The nutrient-dependent profiling and functional characterization of ribosomal RNA modifications in mammalian cells

Project goal: The main goal is to profile and define a nutrient-dependent rRNA modifications in mammalian cells
following the amino acid deprivation condition; identify snoRNAs mediating the nutrient-dependent rRNA modifications; and characterize the roles of the nutrient-dependent rRNA modifications in survival during amino acid starvation.

Project description: This project aims to investigate how nutrient availability influences ribosomal RNA (rRNA) modifications in mammalian cells. Using direct RNA nanopore sequencing, we will profile nutrient-dependent rRNA modifications in both primary and cancer cells under amino acid starvation. We will identify the specific small nucleolar RNAs (snoRNAs) responsible for guiding these modifications and explore their functional roles through loss-of-function studies. Our research will help elucidate how ribosome structure and function adapt to nutrient stress, contributing to cellular survival and potentially uncovering new regulatory mechanisms of translation.

Project facilitators: PI: Dos Sarbassov; Co-PI: Ulykbek Kairov. Bayansulu Ilyassova, Nargiz Rakhimgerey, Aruzhan Dildabek

Realisation period: 2023-2025

Expected results: We expect to identify nutrient-sensitive modifications in rRNA that vary between normal and cancer cells under amino acid starvation. Specific snoRNAs guiding these modifications will be characterized, and their functional roles in regulating translation under stress will be validated. The results may reveal novel mechanisms by which cells adapt their ribosomes to nutrient deprivation, providing insight into stress-responsive translation regulation.

Co-financing: We will use direct RNA sequencing (Oxford Nanopore) to detect rRNA modifications in cells under amino acid starvation. Comparative analyses will be performed between normal and cancer cells. Candidate snoRNAs involved in nutrient-sensitive modifications will be identified through bioinformatics and validated via knockdown experiments. Functional consequences will be assessed by examining changes in translation and cell survival under stress.

Contacts: dos.sarbassov@nu.edu.kz

Nanotechnology enhanced oxidative drug treatment of 3D culture of KRAS mutant colorectal cancer cell lines

Project goal: The main goal is to develop novel therapies for KRAS mutant CRC with nanoforms of oxidative drugs ATO and DVC that work effectively in solid tumor environment simulated by 3D cell culture. We will use enzyme-functionalized nanocarriers that can partially degrade the extracellular matrix and enhance drug penetration.

Project description: We aim to explore the difference in the mitochondrial activity in 3D CRC culture environments as well as cell viability and apoptotic response compared with individual CRC. One would expect to have a radial distribution of the oxidative drug throughout the cell clusteroids, hypoxic conditions reduced glucose intake in the core of the cell cluster which would decrease the therapy effectiveness in solid tumors. We also aim to develop a novel protease-functionalized nanocarrier for delivery of ATO/DVC which is expected to increase the effectiveness of the therapy in solid tumors. Although very efficient in delivery of high local concentrations of anticancer agents, nanocarriers have limited dept of penetration in solid tumor because they get stuck in the collagen network of the extracellular matrix (ECM). Our idea here is to functionalize the surface of ATO/DVC nanocarriers with a collagenase which would help it to digest its way through ECM in between the CRC and allow them to penetrate deeply in the core of the solid tumor. In our studies the solid tumors will be modelled with large cell clusteroids. We will explore the effects of this active nano-formulation of ATO/DVC on both individual CRC and cell clusteroids under identical conditions. This new anticancer nanotechnology is expected to increase the treatment efficacy and reduce the overall concentration of oxidative drug, which is acting indiscriminately and has high toxicity in free form. Similar nanoform can also be applied to other types of solid tumors and help to decrease the side effects in patients and improve outcomes.

