A Study on Preclinical Efficacy, Underlying Mechanisms, and Sensitivity Markers of a Novel Hypomethylating Agent Ntx-301 in Acute Myeloid Leukemia

Introduction: While intensive induction chemotherapy has been standard-or-care for patients with acute myeloid leukemia (AML), intensive regimens have often been limited in elderly patients with comorbidities. As an alternative option, less intensive hypomethylating agents (HMAs), decitabine (DAC) and azacitidine (AZA), are currently being used in these unfit patients. However, their low response rates and adverse events when administered alone suggest a need to develop novel HMAs that elicit enhanced efficacy and reduced mortality. A recent study demonstrated pharmacological improvements of a novel 4′-thio-modified analog, 5-aza-4′-thio-2′-deoxycytidine (hereafter NTX-301), including enhanced chemical stability and incorporation into DNA and improved preclinical efficacy (Thottassery, 2014). However, the mechanism of action (MoA) of NTX-301 has not yet been understood. Herein, we aim to thoroughly investigate the preclinical efficacy and MoAs of NTX-301 in AML through comparative analysis with DAC and AZA. To this end, we used in vitro and in vivo preclinical models of AML and performed multiomics-based analyses. Results: We comprehensively examined viability of 200 cancer cell lines (CCLs) upon treatment with NTX-301. Consistent with the current use of HMAs as therapeutics for hematologic malignancies, this sensitivity profiling displayed the most remarkable potency of NTX-301 toward blood CCLs (OR=3.97, p=0.0003). In addition, phenotypic assays revealed that the anti-leukemic activity of NTX-301 was superior to that of DAC, which is attributed to more effective actions in inducing apoptosis, cell cycle arrest, and differentiation. Supporting the in vitro results, orally administrated NTX-301 led to more potent tumor regression, better tolerability, and survival benefits over DAC and AZA in both systemic (1.5-2.0 mg/kg, qdx5 then 2 days off, 5 days on, and 9 days off, for 3 cycles) and subcutaneous (0.2-1.5 mg/kg) xenograft models. To assess the underlying MoAs, we interrogated global alterations at the transcriptome and methylome levels upon treatment with NTX-301 in three AML cell lines using RNA sequencing and methylation array. Methylome analyses revealed that NTX-301-induced demethylation patterns were distinguished from DAC; DAC triggered stronger global demethylation than NTX-301 did, whereas NTX-301 derived rather selective demethylation, preferentially in early-replicating regions, H3K27ac-marked regions, and non-CpG islands. In transcriptome analyses compared with DAC, NTX-301 more markedly elicited a transcriptional reversal toward a normal myeloid-like signature by increasing a differentiation signature and suppressing a leukemic stem cell signature. NTX-301 also mediated more pronounced activation of DNA damage response and the p53 pathway, which are characterized by marked induction of pH2AX and pChk1 and increased stability of p53, respectively. Given the synthetic lethality of p53 activation and BCL2 inhibition (Rongqing, 2017), stronger p53 stabilization by NTX-301 may confer more benefits in combination with venetoclax. Indeed, the combination of NTX-301 + venetoclax produced a more synergistic combination index compared with DAC + venetoclax. Strikingly, the combined NTX-301 (0.5-2.0 mg/kg) + venetoclax (50 mg/kg) achieved complete tumor remission, no notable toxicity, and prolonged survival benefits over AZA (2.5 mg/kg) + venetoclax (50 mg/kg) in preclinical models of AML. By integrating sensitivity profiles and multiomics data of 200 CCLs, we interrogated molecular determinants associated with sensitivity to NTX-301. Intriguingly, when comparing methylomes between sensitive and resistant CCLs, we found a significantly biased global hypermethylation trend toward sensitive CCLs. A combinatorial set of the most significantly biased 352 differentially methylated regions (FDR<0.05) showed potential as a predictive sensitivity marker for NTX-301, exhibiting a significant correlation (r=0.69, p<0.0001) with sensitivity to NTX-301. Conclusions: Our study demonstrated an improved therapeutic index of NTX-301 over traditional HMAs, providing a rationale for further clinical development of the agent as a single-agent or in combination with other agents. We also believe that our study for MoAs and biomarkers will improve our understanding of NTX-301. Disclosures Lim: Pinotbio: Research Funding. Yoo: Pinotbio: Research Funding. Cho: Pinotbio: Research Funding. Choi: Pinotbio: Research Funding. Jung: Pinotbio: Research Funding. Jung: Pinotbio: Current Employment. Lee: Pinotbio: Current Employment. Chun: Pinotbio: Current Employment. Go: Pinotbio: Current Employment. Lee: Pinotbio: Current Employment. Choi: Pinotbio: Research Funding.

