Search

Inhibitors of anti-apoptotic BCL-2 family proteins in combination with chemotherapy and hypomethylating agents (HMA) are promising therapeutic approaches in acute myeloid leukemia (AML) and high-risk myelodysplastic syndromes (MDS). Alvocidib, a cyclin-dependent kinase 9 (CDK9) inhibitor and indirect transcriptional repressor of the anti-apoptotic factor MCL-1, has previously shown clinical activity in AML. Availability of biomarkers for response to the alvocidib + 5-azacytidine (5-AZA) could also extend the rationale of this treatment concept to high-risk MDS. In this study, we performed a comprehensive in vitro assessment of alvocidib and 5-AZA effects in N=45 high-risk MDS patients. Our data revealed additive cytotoxic effects of the combination treatment. Mutational profiling of MDS samples identified ASXL1 mutations as predictors of response. Further, increased response rates were associated with higher gene expression of the pro-apoptotic factor NOXA in ASXL1-mutated samples. The higher sensitivity of ASXL1 mutant cells to the combination treatment was confirmed in vivo in ASXL1Y588X transgenic mice. Overall, our study demonstrated augmented activity for the alvocidib + 5-AZA combination in higher-risk MDS and identified ASXL1 mutations as a biomarker of response for potential stratification studies.

Somatic mutations in genes coding for splicing factors, e.g., SF3B1, U2AF1, SRSF2, and others are found in approximately 50% of patients with myelodysplastic syndromes (MDS). These mutations have been predicted to frequently occur early in the mutational hierarchy of the disease, therefore, making them particularly attractive potential therapeutic targets. Recent studies in cell lines engineered to carry splicing factor mutations have revealed a strong association with elevated levels of DNA:RNA intermediates (R-loops) and a dependency on proper ATR function. However, data confirming this hypothesis in a representative cohort of primary MDS patient samples have so far been missing. Using CD34+ cells isolated from MDS patients with and without splicing factor mutations as well as healthy controls we show that splicing factor mutation- associated R-loops lead to elevated levels of replication stress and ATR pathway activation. Moreover, splicing factor mutated CD34+ cells are more susceptible to pharmacological inhibition of ATR resulting in elevated levels of DNA damage, cell cycle blockade, and cell death. This can be enhanced by combination treatment with the low-dose splicing modulatory compound Pladienolide B. We further confirm the direct association between R-loops and ATR sensitivity and the presence of a splicing factor mutation using lentiviral overexpression of wild-type and mutant SRSF2 P95H in cord blood CD34+ cells. Collectively, our results from n=53 MDS patients identify replication stress and associated ATR signaling to be critical pathophysiological mechanisms in primary MDS CD34+ cells carrying splicing factor mutations, and provide a preclinical rationale for targeting ATR signaling in these patients.

In myelodysplastic syndromes with a partial deletion of the long arm of chromosome 5, del(5q), lenalidomide is believed to reverse anergic T-cell immunity in the bone marrow resulting in suppression of the del(5q) clone. In this study we used next-generation sequencing of immunoglobulin heavy chain (IGH) and T-cell receptor beta (TRB) rearrangements in bone marrow-residing and peripheral blood-circulating lymphocytes of patients with del(5q) myelodysplastic syndromes to assess the immune architecture and track adaptive immune responses during treatment with lenalidomide. The baseline bone marrow B-cell space in patients was comparable to that of age-matched healthy controls in terms of gene usage and IGH clonality, but showed a higher percentage of hypermutated IGH sequences, indicating an expanded number of antigen-experienced B lineage cells. Bone marrow B lineage clonality decreased significantly and hypermutated IGH clones normalized upon lenalidomide treatment, well in line with the proliferative effect on healthy antigen-inexperienced B-cell precursors previously described for this drug. The T-cell space in bone marrow of patients with del(5q) myelodysplastic syndromes showed higher TRB clonality compared to that of healthy controls. Upon lenalidomide treatment, myelodysplastic syndrome-specific T-cell clusters with low to medium spontaneous generation probabilities emerged; these clusters were shared across patients, indicating a common antigen-driven T-cell response pattern. Hence, we observed B lineage diversification and generation of new, antigen-dependent T-cell clusters, compatible with a model of adaptive immunity induced against the del(5q) clone by lenalidomide. Overall, this supports the concept that lenalidomide not only alters the functional T-cell state, but also the composition of the T- and B-cell repertoires in del(5q) myelodysplastic syndromes.

