Functional characterization of HNF4A gene variants identify promoter and cell line specific transactivation effects.

Hepatocyte nuclear factor-4 alpha (HNF-4A) regulates genes with roles in glucose metabolism and ß-cell development. Although pathogenic HNF4A variants are commonly associated with maturity-onset diabetes of the young (MODY1; HNF4A-MODY), rare phenotypes also include hyperinsulinemic hypoglycemia, renal Fanconi syndrome and liver disease. While the association of rare functionally damaging HNF1A variants with HNF1A-MODY and type 2 diabetes is well established owing to robust functional assays, the impact of HNF4A variants on HNF-4A transactivation in tissues including the liver and kidney is less known, due to lack of similar assays. Our aim was to investigate the functional effects of seven HNF4A variants, located in the HNF-4A DNA binding domain and associated with different clinical phenotypes, by various functional assays and cell lines (transactivation, DNA binding, protein expression, nuclear localization) and in silico protein structure analyses. Variants R85W, S87N and R89W demonstrated reduced DNA binding to the consensus HNF-4A binding elements in the HNF1A promoter (35, 13 and 9%, respectively) and the G6PC promoter (R85W ~10%). While reduced transactivation on the G6PC promoter in HepG2 cells was shown for S87N (33%), R89W (65%) and R136W (35%), increased transactivation by R85W and R85Q was confirmed using several combinations of target promoters and cell lines. R89W showed reduced nuclear levels. In silico analyses supported variant induced structural impact. Our study indicates that cell line specific functional investigations are important to better understand HNF4A-MODY genotype-phenotype correlations, as our data supports ACMG/AMP interpretations of loss-of-function variants and propose assay-specific HNF4A control variants for future functional investigations.

Acute response in circulating microRNAs following a single bout of short-sprint and heavy strength training in well-trained cyclists.

Background: Heavy strength (HS) and short-sprint (SS) are commonly used training methods for competitive road cyclists, with the aim to improve the anaerobic power and short time cycling performance. Knowledge of how such training methods affects biochemical as well as molecular factors, are particularly important for determining individual recovery and long-term adaptations. The primary aim of the current study was to investigate the expression levels of small non-coding RNAs in response to HS and SS training in elite cyclists as potential biomarkers for individual optimal restitution time. Methods: Eleven well trained cyclists performed one session of HS training and one session of SS training on separate days. Blood samples were taken at baseline and 5 min, 1 h and 21 h post training. Along with physiological measurements and biochemical factors (serum creatine kinase, myoglobin, human growth hormone and plasma lactate), real-time quantitative PCR was used to explore whether HS and/or SS training influenced the abundance of 24 circulating miRNAs, in serum, associated with muscle development, angiogenesis, and/or inflammation. Results: Based on complete miRNA profiles from nine cyclists, the miRNAs showing most altered expression after both training sessions included the three striated muscle-specific miRNAs (myomiRs) miR-1-3p, 133a-3p and 133b-3p. While all three miRNAs showed significantly highest expression at 1 h post HS session, the acute effect of the SS session included a significantly higher level of miR-1-3p alone, at 5 min (highest), as well as at 1 h and 21 h post session. Correlation (negative) with biochemical markers was only shown for miR-133a-3p and CK (r = -0.786, p = 0.041) and between miR-133b-3p and [La-] (r = -0.711, p = .032), at 21 h post SS session. Conclusion: Our findings support that unique myomiRs are regulated by HS and SS training. Such knowledge may be important for individually adjusted restitution times.

Modulation of phospho-proteins by interferon-alpha and valproic acid in acute myeloid leukemia.