TAKING INTO ACCOUNT THE MULTIFACTORIAL CHARACTER OF VOICE CHARACTERISTICS IN THE PROBLEMS OF SPEAKER IDENTIFICATION

When testing the most advanced speaker identification systems on specialized databases, their minimum efficiency, estimated by the error probability at the point of intersection of the error curves, is only a few percent. However, many factors are known that affect the variability of the characteristics of the speaker's voice, each of which has its own, different from the others, influence on the results of the speaker's identification by the characteristics of the voice. The complexity of creating and testing speaker identification systems is the need to quantitatively formalize a number of specific factors that affect the characteristics of his voice. The article discusses the proposed method for accounting for a variety of factors affecting the parameters of the characteristics of the speaker's voice, which provides the fundamental possibility of indirectly accounting for their practically unlimited number. According to this method, «atomic» structures are distinguished from speech signals, which depend on the totality of the main factors that affect the speaker's identification process. With this method, all significant factors affecting the characteristics of the voice will be indirectly taken into account at the level of these structures. Subsequent decisions are made on the combinatorial set of a huge number of these «atomic» structures. «Atomic» speech structures are understood as the spectra of any fragments of any vowel sounds allocated in a time window of 20 ms. «Atomic» structures are selected automatically. The proposed method provides a rational consideration of the multifactorial influence of various parameters, since the spectra of these structures are influenced by all the main factors that characterize the individuality of the voice of a particular speaker. The decision on the identity of the voices of the announcers recorded on different phonograms is carried out on the basis of combinatorics of «atomic» spectra of vowel sounds in both phonograms. The method has shown high efficiency in the examination of phonograms of short duration.

A nonseparable invariant extension of Lebesgue measure – A generalized and abstract approach

In this paper, we use some methods of combinatorial set theory, in particular, the ones related to the construction of independent families of sets and also some modified version of the notion of small sets originally introduced by Riečan and Neubrunn, to give an abstract and generalized formulation of a remarkable theorem of Kakutani and Oxtoby related to a nonseparable invariant extension of the Lebesgue measure in spaces with transformation groups.

Using Periodicity Properties to Generate the Combinatorial Configurations

Identifying patterns of the ordering of a certain combinatorial set allows to develop of simple procedures for its generation for an arbitrary value and to strictly prove that this set contains all non-identical combinatorial configurations. A characteristic feature of combinatorial sets is their formation from the base set according to given rules. For this purpose, it is enough to enter the basic set from which elements their formation is carried out, type of these objects and the system of rules of their generation.

Cyclic Sieving for Plane Partitions and Symmetry

The cyclic sieving phenomenon of Reiner, Stanton, and White says that we can often count the fixed points of elements of a cyclic group acting on a combinatorial set by plugging roots of unity into a polynomial related to this set. One of the most impressive instances of the cyclic sieving phenomenon is a theorem of Rhoades asserting that the set of plane partitions in a rectangular box under the action of promotion exhibits cyclic sieving. In Rhoades's result the sieving polynomial is the size generating function for these plane partitions, which has a well-known product formula due to MacMahon. We extend Rhoades's result by also considering symmetries of plane partitions: specifically, complementation and transposition. The relevant polynomial here is the size generating function for symmetric plane partitions, whose product formula was conjectured by MacMahon and proved by Andrews and Macdonald. Finally, we explain how these symmetry results also apply to the rowmotion operator on plane partitions, which is closely related to promotion.

