ALBERT

Description: 112 patients with senile acute myelocytic leukemia (AML), refractory or relapsed AML, and MDS-RAEBt entered this study to receive aclarubicin and low dose Arabinoside Cytosine (Ara-C) in combination with Granulocyte Colony Stimulating Factor (G-CSF) for evaluation of efficacy and tolerance of this CGA regimen. Low dose Ara-C was given at the dosage of 10mg/m2/12h, subcutaneously, D1-14 and aclarubicin 14mg/m2/d, intravenously, D1-4 (regimen A) or 7mg/m2 D1-8 (regimen B). Recombinant G-CSF was given at the dosage of 200μg/m2/d, subcutaneously, D1-14. We proved that overall response rate was 19/26 (73.1%) in senile AML, 48/62 (77.4%) in refractory AML, 12/18 (66.7%) in relapsed AML and 10/13 (76.9%) in MDS-RAEBt; and CR rate was at 8/26 (30.8%) in senile patients, 30/62 (48.4%) in refractory AML, 8/18 (44.4%) in relapsed AML and 5/13 (38.5%) in MDS-RAEBt, which were comparable in four groups of patients. 52 patients were followed up. Median progression free survival (PFS) and overall survival (OS) were 9.0±2.2 months and 11.0±1.6 months, respectively. The Kaplan-Meier estimated PFS and OS rate at 12 months were 40.73±8.15% and 42.85±8.23%, respectively. 1 year OS and PFS rate of the patients with CR were 60%±10.8% and 51.3%±13.7%, respectively, compared with the patients with PR, 17.7%±9.3% and 6.4%±6.1%, respectively. The toxic effects were very rare, mainly manifested as hematological changes, neutropenia and thrombocytopenia due to myelosuppression, which was around 70–80% exceeding NCI grade II. And non-hematological toxicities were not observed in this study. CAG regimen seems to be promising for the treatment of various categories of poor-prognosis AML or MDS-RAEBt, with acceptable toxicity.

Description: Background: Expansion of one or more subclone occurs during progression from myelodysplastic syndrome (MDS) to secondary acute myeloid leukemia (sAML). Existing data suggest that acquired mutations in myeloid transcription factor (e.g., RUNX1, CEBPA, WT1) and signaling genes (e.g., receptor tyrosine kinases or RAS pathway genes) contribute to clonal evolution and the rising blast count that defines progression to sAML. While signaling gene (SG) mutations are typically acquired later in disease progression, our understanding of when transcription factor (TF) mutations occur, in what clone they occur (e.g. founding clone or subclone), and whether TF-mutated clones undergo further clonal evolution remains incomplete. This is largely due to the limited number of paired MDS and sAML samples analyzed, the limitation of current sequencing technology and the lack of serial samples, and incomplete characterization of tumor clonality. Methods: We banked paired MDS and sAML (plus skin) samples from 44 patients who progressed from MDS to sAML (median time to progression 306 days, range 21-3568). We sequenced sAML and skin samples for 285 recurrently mutated genes (RMGs) and genotyped the paired MDS sample in patients with TF and/or SG mutations. Twelve patients were selected for enhanced whole genome sequencing (eWGS) of MDS and sAML samples (plus skin) to characterize tumor clonality. Somatic mutations were validated using error-corrected sequencing and clones were identified in MDS and sAML samples using mutation variant allele frequencies (VAFs). We tracked clonal evolution by sequencing serial samples between MDS and sAML. Results: The frequency of both TF and SG mutations were elevated in the 44 sAML patients compared to our previously sequenced cohort of 150 independent de novo MDS patients (signaling: 36% vs. 15%, transcription factor: 30% vs. 11%, respectively, p