PURPOSE: Valproic acid (VPA) is suggested to be therapeutically beneficial in combination with interferon-alpha (IFNα) in various cancers. Therefore, we examined IFNα and VPA alone and in combinations in selected AML models, examining immune regulators and intracellular signaling mechanisms involved in phospho-proteomics. METHODS: The anti-leukemic effects of IFNα and VPA were examined in vitro and in vivo. We mapped the in vitro phosphoprotein modulation by IFNα-2b and human IFNα-Le in MOLM-13 cells by IMAC/2D DIGE/MS analysis and phospho-flow cytometry, and in primary healthy and AML patient-derived PBMCs by CyTOF. In vivo, IFNα-Le and VPA efficacy were investigated in the immunodeficient NOD/Scid IL2γ-/- MOLM-13Luc+ mouse model and the syngeneic immunocompetent BNML rat model. RESULTS: IFNα-2b and IFNα-Le differed in the modulation of phospho-proteins involved in protein folding, cell stress, cell death and p-STAT6 Y641, whereas VPA and IFNα-Le shared signaling pathways involving phosphorylation of Akt (T308), ERK1/2 (T202/T204), p38 (T180/Y182), and p53 (S15). Both IFNα compounds induced apoptosis synergistically with VPA in vitro. However, in vivo, VPA monotherapy increased survival, but no benefit was observed by IFNα-Le treatment. CyTOF analysis of primary human PBMCs indicated that lack of immune-cell activation could be a reason for the absence of response to IFNα in the animal models investigated. CONCLUSIONS: IFNα-2b and IFNα-Le showed potent and synergistic anti-leukemic effects with VPA in vitro but not in leukemic mouse and rat models in vivo. The absence of IFNα immune activation in lymphocyte subsets may potentially explain the limited in vivo anti-leukemic effect of IFNα-monotherapy in AML.

Clinical proteomics of myeloid leukemia.

Myeloid leukemias are a heterogeneous group of diseases originating from bone marrow myeloid progenitor cells. Patients with myeloid leukemias can achieve long-term survival through targeted therapy, cure after intensive chemotherapy or short-term survival because of highly chemoresistant disease. Therefore, despite the development of advanced molecular diagnostics, there is an unmet need for efficient therapy that reflects the advanced diagnostics. Although the molecular design of therapeutic agents is aimed at interacting with specific proteins identified through molecular diagnostics, the majority of therapeutic agents act on multiple protein targets. Ongoing studies on the leukemic cell proteome will probably identify a large number of new biomarkers, and the prediction of response to therapy through these markers is an interesting avenue for future personalized medicine. Mass spectrometric protein detection is a fundamental technique in clinical proteomics, and selected tools are presented, including stable isotope labeling with amino acids in cell culture (SILAC), isobaric tags for relative and absolute quantification (iTRAQ) and multiple reaction monitoring (MRM), as well as single cell determination. We suggest that protein analysis will play not only a supplementary, but also a prominent role in future molecular diagnostics, and we outline how accurate knowledge of the molecular therapeutic targets can be used to monitor therapy response.

Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells.

BACKGROUND: Acute myeloid leukemia (AML) cells are characterized by non-mutated TP53, high levels of Hdm2, and frequent mutation of the Flt3 receptor tyrosine kinase. The juxtamembrane mutation of FLT3 is the strongest independent marker for disease relapse and is associated with elevated Bcl-2 protein and p53 hyper-phosphorylation in AML. DNA damage forms the basic mechanism of cancer cell eradication in current therapy of AML. Hdm2 and pro-apoptotic Bcl-2 members are among the most intensely induced genes immediately after chemotherapy and Hdm2 is proposed a role in receptor tyrosine kinase regulation. Thus we examined the DNA damage related modulation of these proteins in relation to FLT3 mutational status and induction of apoptosis. RESULTS: Within one hour after exposure to ionizing radiation (IR), the AML cells (NB4, MV4-11, HL-60, primary AML cells) showed an increase in Flt3 protein independent of mRNA levels, while the Hdm2 protein decreased. The FLT3 mutant MV4-11 cells were resistant to IR accompanied by presence of both Mcl-1 and Hdm2 protein three hours after IR. In contrast, the FLT3 wild type NB4 cells responded to IR with apoptosis and pre-apoptotic Mcl-1 down regulation. Daunorubicin (DNR) induced continuing down regulation of Hdm2 and Mcl-1 in both cell lines followed by apoptosis. CONCLUSION: Both IR and DNR treatment resulted in concerted protein modulations of Mcl-1, Hdm2 and Flt3. Cell death induction was associated with persistent attenuation of Mcl-1 and Hdm2. These observations suggest that defining the pathway(s) modulating Flt3, Hdm2 and Mcl-1 may propose new strategies to optimize therapy for the relapse prone FLT3 mutated AML patients.

