Protein Expression Signatures Predict Response, Relapse, and Survival after Venetoclax Containing Therapy in Adults with Acute Myeloid Leukemia

Introduction When added to cytarabine (Ara-c) or hypomethylating agents (HMA), the BCL2 inhibitor, venetoclax (VTX), has been reported to improve response and overall survival (OS) rates. However, resistance and relapse still occur in the majority, and, although alterations in MCL1 and BCLXL are noted at relapse, identification of prognostic features remain unknown, notably not correlating with expression of the BCL2 target. Identification of prognostic markers could guide VTX use in patients and/or post-remission therapy. We searched for protein expression targets individually and collectively to predict VTX response and relapse in AML. Methods Reverse Phase Protein Array (RPPA) was performed on diagnostic leukemia samples of 818 adults with AML, of which 143 received VTX including 33 in combination with high dose Ara-C, 5 with standard dose Ara-C, 50 with HMA, and 13 with HMA and targeted therapy. Protein expression levels were evaluated using 390 validated antibodies were analyzed in the context of clinical data compiled by retrospective chart review. Pearson correlation was used to identify significant protein-protein correlations. Survival curves were generated by the Kaplan-Meier method and survival data was analyzed by multivariate cox regression model. Protein expression signatures were identified by hierarchical clustering and predictive models of classifiers were determined by classification and regression trees (CART) analysis. Results We queried the 390 proteins assayed in the 143 VTX treated patients to identify proteins individually prognostic (p<0.01) for OS (n=27) or remission duration (RD, n=44). Notably, neither MCL1, BCLXL nor BCL2 expression at diagnosis were prognostic of OS or RD. From these, unbiased hierarchical clustering revealed two cohorts (N=102 & 41 patients) for OS and RD. The clusters were similar for clinical features with no significant differences noted for, age, gender, performance status, cytogenetics, or the presence of molecular mutation markers FLT3.ITD, IDH1/2, NPM1 or TP53. The groups did not differ by therapy combination. Remission rates were insignificantly less in cluster 1 (61% vs 77%). Clear differences were observed for OS with estimated 3-yr overall survival 27% vs 66% (p=0.009, Figure 1) and relapse risk (RR) at 1-yr 45% vs 18% (p=0.001, Figure 2) in cluster 1 vs 2, respectively. In multivariate analysis, protein cluster membership was in independent prognostic factor for OS (along with TP53 and NPM1 mutations) but unfavorable cytogenetics was not. Prognostication did not vary based on cytogenetics or therapy received. For RD, protein cluster membership and unfavorable cytogenetics were the only independent predictors. Of the 44 proteins in the protein signature, CART modeling identified 3 - SPI1, NOTCH1.cle, and PTPN12 - that could predict clustering with a computed accuracy of 94.3%. Similarly, when these three proteins were used as training variables for random forest classification, the error rate was 3.7%. Several previously unrecognized potential therapeutic targets for preventing VTX resistance were also identified. Discussion Protein expression patterns, individually and in combination, were very highly predictive of outcome to VTX containing combination chemotherapy. A group with lower response rates, higher relapse rates, shorter RD and inferior OS was defined. A kit to prospectively determine cluster membership is in development. If validated this could be used to triage high-risk patients to alternate therapies, such as transplant, in CR1. Many new targets for combination therapy to prevent VTX resistance were identified and need to be tested in the laboratory for clinical relevance. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

Investigation of the complex biocatalytic and ultrafiltration action on the whey protein cluster in the production of biofilms

Для изготовления биоразлагаемых пленок и покрытий широко используются сырьевые материалы на основе белков растительного и животного происхождения. Белки молочной сыворотки, а также их гидролизаты могут успешно использоваться для создания пищевых пленок с функциональными свойствами. Существующие недостатки в отношении механических свойств упаковочных пленок и покрытий могут быть преодолены с помощью физических или биохимических методов воздействия на белковый кластер благодаря способности глобулярных белков разворачиваться и связываться с новыми полимерными структурами при определенных условиях. Согласно литературным источникам, это происходит благодаря способности тиоловых групп остатков цистеина образовывать как внутри, так и межмолекулярные дисульфидные мостики. В статье приведены результаты исследования зависимости прочностных свойств пленок на основе модифицированных сывороточных белков от профиля молекулярно-массового распределения пептидов. Приведены данные по способу получения гидролизата сывороточных белков с высоким содержанием низкомолекулярных пептидов размером менее 5 кДа. Получены зависимости глубины гидролиза сывороточных белков от продолжительности ферментативной конверсии и дальнейшего ультрафильтрационного концентрирования. Последовательное воздействие ферментативной обработки и ультрафильтрационного концентрирования на белковый кластер способствовало получению пермеата с содержанием белка 2 % и долей низкомолекулярных пептидов 95 %. Показано, что наличие в составе пленок низкомолекулярных пептидов способствует повышению разрушающего напряжения при разрыве и относительного удлинения на 86 и 50 % соответственно по сравнению с пленкой на основе негидролизованных сывороточных белков. For the manufacture of biodegradable films and coatings, raw materials based on plant and animal proteins are widely used. Whey proteins, as well as their hydrolysates, can be successfully used to create food films with functional properties. The existing disadvantages with respect to the mechanical properties of packaging films and coatings can be overcome using physical or biochemical methods of influencing the protein cluster due to the ability of globular proteins to unfold and bind to new polymer structures under certain conditions. According to the literature, this is due to the ability of thiol groups of cysteine residues to form both intra- and intermolecular disulfide bridges. The article presents the results of a study of the dependence of the strength properties of films based on modified whey proteins on the molecular weight distribution profile of peptides. The data on the method of obtaining a hydrolyzate of whey proteins with a high content of low molecular weight peptides less than 5 kDa in size are presented. The dependences of the depth of hydrolysis of whey proteins on the duration of enzymatic conversion and subsequent ultrafiltration concentration were obtained. The successive effect of enzymatic treatment and ultrafiltration concentration on the protein cluster resulted in the production of a permeate with a protein content of 2 % and a fraction of peptides with a low molecular weight of 95 %. It was shown that the presence of low molecular weight peptides in the films promotes an increase in the breaking stress at break and elongation by 86 and 50 %, respectively, in comparison with the film based on non-hydrolyzed whey proteins.