Description: Somatic mutations in U2AF1, a spliceosome gene involved in pre-mRNA splicing, occur in up to 11% of MDS patients. While we reported that mice expressing mutant U2AF1(S34F) have altered hematopoiesis and RNA splicing, similar to mutant MDS patients, the role of wild-type U2AF1 in normal hematopoiesis has not been studied. U2AF1mutations are always heterozygous and the wild-type allele is expressed, suggesting that mutant cells require the residual wild-type (WT) allele for survival. A complete understanding of the role of wild-type U2AF1 on hematopoiesis and RNA splicing will enhance our understanding of how mutant U2AF1 contributes to abnormal hematopoiesis and splicing in MDS. In order to understand the role of wild-type U2af1 in normal hematopoiesis, we created a conditional U2af1 knock-out (KO) mouse (U2af1flox/flox). Homozygous embryonic deletion of U2af1using Vav1-Cre was embryonic lethal and led to reduction in fetal liver hematopoietic stem and progenitor cells (KLS and KLS-SLAM, p ≤ 0.05) at embryonic day 15, suggesting that U2af1 is essential for hematopoiesis during embryonic development. To study the hematopoietic cell-intrinsic effects of U2af1 deletion in adult mice, we performed a non-competitive bone marrow transplant of bone marrow cells from Mx1-Cre/U2af1flox/flox, Mx1-Cre/U2af1flox/wtor Mx1-Cre/U2af1wt/wtmice into lethally irradiated congenic recipient mice. Following poly I:C-induced U2af1deletion, homozygous U2af1 KOmice, but not other genotypes (including heterozygous KO mice), became moribund. Analysis of peripheral blood up to 11 days post poly I:C treatment revealed anemia (hemoglobin decrease 〉1.7 fold) and multilineage cytopenias in homozygous U2af1 KOmice compared to all other genotypes(p ≤ 0.001, n=5 each).Deletion of U2af1 alsoled to rapid bone marrow failure and a reduction in the absolute number of bone marrow neutrophils (p ≤ 0.001), monocytes (p ≤ 0.001), and B-cells (p ≤ 0.05), as well as a depletion of hematopoietic progenitor cells (KL, and KLS cells, p ≤ 0.001, n=5 each). Next, we created mixed bone marrow chimeras (i.e., we mixed equal numbers of homozygous KO and wild-type congenic competitor bone marrow cells and transplanted them into lethally irradiated congenic recipient mice) to study the effects of U2af1 deletion on hematopoietic stem cell (HSC) function. As early as 10 days following Mx1-Cre-induction, we observed a complete loss of peripheral blood neutrophil and monocyte chimerism of the U2af1 KOcells, but not U2af1 heterozygous KO cells, and at 10 months there was a complete loss of homozygous U2af1 KObone marrow hematopoietic stem cells (SLAM, ST-HSCs, and LT-HSCs), neutrophils, and monocytes, as well as a severe reduction in B-cells and T-cells (p ≤ 0.001, n=3-4 for HSCs. p ≤ 0.001, n=9-10 for all other comparisons). The data indicate that normal hematopoiesis is dependent on wild-type U2af1expression, and that U2af1 heterozygous KO cells that retain one U2af1 allele are normal. Next, we tested whether mutant U2AF1(S34F) hematopoietic cells require expression of wild-type U2AF1 for survival. To test this, we used doxycycline-inducible U2AF1(S34F) or U2AF1(WT) transgenic mice. We generated ERT2-Cre/U2af1flox/flox/TgU2AF1-S34F/rtTA(S34F/KO), and ERT2-Cre/U2af1flox/flox/TgU2AF1-WT/rtTA,(WT/KO) mice, as well as all other single genotype control mice. We then created 1:1 mixed bone marrow chimeras with S34F/KO or WT/KO test bone marrow cells and wild-type competitor congenic bone marrow cells and transplanted them into lethally irradiated congenic recipient mice. Following stable engraftment, we induced U2AF1(S34F) (or WT) transgene expression with doxycycline followed by deletion of endogenous mouse U2af1 using tamoxifen. As early as 2 weeks post-deletion of U2af1, S34F/KO neutrophil chimerism dropped to 5.4% indicating loss of mutant cells, while WT/KO neutrophil chimerism remained elevated at 31.6% (p = 0.01, n=6-8). The data suggest that mutant U2AF1(S34F) hematopoietic cells are dependent on expression of wild-type U2af1 for survival. Since U2AF1mutant cells are vulnerable to loss of the residual wild-type U2AF1allele, and heterozygous U2af1KO cells are viable, selectively targeting the wild-type U2AF1allele in heterozygous mutant cells could be a novel therapeutic strategy. Disclosures No relevant conflicts of interest to declare.

