Exostosin glycosyltransferase 1 (EXT1) is a glycosyltransferase involved in the elongation of the saccharide chain of heparan sulfate proteoglycans (HSPGs). HSPGs, also known as Heparanosome, are important components of the extracellular matrix (ECM) and the cell surface. The ECM profile has been extensively studied in high-grade serous ovarian cancer (HGSOC), but data on the involvement of HPSGs in platinum resistance are still incomplete.

HGSOC patients from the TCGA dataset (73 resistant and 107 sensitive) were stratified in terms of their response to platinum-based therapy. The copy number alterations (CNAs) that occurred in the TCGA patients, leading to altered mRNA expression, were analyzed using the PathScore algorithm. The biosynthesis of HSPGs was significantly (p <0.05) enriched in the resistant patients with a high percentage of amplifications of the Ext1 gene. EXT1 protein expression was analyzed by immunohistochemistry on a microarray of 25 resistant and 25 sensitive patients. EXT1 protein levels were elevated in 60% of resistant patients compared to 24% of sensitive patients (p < 0.05). Ovarian cancer cells were transfected with an Ext1-expressing vector (COV318 and MDAH2774) and/or interfered by shRNAs targeting the Ext1 gene (shEXT1 OVCAR-5 and OVCAR-429) to investigate the deposition and distribution of HPSGs and to determine how they affect the signal transduction, cell motility and invasion, and drug sensitivity of cancer cells.

EXT1-overexpressing cells synthesized an increased amount of HSPGs and showed reduced sensitivity to platinum-based drugs, which could be restored by heparinase treatment. In contrast, cells which experienced EXT1 knockdown showed increased sensitivity to platinum-based drugs. Through the transcriptomics analysis of EXT1 knockdown cells, we detected significantly reduced expressions of the Lcn2, Cacna2d1, Edn1, and Tagln genes. In addition, the GO analysis of shExt1 knockdown cells revealed a lower enrichment of the EMT category, as reflected by reduced cell motility in vitro, which was analyzed by time lapse microscopy. These effects could be partly explained by the lower accumulation of INFg and TNFa in cells lacking EXT1. Our preliminary results suggest that Heparanosome architecture can alter the behavior of cancer cells and their response to external stimuli. These phenomena may play an important role in the development of recurrence in patients resistant to platinum-based drugs.

According to the latest global statistics for 2020, breast cancer (BC) has taken first place in the incidence of epithelial tumors, ahead of lung cancer, and is the main cause of mortality from cancer pathology among women around the world. Recently, the critical role of non-coding RNAs (ncRNAs) in the regulation of genes and signaling pathways in cancer pathogenesis, particularly apoptosis, has been identified.

The aim of our work was to identify new aberrantly expressed long non-coding RNAs and mRNAs in BC, as well as to study their possible interactions in BC.

Materials and Methods. Total RNA was isolated from paired breast cancer samples according to the standard method. Reverse transcription was performed using the MMLV RT kit # SK021 (Evrogen, Moscow, Russia), and qPCR was performed on a Bio-Rad CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using the qPCRmix kit -HS SYBR (Evrogen, Moscow, Russia) according to the manufacturer's protocol. Data were analyzed using relative quantification based on the ΔΔCt method. Changes in the expression levels of lncRNAs and mRNAs were considered less than 2 times (|ΔΔCt|≤2) as ‘no changes’. qPCR was performed in three technical replicas.

Results. Changes in the expression level of the lncRNA ADAMTS9-AS1, OIP5-AS1, and mRNA of genes associated with apoptosis, such as APAF1, BAX, BAK1, BIM, and BCL2, were studied in breast cancer. We found decreased expression levels of ADAMTS9-AS1, OIP5-AS1, and APAF1 and BAX in 60 breast tumor samples compared with paired norms (p<0.05). The level of BCL2 expression is significantly (p<0.001) higher in breast tumors compared to the paired norm. A high statistically significant correlation was established between the expression of BIM and BAX (Rs=0.48, p=0.01). This result may indicate that the mRNA pairs may be involved in common processes of breast carcinogenesis. A highly significant positive correlation of expression was established for the pairs ADAMTS9-AS1–BAX (Rs=0.67, p=0.01), OIP5-AS1–BAK1 (Rs=0.52, p<0.01), and OIP5-AS1–BIM (Rs =0.51, p<0.01), suggesting a direct or indirect activating interaction of these pairs in breast cancer. The obtained data on coexpression are confirmed by the data obtained from correlation analysis carried out on a dataset showing differential expression levels of lncRNAs in breast cancer, selected from and complementary to the TCGA Tumor/TCGA Normal libraries.

Conclusions. LncRNAs and mRNAs that are differentially expressed in breast cancer have been identified; a positive correlation has been established in mRNA-mRNA and mRNA-lncRNA pairs in breast cancer, which indicates their participation in common signaling pathways in breast cancer. Determining new lncRNAs involved in the pathogenesis of BC and in the deregulation of apoptosis genes is one of the priority tasks of modern molecular oncology.

This work was supported by the Russian Science Foundation grant no. 22-75-00132.

Introduction.