Preparation and use of the whey protein microparticulate in the sour cream production technology

One of the promising directions in the application of the valuable whey protein cluster is production of the whey protein microparticulate. The aim of the study was modification of the whey protein cluster to obtain the whey protein microparticulate and its use in the sour cream production technology. Cheese whey, whey protein microparticulate and sour cream were used as the objects of the research. The microparticulate production technology included purification of cheese whey from casein and fat, ultrafiltration, thermomechanical treatment. The composition of the objects of the research, their physico-chemical properties were determined according to the Russian standards. During investigations, the bifidogenic properties of the microparticulate, its high antioxidant activity, water- and fat-binding capacities as well as emulsifying capacity were confirmed. The data about an effect of the microparticulate on the physico-chemical and biochemical processes in sour cream production are presented. Addition of the microparticulate stimulated lactose fermentation, influenced sour cream consistency increasing product viscosity and reducing syneresis. The recommended dose of the microparticulate in the sour cream composition was 15%. Quality indicators of sour cream corresponded to the requirements of the normative documentation. Addition of the microparticulate imparted more pronounced aroma to the product. Higher content of lactic acid microorganisms was observed in the finished product compared to the control sample. The prebiotic properties of the microparticulate improved viability of starter cultures during storage. Formation of free fatty acids in the experimental sample during storage was more intensive compared to the control, while fat oxidation was less pronounced. The use of the whey protein microparticulate in sour cream production is characterized by many advantages: it allows achieving an increase in the effectiveness of milk processing, facilitates sustainability, production profitability, improves finished product quality.

Influence of the Milk-Vegetable Mixture Components on the Quality Characteristics of the Fermented Product

The article concerns the specifics of combining in the formulation animal and vegetable raw materials in order to create a food product with a balanced amino acid and fatty acid composition. It presents the results of studying the influence of plant and animal components on the quality indicators of the fermented product. During preliminary tests a man revealed the mixture composition as the basis for the fermented product: the milk component – skimmed milk; the vegetable component – soy food base; polysaccharide (arabinogalactan). The authors used standard methods of research and data processing for a comprehensive quality assessment of a protein cluster obtained during milk-vegetable mixture fermentation. According to the research results, they determined the organoleptic, physical and chemical, microbiological and biochemical characteristics of the product. A man concluded that the soy food base as one of the mixture components influenced the product taste and smell; arabinogalactan had a positive effect on the number of viable cells of lacto - and bifidobacteria and on the moisture-retaining ability of the resulting protein cluster. The quantitative and qualitative content of essential and non-essential amino acids in the product indicated its amino acid composition balance. The fatty acid composition is represented by saturated, monounsaturated and polyunsaturated fatty acids in an optimal ratio. The results analysis of a comprehensive quality assessment indicates the possibility of using a milk-vegetable mixture as a basis for the fermented products development.

Impact of two neighbouring ribosomal protein clusters on biogenesis factor binding and assembly of yeast late small ribosomal subunit precursors

Many of the small ribosomal subunit proteins are required for the stabilisation of late small ribosomal subunit (SSU) precursors and for final SSU rRNA processing in S. cerevisiae. Among them are ribosomal proteins (r-proteins) which form a protein cluster around rpS0 (uS2) at the “neck” of the SSU (S0-cluster) and others forming a nearby protein cluster around rpS3 (uS3) at the SSU “beak”. Here we applied semi-quantitative proteomics together with complementary biochemical approaches to study how incomplete assembly of these two r-protein clusters affects binding and release of SSU maturation factors and assembly of other r-proteins in late SSU precursors in S. cerevisiae. For each of the two clusters specific impairment of the local r-protein assembly state was observed. Besides, cluster-specific effects on the association of biogenesis factors were detected.These suggested a role of S0-cluster formation for the efficient release of the two nuclear export factors Rrp12 and Slx9 from SSU precursors and for the correct incorporation of the late acting biogenesis factor Rio2. Based on our and on previous results we propose the existence of at least two different r-protein assembly checkpoints during late SSU maturation in S. cerevisiae. We discuss in the light of recent SSU precursor structure models how r-protein assembly states might be sensed by biogenesis factors at the S0-cluster checkpoint.