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Splicing factor (SF) mutations are important contributors to the pathogenesis of hematological malignancies; however, their relevance in risk classification of acute myeloid leukemia (AML) warrants further investigation. To gain more insight into the characteristics of patients with AML carrying SF mutations, we studied their association with clinical features, cytogenetic and molecular abnormalities, and clinical outcome in a large cohort of 1447 patients with AML and high-risk myelodysplastic syndrome. SF mutations were identified in 22% of patients and were associated with multiple unfavorable clinical features, such as older age, antecedent myeloid disorders, and adverse risk factors (mutations in RUNX1 and ASXL1). Furthermore, they had significantly shorter event-free and overall survival. Notably, in European LeukemiaNet (ELN) 2017 favorable- and intermediate-risk groups, SF3B1 mutations were indicative of relatively poor prognosis. In addition, patients carrying concomitant SF mutations and RUNX1 mutations had a particularly adverse prognosis. In patients without any of the 4 most common SF mutations, RUNX1 mutations were associated with relatively good outcome, which was comparable to that of intermediate-risk patients. In this study, we propose that SF mutations be considered for incorporation into prognostic classification systems. First, SF3B1 mutations could be considered an intermediate prognostic factor when co-occurring with favorable risk features and as an adverse prognostic factor for patients currently categorized as having intermediate risk, according to the ELN 2017 classification. Second, the prognostic value of the current adverse factor RUNX1 mutations seems to be limited to its co-occurrence with SF mutations.

Background Computational tools analyzing RNA-sequencing data have boosted alternative splicing research by identifying and assessing differentially spliced genes. However, common alternative splicing analysis tools differ substantially in their statistical analyses and general performance. This report compares the computational performance (CPU utilization and RAM usage) of three event-level splicing tools; rMATS, MISO, and SUPPA2. Additionally, concordance between tool outputs was investigated. Results Log-linear relations were found between job times and dataset size in all splicing tools and all virtual machine (VM) configurations. MISO had the highest job times for all analyses, irrespective of VM size, while MISO analyses also exceeded maximum CPU utilization on all VM sizes. rMATS and SUPPA2 load averages were relatively low in both size and replicate comparisons, not nearing maximum CPU utilization in the VM simulating the lowest computational power (D2 VM). RAM usage in rMATS and SUPPA2 did not exceed 20% of maximum RAM in both size and replicate comparisons while MISO reached maximum RAM usage in D2 VM analyses for input size. Correlation coefficients of differential splicing analyses showed high correlation (β > 80%) between different tool outputs with the exception of comparisons of retained intron (RI) events between rMATS/MISO and rMATS/SUPPA2 (β Conclusions Prior to RNA-seq analyses, users should consider job time, amount of replicates and splice event type of interest to determine the optimal alternative splicing tool. In general, rMATS is superior to both MISO and SUPPA2 in computational performance. Analysis outputs show high concordance between tools, with the exception of RI events.

To answer the increasing need for detecting and validating protein biomarkers in clinical specimens, proteomic techniques are required that support the fast, reproducible and quantitative analysis of large clinical sample cohorts. Targeted mass spectrometry techniques, specifically SRM, PRM and the massively parallel SWATH/DIA technique have emerged as a powerful method for biomarker research. For optimal performance, they require prior knowledge about the fragment ion spectra of targeted peptides. In this report, we describe a mass spectrometric (MS) pipeline and spectral resource to support data-independent acquisition (DIA) and parallel reaction monitoring (PRM) based biomarker studies. To build the spectral resource we integrated common open-source MS computational tools to assemble an open source computational workflow based on Docker. It was then applied to generate a comprehensive DIA pan-human library (DPHL) from 1,096 data dependent acquisition (DDA) MS raw files, and it comprises 242,476 unique peptide sequences from 14,782 protein groups and 10,943 SwissProt-annotated proteins expressed in 16 types of cancer samples. In particular, tissue specimens from patients with prostate cancer, cervical cancer, colorectal cancer, hepatocellular carcinoma, gastric cancer, lung adenocarcinoma, squamous cell lung carcinoma, diseased thyroid, glioblastoma multiforme, sarcoma and diffuse large B-cell lymphoma (DLBCL), as well as plasma samples from a range of hematologic malignancies were collected from multiple clinics in China, the Netherlands and Singapore and included in the resource. This extensive spectral resource was then applied to a prostate cancer cohort of 17 patients, consisting of 8 patients with prostate cancer (PCa) and 9 with benign prostate hyperplasia (BPH), respectively. Data analysis of DIA data from these samples identified differential expressions of FASN, TPP1 and SPON2 in prostate tumors. Thereafter, PRM validation was applied to a larger PCa cohort of 57 patients and the differential expressions of FASN, TPP1 and SPON2 in prostate tumors were validated. As a second application, the DPHL spectral resource was applied to a patient cohort consisting of samples from 19 DLBCL patients and 18 healthy individuals. Differential expressions of CRP, CD44 and SAA1 between DLBCL cases and healthy controls were detected by DIA-MS and confirmed by PRM. These data demonstrate that the DPHL supported that DIA-PRM MS pipeline enables robust protein biomarker discoveries.