A fine-tuned interplay of three A. thaliana UDP glucosyltransferases orchestrates salicylic acid homeostasis

Salicylic acid (SA) is a central signaling molecule in development and defense, therefore its levels are tightly controlled. One control mechanism is conjugation with sugar moieties by UDP glucosyltransferases (UGTs). In Arabidopsis, UGT76B1, UGT74F1, and UGT74F2 are known to glucosylate SA. We show that these are the main SA UGTs in leaves, since only marginal levels of SA glucosides were found in a triple loss-of-function mutant. Analyzing transcriptomes, metabolite levels, and phenotypes of a full combinatorial set of loss-of-function mutants, we resolved the mutual relationships and the individual roles of these enzymes in SA homeostasis. The strongest gene expression changes were observed for the ugt76b1 ugt74f1 double mutant, which downregulated developmental genes and most pronouncedly upregulated cell death-related genes. Among the single mutants, ugt76b1 specifically exhibited increased production of reactive oxygen species, increased resistance to infection, and early senescence. Likewise, higher-order mutations confirmed the dominant role of UGT76B1 in controlling SA levels and thereby the expression of biotic stress response genes. Both UGT74F1 and UGT74F2 affected UGT76B1 expression. However, while UGT76B1 and UGT74F1 produced SA-2-O-β-glucoside, UGT74F2 did not contribute there substantially. Instead, UGT74F2 acted independently of UGT74F1, decreasing steady-state SA levels by producing salicyloyl glucose ester. Remarkably, this did not restrict defense responses. In contrast, UGT74F1 interacted with UGT76B1 in suppressing defense responses. Nevertheless, a benzothiadiazole-triggered defense scenario induced only UGT76B1, whereas UGT74F1 was linked to controlling abiotic stress responses. All three enzymes form a network that, in concert with other UGTs, regulates expression of developmental and stress-related genes.One sentence summaryThe salicylic acid glucosylating enzymes of Arabidopsis leaves are crucial for salicylic acid homeostasis and combinatorially impact defense responses and developmental processes.

A Harmonized Atlas of Spinal Cord Cell Types and Their Computational Classification

ABSTRACTSingle cell sequencing is transforming many fields of science but the vast amount of data it creates has the potential to both illuminate and obscure underlying biology. To harness the exciting potential of single cell data for the study of the mouse spinal cord, we have created a harmonized atlas of spinal cord transcriptomic cell types that unifies six independent and disparate studies into one common analysis. With the power of this large and diverse dataset, we reveal spinal cord cell type organization, validate a combinatorial set of markers for in-tissue spatial gene expression analysis, and optimize the computational classification of spinal cord cell types based on transcriptomic data. This work provides a comprehensive resource with unprecedented resolution of spinal cord cell types and charts a path forward for how to utilize transcriptomic data to expand our knowledge of spinal cord biology.

Combinatorial Set of Lexico-Grammatical Classes of Nouns in the Russian Language

The article focuses on the changes of nouns, the lexico-grammatical features of which make it impossible to refer them to a specific lexico-grammatical class. The article is based on the assumption, that traditional methodological foundations, generally accepted in native language studies – the grammatical classification, according to which nouns belong to one of the four classes: concrete, abstract, collective or material nouns, – is rather conventional and doesn't cover many transitional phenomena observed in substantive lexis. The expansion of nominal semantic structure is often accompanied by grammatical shifts. Semantic structures of Russian substantives are described as apt to undergo six types of changes, which reflect combinatorics of lexical and grammatical categories of polysemantic nouns: concreteness – abstractness, concreteness – collectiveness, abstractness – concreteness, abstractness – collectiveness, collectiveness – abstractness, collectiveness – concreteness. The considered polysemants demonstrate different lexico-grammatical features depending on the meaning in which they are used. The applied quantitative analysis has enabled the authors to conclude that the prevalent changes occur in nominal structures of abstraction – concreteness type, while the changes of collectivity – abstraction type are less prominent in the Russian language. The article justifies the use of the term "lexical-grammatical class" in relation to a lexical-semantic variant of the word, which refers to nouns by morphological characteristics.

Method for Developing Combinatorial Generation Algorithms Based on AND/OR Trees and Its Application

In this paper, we study the problem of developing new combinatorial generation algorithms. The main purpose of our research is to derive and improve general methods for developing combinatorial generation algorithms. We present basic general methods for solving this task and consider one of these methods, which is based on AND/OR trees. This method is extended by using the mathematical apparatus of the theory of generating functions since it is one of the basic approaches in combinatorics (we propose to use the method of compositae for obtaining explicit expression of the coefficients of generating functions). As a result, we also apply this method and develop new ranking and unranking algorithms for the following combinatorial sets: permutations, permutations with ascents, combinations, Dyck paths with return steps, labeled Dyck paths with ascents on return steps. For each of them, we construct an AND/OR tree structure, find a bijection between the elements of the combinatorial set and the set of variants of the AND/OR tree, and develop algorithms for ranking and unranking the variants of the AND/OR tree.