Michael A. Morgan

Frequency and prognostic impact of mutations in SRSF2 , U2AF1 , and ZRSR2 in patients with myelodysplastic syndromes

Mutations in genes of the splicing machinery have been described recently in myelodysplastic syndromes (MDS). In the present study, we examined a cohort of 193 MDS patients for mutations in SRSF2, U2AF1 (synonym U2AF35), ZRSR2, and, as described previously, SF3B1, in the context of other molecular markers, including mutations in ASXL1, RUNX1, NRAS, TP53, IDH1, IDH2, NPM1, and DNMT3A. Mutations in SRSF2, U2AF1, ZRSR2, and SF3B1 were found in 24 (12.4%), 14 (7.3%), 6 (3.1%), and 28 (14.5%) patients, respectively, corresponding to a total of 67 of 193 MDS patients (34.7%). SRSF2 mutations were associated with RUNX1 (P < .001) and IDH1 (P = .013) mutations, whereas U2AF1 mutations were associated with ASXL1 (P = .005) and DNMT3A (P = .004) mutations. In univariate analysis, mutated SRSF2 predicted shorter overall survival and more frequent acute myeloid leukemia progression compared with wild-type SRSF2, whereas mutated U2AF1, ZRSR2, and SF3B1 had no impact on patient outcome. In multivariate analysis, SRSF2 remained an independent poor risk marker for overall survival (hazard ratio = 2.3; 95% confidence interval, 1.28-4.13; P = .017) and acute myeloid leukemia progression (hazard ratio = 2.83; 95% confidence interval, 1.31-6.12; P = .008). These results show a negative prognostic impact of SRSF2 mutations in MDS. SRSF2 mutations may become useful for clinical risk stratification and treatment decisions in the future.

Mutations affecting mRNA splicing define distinct clinical phenotypes and correlate with patient outcome in myelodysplastic syndromes

A cohort of MDS patients was examined for mutations affecting 4 splice genes (SF3B1, SRSF2, ZRSR2, and U2AF35) and evaluated in the context of clinical and molecular markers. Splice gene mutations were detected in 95 of 221 patients. These mutations were mutually exclusive and less likely to occur in patients with complex cytogenetics or TP53 mutations. SF3B1(mut) patients presented with lower hemoglobin levels, increased WBC and platelet counts, and were more likely to have DNMT3A mutations. SRSF2(mut) patients clustered in RAEB-1 and RAEB-2 subtypes and exhibited pronounced thrombocytopenias. ZRSR2(mut) patients clustered in International Prognostic Scoring System intermediate-1 and intermediate-2 risk groups, had higher percentages of bone marrow blasts, and more often displayed isolated neutropenias. SRSF2 and ZRSR2 mutations were more common in TET2(mut) patients. U2AF35(mut) patients had an increased prevalence of chromosome 20 deletions and ASXL1 mutations. Multivariate analysis revealed an inferior overall survival and a higher AML transformation rate for the genotype ZRSR2(mut)/TET2(wt) (overall survival: hazard ratio = 3.3; 95% CI, 1.4-7.7; P = .006; AML transformation: hazard ratio = 3.6; 95% CI, 2-4.2; P = .026). Our results demonstrate that splice gene mutations are among the most frequent molecular aberrations in myelodysplastic syndrome, define distinct clinical phenotypes, and show preferential associations with mutations targeting transcriptional regulation.

Gene expression profiling in MDS and AML: potential and future avenues.

Today, the classification systems for myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) already incorporate cytogenetic and molecular genetic aberrations in an attempt to better reflect disease biology. However, in many MDS/AML patients no genetic aberrations have been identified yet, and even within some cytogenetically well-defined subclasses there is considerable clinical heterogeneity. Recent advances in genomics technologies such as gene expression profiling (GEP) provide powerful tools to further characterize myeloid malignancies at the molecular level, with the goal to refine the MDS/AML classification system, incorporating as yet unknown molecular genetic and epigenetic pathomechanisms, which are likely reflected by aberrant gene expression patterns. In this study, we provide a comprehensive review on how GEP has contributed to a refined molecular taxonomy of MDS and AML with regard to diagnosis, prediction of clinical outcome, discovery of novel subclasses and identification of novel therapeutic targets and novel drugs. As many challenges remain ahead, we discuss the pitfalls of this technology and its potential including future integrative studies with other genomics technologies, which will continue to improve our understanding of malignant transformation in myeloid malignancies and thereby contribute to individualized risk-adapted treatment strategies for MDS and AML patients.

