXCR4 by AMD3100 overcomes mobilization failure to G-CSF in LEN-treated patients, we analyzed the effects of LEN on CXCR4 expression and function in CD34 þ cells.

CONFLICT OF INTEREST
We first analyzed CXCR4 expression in CD34 þ cells treated with LEN and POM. Flow cytometry analysis indicated that the CD34 þ cell surface expression of CXCR4 began to increase after 48 h of LEN (1 or 10 mm) or POM (1 or 10 mm) treatment and remained increased with continuous treatment (Figure 1a left). CD34 þ cell number did not change under LEN or POM treatment (Figure 1a right). In contrast, neither thalidomide (1 mm) nor bortezomib (1 nm) showed effects on CXCR4 membrane expression on CD34 þ Accepted article preview online 9 November 2012; advance online publication, 4 December 2012