Description: Myelodysplastic syndromes (MDS) are the most common myeloid malignancies among the elderly. Patients present with bone marrow (BM) failure manifested by low peripheral blood (PB) counts and are at increased risk of developing acute myeloid leukemia. Mutations of U2AF1, a gene that encodes a spliceosome protein, are identified in 11% of MDS patients. The two most common U2AF1 mutants, S34F and Q157P, alter the splicing of two distinct sets of pre-mRNA targets in vitro and each co-occur with unique gene mutations in MDS patients, suggesting these mutants may affect MDS pathogenesis differently. In mice, U2AF1S34F expression leads to altered splicing, reduced B-cell counts, and features of MDS. Similar studies have not been performed for U2AF1Q157P. To study the impact of U2AF1Q157P expression on splicing and hematopoiesis in vivo, we created a doxycycline (DOX)-inducible ("Tet-On") transgenic mouse that expresses mutant U2AF1Q157P and is isogenic to our previously reported U2AF1S34F and U2AF1WT transgenic mice. First, we confirmed DOX-inducible expression of the U2AF1Q157P transgene in BM by RT-PCR-seq. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P, we performed non-competitive BM transplants into lethally irradiated congenic recipient mice. Donor BM from U2AF1WT or U2AF1S34F mice was also transplanted for comparison. Six weeks after transplant, mice were maintained on DOX chow to induce U2AF1 transgene (U2AF1WT, U2AF1S34F, or U2AF1Q157P) expression (n = 10 mice per genotype). After six weeks on DOX, there were no significant changes in PB counts for U2AF1Q157P mice compared to U2AF1WT controls. In contrast, white blood cell (WBC) and B-cell counts were significantly reduced in U2AF1S34F mice, as reported previously. Assessment of the BM revealed increased numbers (per five leg bones) of hematopoietic stem and progenitor cells (LSK [Lin− Sca-1+ c-kit+] and LK [Lin− Sca-1− c-kit+]) in U2AF1S34F mice (1.33×105 LSK and 7.13×105 LK cells) compared to U2AF1WT (1.04×105 LSK and 5.69×105 LK cells; p 〈 0.05 for LSK and LK), as reported previously. In contrast, there was no change in LSK cells (1.03×105, p = 0.9668) and a non-significant increase in LK cells (6.84×105, p = 0.0547) in U2AF1Q157P mice compared to U2AF1WT. Both U2AF1S34F and U2AF1Q157P mice shared a significant increase in the number of common myeloid progenitors (CMP) compared to U2AF1WT (2.43×105 and 2.39×105 vs. 1.66×105 cells; p 〈 0.001 and p 〈 0.01, respectively), although CFU-C interrogated by methylcellulose assay were significantly increased only for U2AF1S34F mice. To study the hematopoietic cell-intrinsic effects of U2AF1Q157P on stem cell function, we mixed equal numbers of whole BM test cells (CD45.2+; U2AF1Q157P or U2AF1WT) with congenic control wild-type BM competitor cells (CD45.1+/CD45.2+) and transplanted them into lethally irradiated congenic recipient mice (CD45.1+/CD45.2+ ; n = 6 per genotype). As in non-competitive transplants, DOX chow was administered six weeks after transplant. After six weeks on DOX, we observed a relative multi-lineage competitive disadvantage by analysis of peripheral blood chimerism (%CD45.2+ WBC) for U2AF1Q157P test compared to U2AF1WT test cells (49.5% vs. 71.7%, respectively, p 〈 0.001). In addition, stem and progenitor cells were all significantly reduced in the BM of U2AF1Q157P competitive transplant mice compared to U2AF1WT after 18 weeks of DOX (LSK, 36.1% vs. 92.2%, respectively, p 〈 0.001; LK, 53.1% vs. 92.0%, p 〈 0.001). Lastly, using a