Expression of apoptosis-regulating proteins (B-cell CLL/lymphoma 2 - BCL-2, Myeloid Cell Leukemia 1 - MCL-1, BCL-2 like 1 - BCL-X and BCL-2-associated X protein - BAX) in acute myeloid leukemia (AML) blasts at diagnosis is associated with disease-free survival. We previously found that the initially high apoptosis-resistance of AML cells decreased after therapy, while regaining high levels at relapse. Herein, we further explored this aspect of dynamic apoptosis regulation in AML. First, we showed that the intraindividual ex vivo apoptosis-related profiles of normal lymphocytes and AML blasts within the bone marrow of AML patients were highly correlated. The expression values of apoptosis-regulating proteins were far beyond healthy control lymphocytes, which implicates the influence of microenvironmental factors. Second, we demonstrated that apoptosis-resistant primary AML blasts, as opposed to apoptosis-sensitive cells, were able to up-regulate BCL-2 expression in sensitive AML blasts in contact cultures (p = 0.0067 and p = 1.0, respectively). Using secretome proteomics, we identified novel proteins possibly engaged in apoptosis regulation. Intriguingly, this analysis revealed that major functional protein clusters engaged in global gene regulation, including mRNA splicing, protein translation, and chromatin remodeling, were more abundant (p = 4.01E-06) in secretomes of apoptosis-resistant AML. These findings were confirmed by subsequent extracellular vesicle proteomics. Finally, confocal-microscopy-based colocalization studies show that splicing factors-containing vesicles secreted by high AAI cells are taken up by low AAI cells. The current results constitute the first comprehensive analysis of proteins released by apoptosis-resistant and sensitive primary AML cells. Together, the data point to vesicle-mediated release of global gene regulatory protein clusters as a plausible novel mechanism of induction of apoptosis resistance. Deciphering the modes of communication between apoptosis-resistant blasts may in perspective lead to the discovery of prognostic tools and development of novel therapeutic interventions, aimed at limiting or overcoming therapy resistance.

Defects in apoptosis regulation are known to impact chemotherapy resistance and consequently refractoriness and relapse of acute myeloid leukemia (AML). We previously showed that apoptosis-resistant protein profile of AML blasts at diagnosis is associated with shorter disease-free survival. Specifically, by flow cytometry, we measured the expression of Bcl-2, Bcl-xL, Mcl-1 and Bax in leukemic cells and combined these parameters to define their anti-apoptosis index (AAI). Interestingly, the AAI of normal lymphocytes in the AML patients corresponded to the AAI of AML blasts obtained from the same patient, reaching values far outside the normal AAI range of lymphocytes. In addition, the AAI in both cell types displayed parallel changes during the course of therapy. This points to a role of microenvironment in regulation of apoptosis in bone marrow cells of AML patients. Therefore, the aim of the current study was to assess if apoptosis-resistant AML cells are able to regulate the AAI of apoptosis-sensitive cells by influencing the microenvironment, as well as to perform molecular dissection of microenvironment, to identify novel proteins that regulate apoptosis. First, we showed that apoptosis-resistant AML blasts (high AAI) release factors that modulate sensitive AML blasts (low AAI) to upregulate Bcl-2 and become apoptosis-resistant. In the majority of cases (10 out of 14), Bcl-2 expression was significantly increased in apoptosis-sensitive AML blasts upon contact culture with apoptosis-resistant AML blasts (1.7-fold; p=0.0067). To characterize the AML microenvironment, conditioned medium (18 hrs) from patient samples displaying either apoptosis-resistant (n=5) or apoptosis-sensitive profile (n=6) were collected. Using mass spectrometry-based proteomics, comparative analysis was performed on these secretomes. Strikingly, we found that the major functional protein clusters upregulated in secretomes of the apoptosis-resistant AML were involved in mRNA splicing, protein translation and chromatin remodeling/chromosome organization. We further compared protein profiles of the soluble secretome and the extracellular vesicle fraction of a high AAI patient to those of a low AAI patient. Proteomic analysis of these fractions of the conditioned medium showed that the functional protein networks found in the whole secretome are well-represented in extracellular vesicles that are enriched for exosome markers. Transfer of functional proteins between cells by extracellular vesicles is a well documented phenomenon. Therefore, it is conceivable that the regulatory protein networks detected in the vesicles excreted by AML blasts are involved in regulation of apoptosis-related proteins in recipient AML blasts and other cells residing in the bone marrow, thereby contributing to therapy resistance. Funded by STR and KiKa - Children cancer-free Citation Format: Anna Wojtuszkiewicz, Jacqueline Cloos, Floortje L. Kessler, Sander Piersma, Jako Knol, Gerrit Jansen, Yehuda G. Assaraf, Gertjan L. Kaspers, Sonja Zweegman, Gerrit J. Schuurhuis, Connie R. Jimenez. Transfer of regulatory protein networks via extracellular vesicles as a candidate mechanism of apoptosis-resistance in acute myeloid leukemia. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4764. doi:10.1158/1538-7445.AM2014-4764