Prognostic Importance of Histone Methyltransferase MLL5 Expression in Acute Myeloid Leukemia

PURPOSE To assess the prognostic importance of mixed lineage leukemia 5 (MLL5) expression in acute myeloid leukemia (AML). PATIENTS AND METHODS MLL5 transcript levels from 509 patients with AML who were treated in multicenter trials AML SHG 0199 and AML SHG 0295 and 48 healthy volunteers were analyzed by real-time reverse-transcription polymerase chain reaction in the context of other molecular markers (NPM1, FLT3, CEBPA, IDH1/IDH2, NRAS, KIT, MN1, BAALC, ERG, and WT1). RESULTS Patients with high (n = 127) compared with low (n = 382) MLL5 expression had a higher complete response rate in multivariate analysis (odds ratio, 1.87; 95% CI, 1.08 to 3.24; P = .026). In multivariate analysis, high MLL5 expression was a favorable prognostic marker for overall survival (OS; hazard ratio [HR], 0.66; 95% CI, 0.49 to 0.89; P = .007) and relapse-free survival (RFS; HR, 0.72; 95% CI, 0.52 to 1.01; P = .057). Patient characteristics, cytogenetic aberrations, and gene mutations were similarly distributed between patients with high and low MLL5 expression except for a higher platelet count in those with high MLL5 expression. MLL5 expression independently predicted prognosis in cytogenetically normal AML patients (n = 268; OS: HR, 0.53; 95% CI, 0.33 to 086; P = .011; RFS: HR, 0.61; 95% CI, 0.38 to 0.99; P = .05) and in patients with core-binding factor leukemias (n = 81; OS: HR, 0.12; 95% CI, 0.02 to 0.91; P = .04; RFS: HR, 0.18; 95% CI, 0.04 to 0.77; P = .02). The prognostic importance of high MLL5 expression was independently validated in 167 patients treated in the AMLSG 07/04 trial (OS: HR, 0.5; 95% CI, 0.27 to 0.92; P = .023; RFS: HR, 0.49; 95% CI, 0.25 to 0.96; P = .033). CONCLUSION High MLL5 expression levels are associated with a favorable outcome and may improve risk and treatment stratification in AML.

A Tagging-via-substrate Approach to Detect the Farnesylated Proteome Using Two-dimensional Electrophoresis Coupled with Western Blotting

Prenylation is a post-translational modification critical for the proper function of multiple physiologically important proteins, including small G-proteins, such as Ras. Methods allowing rapid and selective detection of protein farnesylation and geranylgeranylation are fundamental for the understanding of prenylated protein function and for monitoring efficacy of drugs such as farnesyltransferase inhibitors (FTIs). Although the natural substrates for prenyltransferases are farnesyl pyrophosphate and geranylgeranyl pyrophosphate, farnesyltransferase has been shown to incorporate isoprenoid analogues into protein substrates. In this study, protein prenyltransferase targets were labeled using anilinogeraniol, the alcohol precursor to the unnatural farnesyl pyrophosphate analogue 8-anilinogeranyl diphosphate in a tagging-via-substrate approach. Antibodies specific for the anilinogeranyl moiety were used to detect the anilinogeranyl-modified proteins. Coupling this highly effective labeling/detection method with two-dimensional electrophoresis and subsequent Western blotting allowed simple, rapid analysis of the complex farnesylated proteome. For example, this method elucidated the differential effects induced by two chemically distinct FTIs, BMS-214,662 and L-778,123. Although both FTIs strongly inhibited farnesylation of many proteins such as Lamins, NAP1L1, N-Ras, and H-Ras, only the dual prenylation inhibitor L-778,123 blocked prenylation of Pex19, RhoB, K-Ras, Cdc42, and Rap1. This snapshot approach has significant advantages over traditional techniques, including radiolabeling, anti-farnesyl antibodies, or mass spectroscopy, and enables dynamic analysis of the farnesylated proteome.

Dinucleoside polyphosphates: strong endogenous agonists of the purinergic system

The purinergic system is composed of mononucleosides, mononucleoside polyphosphates and dinucleoside polyphosphates as agonists, as well as the respective purinergic receptors. Interest in the role of the purinergic system in cardiovascular physiology and pathophysiology is on the rise. This review focuses on the overall impact of dinucleoside polyphosphates in the purinergic system. Platelets, adrenal glands, endothelial cells, cardiomyocytes and tubular cells release dinucleoside polyphosphates. Plasma concentrations of dinucleoside polyphosphates are sufficient to cause direct vasoregulatory effects and to induce proliferative effects on vascular smooth muscle cells and mesangial cells. In addition, increased plasma concentrations of a dinucleoside polyphosphate were recently demonstrated in juvenile hypertensive patients. In conclusion, the current literature accentuates the strong physiological and pathophysiological impact of dinucleoside polyphosphates on the cardiovascular system.