Realisation period: 2023-2025

Expected results: 1) Our team expects to deliver better understanding of the differences in the ATO/DVC mechanism in 2D and 3D cell culture environment which would bring us closer to revealing the mechanism of resistance and toxic (side) effects in this anticancer therapy for CRC. The project will bring us closer in the understanding the mechanism of suicidal ROS generation upon application of oxidative drugs.
2) Development of novel therapies for CRC requires new drugs development (which are costly to get through clinical trials) or reformulation and novel methods of delivery of already approved drugs (as ATO/VC) as proposed in this project. We expect to produce novel class of smart nanocarriers of ATO/DVC which can penetrate CRC tissue (3D cell culture) and make the therapy more successful with lower side effects. This will be achieved by lowering the overall amount of ATO required for the nanoform therapy compared with free ATO administration. This may result in a revolutionary new anticancer therapy with potential application for CRC but also for other types of tumors.
3) Based on our results of study of the ATO/VC mechanisms of action in 2D and 3D cell culture as well as the development nanoform of delivery of these drugs the results will be published:
- at least 3 articles and/or reviews in peer-reviewed scientific journals, included in the Q1,Q2 and/or Q3 according to the IF in the Web of Science(WoS) and/or having a CiteScore percentile in the Scopus ≥50;
- either at least 2 articles and/or reviews in peer-reviewed journals included in the Q1,Q2 and/or Q3 by IF in the WoS and/or having a percentile in CiteScore in the Scopus ≥65;
- either at least 1 article or review in a peer-reviewed journals in the Q1 in the WoS or a CiteScore percentile in the Scopus ≥95. Open access policy endorsed by Nazarbayev University(NU) will be followed. The project has a planned budget for open access article processing fees. All manuscripts and published conference abstracts arising from this work will be deposited in the NU Digital Repository (NUR, nur.nu.edu.kz).
4) The project will also allow NU and NLA to further develop novel anticancer team capabilities, train a new generation of Kazakhstani researchers in this field and advance biomedical knowledge and the creation of modern nanotechnology-based therapies for colorectal cancer.
5) The project team will ensure that the results are disseminated by participation and presentations at one international and one national conference per year over the duration of the project. The team will also participate in organizing the 3rd and 4th Eurasian International Conferences on Antimicrobial and Biomedical Nanotechnologies, in 2023 and 2024 at NU, where our research assistants and the PI will be delivering research talks.

Methodology: We will employ 3D culture models of KRAS mutant CRC, . 3D culture of cell clusteroids will be fabricated by using a methodology recently developed by Prof Paunov group (see Task 2.2 below). 2D-cultured cells lose their polarity and have equal access to various compounds in the medium in disparity with in vivo circumstances as CRC in the colon do not grow as flat 2D cell structures in the human body. The key hypothesis here is that in 3D culture microenvironment can be mimicked in tumour cell clusteroids (tumoroids), where the nutrients and oxygen deficiency is observed in the cores of the clusters similarly to the 3D solid tumours found in colon. Cells in the tumours may have different responses to oxidative stress agents such as ATO and DVC, depending on their position within the tumour structure i.e. surface vs. tumour core. So far, most of studies in this area which focused on novel anticancer drugs development were typically performed using monolayer cultures (2D) of cancer cells and frequently leading to the failure of subsequent in vivo models because may not be representative of the real outcome. The proposed technique of generation of CRC clusteroids (tumoroids) is novel approach in the testing the efficiency of the oxidative drug. For this purpose, we will: (a) Produce 3D cell culture of HCT-116 and SW480 CRC cell lines. (b) 3D tumoroids and cells grown in 2D monolayers will be treated with ATO/DVC. After the treatment the 2D cell culture and the tumoroids will then be dispersed down to individual cells using trypsin and studied by flow cytometry to find the distribution of the live/dead cells in the treated clusters and assess mitochondrial membrane potential. This experimental set up would allow us to test bioactivity of ATO, DVC and combination of these oxidative compounds in core, hypoxic, regions of this 3D culture of cancer model. Results would provide answers to whether tumour cells in metabolically compromised microenvironments are able or not or to which extend to respond to this specific anticancer treatment combination.