Adaptive contrast enhancement of two-dimensional electrophoretic protein gel images facilitates visualization, orientation and alignment.

2-DE is a powerful technique to discriminate post-translationally modified protein isoforms. However, all steps of 2-DE preparation and gel-staining may introduce unwanted artefacts, including inconsistent variation of background intensity over the entire 2-DE gel image. Background intensity variations limit the accuracy of gel orientation, overlay alignment and spot detection methods. We present a compact and efficient denoising algorithm that adaptively enhances the image contrast and then, through thresholding and median filtering, removes the gray-scale range covering the background. Applicability of the algorithm is demonstrated on immunoblots, isotope-labeled gels, and protein-stained gels. Validation is performed in contexts of (i) automatic gel orientation based on Hough transformation, (ii) overlay alignment based on cross correlation and (iii) spot detection. In gel stains with low background variability, e.g. Sypro Ruby, denoising will lower the spot detection sensitivity. In gel regions with high background levels denoising enhances spot detection. We propose that the denoising algorithm prepares images with high background for further automatic analysis, without requiring manual input on a gel-to-gel basis.

Proteomic strategies for individualizing therapy of acute myeloid leukemia (AML).

Acute myeloid leukemia (AML) is an aggressive hematological malignancy characterized by accumulating myeloid precursor cells in the bone marrow, with approximately 2-3 months 50% survival if left untreated. With current treatment modalities the five years overall survival hardly exceeds 50%. Cytogenetics and molecular diagnostics guide the clinician to select individualized therapy in certain subsets of AML, achieving long-term survival above 70% of these cases. However, approximately half of the AML patients have no risk stratifying features, and early reports indicate that proteomic approaches may be utilized for disease classification as well as development of novel biomarkers related to prognosis, diagnosis, and choice of therapeutic regimen. Proteomics, here defined as the analysis of all proteins in a cell, in a cell compartment or in a signaling pathway, has probably its greatest potential in investigating pathways that are easily targeted by small molecules or therapeutic antibodies. The major methodological challenges include detection sensitivity in a limited clinical material, a problem that in some cases can be solved through designated multiplexed protein assays based on single cells or cell extracts. In this review we will discuss pharmacoproteomic studies of drugs regulating leukemia specific targets like all-trans retinoic acid, histone deacetylase inhibitors, proteasome inhibitors and tyrosine kinase inhibitors, as well as studies on drug resistance and graft-versus-host studies during stem cell transplantations. These studies indicate new avenues in AML diagnostics, individualized therapy design and therapy response surveillance for the clinician.

Proteomics in acute myelogenous leukaemia (AML): methodological strategies and identification of protein targets for novel antileukaemic therapy.

Enduring efforts into determination of the molecular biological status of acute myelogenous leukaemia (AML), a stem cell disease characterised by distinct blastic differentiation blocks and their extensive growth, continue to provide us with prognostically important information for more than half of all patients. In subsets of AML, molecular diagnostics rigorously guide the clinician toward the choice of optimal therapy. The in-depth characterization of leukemogenesis associated genetic alterations, such as the combined presence of activating mutations of tyrosine kinases together with altered transcription factors, and the documented impact of these mutations upon prognosis of AML, suggests AML as a primary candidate for pioneering proof-of-principle studies with new high throughput protein analysis techniques. This review aims to introduce the reader to proteomic methodology, e.g. two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, SELDI and protein arrays. Examples of its use, including single cell phosphoprotein profiling in risk stratification, the probing of cellular effects of conventional chemotherapeutics and novel target determination are presented. Based on original proteomic analysis of AML, molecular characteristics of AML, in addition to knowledge of conventional therapeutics and novel drugs, we attempt to forecast the influence of proteomics in therapy development for AML.