Nanostring array, we identified consensus 3' splice sites of cassette exons that were increased or decreased in RNA from c-kit enriched mutant (U2AF1S34F or U2AF1Q157P) BM cells relative to U2AF1WT (FDR 〈 0.1). As expected, we observed altered consensus 3' splice sites at the −3 position (for U2AF1S34F) and +1 position (for U2AF1Q157P) of differentially spliced exons, indicating altered but different pre-mRNA splicing induced by either U2AF1 mutant. In aggregate, hematopoietic expression of U2AF1Q157P causes a multi-lineage competitive disadvantage of BM stem cells and expanded myeloid progenitors in the non-competitive transplant setting, like U2AF1S34F. However, PB counts and lineage distribution are not affected, indicating that the two common U2AF1 mutants, Q157P and S34F, are associated with different hematopoietic phenotypes and alterations to splicing, and may have different roles in MDS pathogenesis. Disclosures No relevant conflicts of interest to declare.

Description: Topotecan [(S)-9-dimethylaminomethyl-10-hydroxycamptothecin hydrochloride; SK&F 104864-A, NSC 609699], a water soluble semisynthetic analogue of the alkaloid camptothecin, is a potent topoisomerase I inhibitor. Here we show that topotecan stabilizes topoisomerase I/DNA cleavable complexes in radiation-resistant human B-lineage acute lymphoblastic leukemia (ALL) cells, causes rapid apoptotic cell death despite high-level expression of bcl-2 protein, and inhibits ALL cell in vitro clonogenic growth in a dose-dependent fashion. Furthermore, topotecan elicited potent antileukemic activity in three different severe combined immunodeficiency (SCID) mouse models of human poor prognosis ALL and markedly improved event-free survival of SCID mice challenged with otherwise fatal doses of human leukemia cells at systemic drug exposure levels that can be easily achieved in children with leukemia.

Description: Background: Previous studies indicate that mutations in signaling (e.g., receptor tyrosine kinases and RAS pathway members) and transcription factor genes are more common in secondary acute myeloid leukemia (sAML) than myelodysplastic syndrome (MDS), suggesting a role in disease progression. However, our understanding of the timing and order of mutation acquisition in these genes remains incomplete without analyzing paired MDS and sAML samples from the same patient. Defining the role of signaling gene mutations during progression should provide biologic insight into clonal evolution and help define prognostic markers for MDS progression. Methods: We banked paired MDS and sAML (and matched skin) samples from 44 patients (median time to progression: 306 days, range 21-3568). We sequenced 44 sAML (+ skin) samples for 285 recurrently mutated genes (RMGs) and 12 samples were selected for enhanced whole genome sequencing (eWGS, genome with deep exome coverage) of MDS and sAML samples (+ skin) to determine clonal hierarchy. Somatic mutations in these 12 samples were validated with high coverage error-corrected sequencing, and clonality was defined in MDS and sAML samples using mutation variant allele frequencies (VAFs). Additionally, error-corrected sequencing for all sAML RMG mutations, plus 40 additional genes, was performed on 43 of the MDS samples. Single cell DNA sequencing (scDNAseq, Mission Bio) was performed on 6 samples. Results: We identified 32 signaling gene mutations in 15 of the 44 sAML samples, with only 11 of 32 mutations (34%) detected in the initial, paired MDS sample (limit of detection;