The development of nanoparticle carriers filled with ATO would require collaboration with nanotechnology experts to design nano-carriers for mitochondria targeted controlled-release of ATO. The approach will be based on research from for anticancer therapy instead of antibiotic treatment. ATO will be conjugated or co-precipitated with biodegradable polymers like polylactic acid, PLGA or shellac, following coating with polyethylene glycol-derivatives for suppressing the immune response towards the nanocarriers and increasing blood circulation time. ATO/DVC-loaded P407-stabilized shellac NPs will be produced by co-precipitation with pH drop methods and further coated with Sainase or Alcalase (proteases). The formulated nanoparticles will be characterised by dynamic light scattering (DLS) and electron microscopy (TEM, SEM), EDS and the ATO encapsulation efficiency and release kinetics from the nanocarrier will be determined along with the enzymatic coating efficiency. irst exploratory experiments with novel nanoparticles will be tested in 2D and 3D models of CRC cell lines (HCT-116 and SW620). We will stain the cells with cell tracker dye and the nanoparticles will be also fluorescently tagged to allow us to colocalise them within the 3D cell cultures and examine their ability to penetrate in between the cells. We will optimise the nanocarrier ATO payload and the enzyme coating to ensure more uniform treatment of the CRC tissue with the oxidative drugs combination. The safety and efficacy in vitro will be investigated by assessing the ATO nano-formulation cytotoxicity, cell proliferation, migration, apoptosis, and other changes in the 3D culture cell behaviour. We will compare the effect of ATO/DVC in nanoparticle form at the same concentrations and treatment times without loading into nanoparticles and without protease coating. This will allow us to evaluate how much the protease coating improves the oxidative drug penetration within the cell clusteroids. This will be done by using CLSM as well as by dispersing the 3D cell culture by tripsin/EDTA treatment and using flow cytometry. Various markers will be evaluated to track cell viability (MTS assay, FDA assay), cell apoptosis (Annexin-V) as well as ROS production in mitochondria (MitoSox-Red) and overall (DCFDA assay).

Defining of preapoptotic nuclear condensation mediated by ROS in KRAS mutant cancer cells

Project goal: The main goal of this grant proposal is to define the mechanisms of a pre-apoptotic nuclear condensation by characterizing the induction of histone deacetylation and formation of heterochromatin.

Project description: The KRAS mutant cancers represent highly malignant oncologic disorders with a poor clinical outcome that are common in pancreatic, colorectal, and lung human cancers. There are no effective therapies have been developed to treat the KRAS mutant cancers, because it encodes a small GTPase that does not provide any distinctive “druggable” pocket for targeting. A specific and potent targeting of this highly malignant oncogenic pathway is one of the most challenging and demanding tasks in oncology. Hypothesis: Our main hypothesis is that glucose deprivation of KRAS mutant cancer cells causes a severe metabolic stress leading to generation of a mitochondrial ROS and priming the cancer cells for apoptotic event by inducing nuclear condensation and formation of heterochromatin. Scientific novelty: Study of the redox-dependent mechanism of preapoptotic nuclear condensation: the formation of heterochromatin on the periphery of the nucleus. As a model, we used KRAS mutant cancer cells that are sensitive to glucose deficiency. We determined that redox-dependent heterochromatin formation is mediated by functional activation of a chromatin repressive complex known as Nucleosome Remodeling Deacetylase (NuRD). Intense ROS generation caused by a lack of glucose in KRAS mutant cancer cells induces histone deacetylation via redox-dependent recruitment of the histone deacetylase complex (NuRD) to chromatin. As evidence, we demonstrate that oxidizing compounds mimic glucose-dependent histone deacetylation by inducing active recruitment of the histone deacetylase complex to chromatin. Targeting oncogenic KRAS oncology through the mechanism of redox-dependent nuclear condensation. Oncogenic KRAS is an "indestructible" target. Considering the sensitivity of KRAS mutant cancer cells to oxidative stress leading to pre-apoptotic nuclear condensation, we designed an oxidative combination that selectively acts on mutant cancer cells to induce cytotoxic oxidative stress. This discovery directs the redox-dependent activation of NuRD as a powerful tool for the process of pre-apoptotic chromatin/nucleus condensation and suppression of malignant KRAS mutant tumors.