Combining prenylation inhibitors causes synergistic cytotoxicity, apoptosis and disruption of RAS‐to‐MAP kinase signalling in multiple myeloma cells

The high incidence of activating RAS mutations, coupled with accumulating evidence linking RAS to multiple myeloma (MM) pathogenesis, indicate that novel therapies utilising inhibitors of RAS prenylation and signalling may be successful in the management of this disease. While preclinical studies investigating prenylation inhibitors, such as lovastatin, farnesyltransferase inhibitors (FTI) and geranylgeranyltransferase inhibitors (GGTI), have been promising, recent phase I/II clinical trials with FTI R115777 were disappointing, suggesting resistance to FTI monotherapy. To address this issue, the effects of FTI, GGTI and lovastatin alone and in combination were analysed in MM cell lines and primary cells. FTI treatment blocked H‐RAS processing, but was ineffective at inhibiting K‐ and N‐RAS prenylation because of alternative geranylgeranylation of these isoforms. However, combinations of FTI and GGTI or lovastatin were found to synergistically inhibit MM cell proliferation, migration, K‐ and N‐RAS processing, RAS‐to‐mitogen‐activated protein kinase signalling and to induce apoptosis. In contrast to FTI, lovastatin and some GGTI were found to cause intracellular accumulation of Rho proteins. Our results suggest that clinical efficacy of prenylation inhibitors in MM are limited by alternative prenylation of several small G‐proteins, such as RhoB, K‐ and N‐RAS. Furthermore, strategies combining FTI with GGTI or statins may provide greater efficacy in MM treatment.

Molecularly targeted therapies in myelodysplastic syndromes and acute myeloid leukemias

Although there has been significant progress in acute myeloid leukemia (AML) treatment in younger adults during the last decade, standard induction therapy still fails to induce remission in up to 40% of AML patients. Additionally, relapses are common in 50–70% of patients who achieve a complete remission, and only 20–30% of patients enjoy long-term disease-free survival. The natural history of myelodysplastic syndrome (MDS) is variable, with about half of the patients dying from cytopenic complications, and an additional 20–30% transforming to AML. The advanced age of the majority of MDS patients limits the therapeutic strategies often to supportive care. To address these shortcomings, much effort has been directed toward the development of novel treatment strategies that target the evolution and proliferation of malignant clones. Presented here is an overview of molecularly targeted therapies currently being tested in AML and MDS patients, with a focus on FMS-like tyrosine kinase 3 inhibitors, farnesyltransferase inhibitors, antiangiogenesis agents, DNA hypomethylation agents, and histone deacetylase inhibitors.

Synergistic cytotoxic effects in myeloid leukemia cells upon cotreatment with farnesyltransferase and geranylgeranyl transferase-I inhibitors

As deregulation of RAS signaling is important in the pathogenesis of myeloid leukemias, molecular targeting of RAS signaling may be a promising therapeutic strategy. Farnesyl transferase inhibitors (FTIs) are the most promising class of these new cancer therapeutics. Several FTIs have entered phase II clinical trials in acute myeloid leukemia (AML). Since geranylgeranylation of K-RAS and N-RAS in the presence of FTIs may represent an important mechanism of FTI resistance, 6 geranylgeranyl transferase-I inhibitors (GGTIs) were screened alone and in combination with FTI for growth inhibition of myeloid leukemia cells. Significant growth inhibition (>70%) in myeloid cell lines was observed for GGTI-286 (9/19), GGTI-298 (14/19), GGTI-2147 (16/19) and FTI L-744,832 (17/17). GGTI treatment of NB-4 cells resulted in an accumulation of cells in G0/G1, whereas FTI L-744,832 primarily caused an increase in G2/M. FTI and GGTIs both induced apoptosis. In all cases, FTI/GGTI cotreatment led to synergistic cytotoxic effects in both myeloid cell lines (5/5) and primary AML cells (6/6). This synergy coincided with increased apoptosis. FTI/GGTI cotreatment caused an accumulation of unprocessed N-RAS and inactive N-RAS–RAF complexes. Our results suggest that alternative geranylgeranylation of N-RAS may represent an important mechanism of resistance to FTI monotherapy in myeloid leukemia cells.

Therapeutic efficacy of prenylation inhibitors in the treatment of myeloid leukemia

Farnesyltransferase inhibitors (FTIs) represent a new class of anticancer agents that specifically target post-translational farnesylation of various proteins that mediate several cellular processes such as signal transduction, growth, differentiation, angiogenesis and apoptosis. These compounds were originally designed to block oncogenic RAS-induced tumor growth by impeding RAS localization to the membrane, but it is now evident that FTIs also affect processing of several other proteins. The need for novel therapies in myeloid leukemia is underscored by the high rate of treatment failure due to high incidences of relapse- and treatment-related toxicities. As RAS deregulation is important in the pathogenesis of myeloid leukemias, targeting of RAS signaling may provide a new therapeutic strategy. Several FTIs (eg BMS-214662, L-778,123, R-115777 and SCH66336) have entered phase I and phase II clinical trials in myeloid leukemias. This review discusses recent clinical results, potential combination therapies, mechanisms of resistance and the clinical challenges of toxicities associated with prenylation inhibitors.