Background Differences in survival have been reported between pediatric and adult acute lymphoblastic leukemia. The inferior prognosis in adult acute lymphoblastic leukemia is not fully understood but could be attributed, in part, to differences in genomic alterations found in adult as compared to in pediatric acute lymphoblastic leukemia.Design and Methods We compared two different sets of high-density single nucleotide polymorphism array genotyping data from 75 new diagnostic adult and 399 previously published diagnostic pediatric acute lymphoblastic leukemia samples. The patients’ samples were randomly acquired from among Caucasian and Asian populations and hybridized to either Affymetrix 50K or 250K single nucleotide polymorphism arrays. The array data were investigated with Copy Number Analysis for GeneChips (CNAG) software for allele-specific copy number analysis.Results The high density single nucleotide polymorphism array analysis of 75 samples of adult acute lymphoblastic leukemia led to the identification of numerous cryptic and submicroscopic genomic lesions with a mean of 7.6 genomic alterations per sample. The patterns and frequencies of lesions detected in the adult samples largely reproduced known genomic hallmarks detected in previous single nucleotide polymorphism-array studies of pediatric acute lymphoblastic leukemia, such as common deletions of 3p14.2 (FHIT), 5q33.3 (EBF), 6q, 9p21.3 (CDKN2A/B), 9p13.2 (PAX5), 13q14.2 (RB1) and 17q11.2 (NF1). Some differences between adult and pediatric acute lymphoblastic leukemia were identified when the pediatric data set was partitioned into hyperdiploid and non-hyperdiploid cases and then compared to the nearly exclusively non-hyperdiploid adult samples. In this analysis, adult samples had a higher rate of deletions of chromosome 17p (TP53) and duplication of 17q.Conclusions Our analysis of adult acute lymphoblastic leukemia cases led to the identification of new potential target lesions relevant for the pathogenesis of acute lymphoblastic leukemia. However, no unequivocal pattern of submicroscopic genomic alterations was found to separate adult acute lymphoblastic leukemia from pediatric acute lymphoblastic leukemia. Therefore, apart from different therapy regimen, differences of prognosis between adult and pediatric acute lymphoblastic leukemia are probably based on genetic subgroups according to cytogenetically detectable lesions but not focal genomic copy number microlesions.

Background T-cell prolymphocytic leukemia is a rare aggressive lymphoproliferative disease with a mature T-cell phenotype and characteristic genomic lesions such as inv(14)(q11q34), t(14;14)(q11;q32) or t(X;14)(q28;q11), mutation of the ATM gene on chromosome 11 and secondary alterations such as deletions of chromosome 8p and duplications of 8q.Design and Methods We analyzed malignant cells from 18 patients with T-cell prolymphocytic leukemia using high density 250K single nucleotide polymorphism arrays and molecular allelokaryotyping to refine understanding of known alterations and identify new target genes.Results Our analyses revealed that characteristic disruptions of chromosome 14 are frequently unbalanced. In the commonly deleted region on chromosome 11, we found recurrent microdeletions targeting the microRNA 34b/c and the transcription factors ETS1 and FLI1. On chromosome 8, we identified genes such as PLEKHA2, NBS1, NOV and MYST3 to be involved in breakpoints. New recurrent alterations were identified on chromosomes 5p, 12p, 13q, 17 and 22 with a common region of acquired uniparental disomy in four samples on chromosome 17q. Single nucleotide polymorphism array results were confirmed by direct sequencing and quantitative real-time polymerase chain reaction.Conclusions The first high density single nucleotide polymorphism array allelokaryotyping of T-cell prolymphocytic leukemia genomes added substantial new details about established alterations in this disease and moreover identified numerous new potential target genes in common breakpoints, deletions and regions of acquired uniparental disomy.

Hematide is a synthetic peptide-based, pegylated erythropoiesis stimulating agent in clinical development for treatment of anemia. To support chronic clinical dosing requirements, a 9-month repeat dose IV monkey safety study was undertaken. Animals received 0, 0.2, 2 or 20 mg/kg hematide IV every three weeks for nine months followed by a 14-week recovery. Hematide administration was associated with time and dose-dependent polycythemia. Histological findings were related to exaggerated pharmacology that was secondary to the administration of an erythropoiesis stimulating agent to a normocythemic animal. In conclusion, these results support the use of repeated administration of hematide for the correction of anemia.