Project facilitators: PI: Dinara Begimbetova. Dos Sarbassov, Bakhytgul Yermekbayeva, Assiya Kukanova, Zhansaya Kanketayeva

Realisation period: 2023-2025

Expected results: Types of oxidizing agents that mimic the action of reactive oxygen species and effectively cause the death of cancer cells due to nuclear condensation will be determined.
Nuclear condensation mediated by oxidants mimicking the action of reactive oxygen species will be determined.
The types of rigidity of the nuclei will be determined by AFM (atomic force microscopy).
Nuclear deacetylation will be determined by the action of oxidizing agents in imitation of glucose-dependent induction of pre-apoptotic nuclear condensation in cancer cells.
Areas of redox-dependent recruitment of the histone deacetylase complex (NuRD) to chromatin will be explored.
Histone deacetylation under the action of oxidizing agents that mimic the action of reactive oxygen species will be tested.
The role of inhibition of NURD activity in proapoptotic nuclear condensation will be determined.

Methodology: Microscopy (light, fluorescence atomic force microscopy, etc.), flow cytometry and spectrometry are analyzed to investigate the factors causing nuclear condensation and cancer cell death through the formation of reactive oxygen species (ROS) and to investigate histone deacetylation and activation of NuRD complex during pre-apoptotic nuclear condensation.

Co-financing: no

Contacts: dinara.begimbetova@nu.edu.kz

Employment of NKG2D as chimeric receptor of immune cells and chemokines CXCL9/10 for enhancement of therapeutic effect on colorectal cancer cells

Project goal: Objective 1: To evaluate the cytotoxic potential of immune cells expressing the chimeric NKG2D receptor against colorectal cancer (CRC) cells.

Objective 2: To perform a comparative analysis of the contribution of different chemokines to the enhancement of CAR-NKG2D-T cell infiltration efficiency using various colorectal cancer cell models.

Project description: This project focuses on the development of a novel CAR-T cell-based strategy for colorectal cancer (CRC) by combining NKG2D-directed cytotoxicity with enhanced tumor infiltration mediated by chemokines CXCL9 and CXCL10. The approach targets immunologically “cold” CRC subtypes that are poorly responsive to standard immunotherapies, aiming to improve immune cell recruitment and tumor eradication.

Project facilitators: PI: Nikolai Barlev, Co-PI: Dinara Begimbetova. Aleksei Petukhov, Shynggys Tursymbek, Alina Ershova, Aliya Kabasheva

Realisation period: 2024-2026

Expected results: The project is expected to generate CAR-NKG2D-T cells with enhanced cytotoxicity and tumor infiltration capacity. It will also identify the most effective chemokines for improving CAR-T cell migration into CRC models and clarify the influence of p53 status on ligand expression.

Methodology: The study employs genetic engineering of T cells using in vitro–transcribed mRNA, followed by co-culture with CRC models. Cytotoxicity will be assessed using RTCA assays, while gene expression of NKG2D ligands and chemokines will be quantified by qPCR and flow cytometry under genotoxic stress. Cell migration will be evaluated using soft agar–based assays.

Contacts: Nikolai Barlev, nikolai.barlev@nu.edu.kz

Defining the adaptive biological pathways mediating survival of starved KRAS mutant cancer cells and accommodating tumorigenesis

Project goal: Considering an altered cancer cell metabolism that is highly evident in KRAS mutant cancer cells, we propose to define their adaptive and survival pathways turned on in response to starvation conditions by performing the polysomal profiling and transcriptomic studies

Project description: This project aims to investigate the regulation of translation under metabolic stress in cancer cells, focusing on how serum deprivation and nutrient limitations affect polysome loading and selective mRNA translation. Using ribosome profiling, RNA-seq, and biochemical validation, we will identify translationally regulated genes and pathways involved in adaptive responses. The study may reveal new molecular targets that help cancer cells survive metabolic challenges

Project facilitators: PI: Dos Sarbassov; Co-PI: Ulykbek Kairov. Bayansulu Ilyassova, Nargiz Rakhimgerey, Aruzhan Dildabek

Realisation period: 2024-2026

Expected results: We expect to identify mRNAs that are selectively translated under serum and nutrient deprivation in cancer cells. The results will highlight key adaptive translational responses and reveal candidate genes and pathways that support survival under metabolic stress

Methodology: We will perform ribosome profiling and RNA sequencing to compare translational and transcriptional responses in cancer cells under normal and stress conditions (serum or nutrient deprivation). Bioinformatics analysis will be used to uncover enriched pathways and regulatory mechanisms

Contacts: dos.sarbassov@nu.edu.kz