Bone marrow necrosis (BMN) is a relatively rare discovery in bone marrow biopsies (BMB); it is estimated to be present in less than 1% of cases. BMN is defined as the destruction of both the myeloid tissue and the medullary stroma, with the preservation of bone structures.

BMN is most commonly attributed to malignancy and chemotherapy. That being said, heterologous causes such as medication and infection have been incriminated in BMN.

We describe the case of a patient diagnosed with multiple myeloma (MM) presenting initially with extensive necrosis of the bone marrow in their biopsy. A finding that, to the best of our knowledge, remains an exceptional manifestation of this condition, only being described in five cases in the literature previously.

Objective

The particular nature of our case lies in the rarity of this presentation, which renders BMB interpretation challenging to pathologists who are unfamiliar with this complication.

Materials and Methods

We report the case of Mrs A, a 67-year-old patient with a medical history of repeated transfusions for iron deficiency anaemia and a fracture of the femoral diaphysis. The patient presented initially with severe anaemic syndrome and an alteration of her general state, associated with ostealgia, epigastric tenderness and polyuria.

Blood count showed a non-regenerative normochromic normocytic anaemia that is associated with thrombopenia and myeloma. The biochemical profile of the patient showed a malignant hypercalcemia with an alteration in the renal function.

Imaging found multiple geographical osteolytic lesions of axial and appendicular topography.

Bone marrow aspiration was performed, showing a sample of poor cellularity and the presence of a population of suspicious lymphoid-like cells.

A bone marrow biopsy was performed and treated for histopathological study.

Results

A histological examination of the fragments obtained showed multiple regular osseous trabeculae bordering marrow spaces that contained extensive tumoral necrosis which took up approximately 80% of the entire sample. We also noted the presence of a tumoral proliferation of cells with hyperchromatic eccentric nuclei and an abundant basophilic cytoplasm evoking a plasmacytoid nature.

An immunohistochemical study was carried out showing a positive marking of plasmacytoid cells by the CD 138 antibody with Lambda monoclonality.

By combining the histopathological and immunohistochemical findings with the patient's history, clinical presentation and imaging results, the diagnosis of multiple myeloma was made.

Discussion and Conclusion

BMN is a rare finding in bone marrow biopsies and it is estimated that only around 300 cases have been reported in the literature. A detailed pathophysiology of this condition is yet to be properly established, with the prevailing hypothesis involving elevated levels of TNF-alpha and interleukin-6 causing damage to endothelial cells in the microenvironment, which results in vascular occlusions and necrosis.

Clinically, patients present with ostealgia, fever, elevated alkaline phosphatase, markedly elevated LDH levels and peripheral cytopenias with typical findings of bone marrow necrosis, the extent of which allows for the grading of BMN: grade I with necrosis involving less than 20% of the bone marrow; necrosis occupies between 20 and 50% of the bone marrow in grade II; and eventually grade III where the necrosis extends beyond 50% of the bone marrow.

A ten-year study by Wool et al. showed that malignancy—both secondary to a metastatic tumour and haemato-lymphoid malignancy – was responsible for 90% of BMN cases. BMN was also associated with poor prognosis, with a mortality rate of 55% during follow-up; however, Zhu et al. reported two cases of BMN with MM who received prompt treatment and showed complete restoration of hematopoietic function.

BMN is an exceptional discovery in BMBs and its aetiology is largely dominated by malignancy. To the best of our knowledge, BMN in MM has only been described in five cases in the literature; as such, the rarity of this finding may pose difficulties for pathologists during interpretation. The importance of pathologists' awareness of this possibility is amplified by the understanding that BMN is not only associated with a poorer prognosis for patients, but also that prompt diagnosis and treatment have introduced the possibility of recovery.

Acute myeloid leukemia (AML) is an aggressive blood cancer where immature stem cells in the bone marrow multiply rapidly, disrupting blood cell production and leading to infections, anemia, and bleeding. This devastating disease claims around 85,000 lives annually and is projected to double by 2040, highlighting its critical importance for research and improved treatment strategies. The RUNX1 transcription factor, a critical gene for hematopoiesis, is highly prevalent in AML and linked to poor patient outcomes. Mutations disrupt RUNX1's function, essentially acting as a "bad switch" that promotes aggressive leukemia growth and significantly reduces survival chances. Targeting this master regulator holds promise for developing novel therapies to improve patient outcomes in AML.

In the current study, we utilized a computational drug discovery approach, involving bioactivity data retrieval for Human RUNX1 “CHEMBL2093862” from the CHEMBL database, where RUNX1 is the target protein. We performed chemical space analysis using Lipinski descriptors to identify whether the compounds have the properties of ideal drugs or not. We calculated molecular descriptors, specifically PubChem fingerprints, to identifythe unique structure of the compounds. We applied machine learning algorithms, like Random Forest, Linear Regression, Decision Tree, and XGBoost, to create a model with the ability to classify whether the compounds are active or inactive. This study uses a user-friendly bioactivity prediction app using Streamlit so that researchers can check for the bioactivity and potency of drugs in real time for the targeting of RUNX1 in AML. This study introduces a comprehensive plan for drug development targeting RUNX1, offering potential interventions for AML mediated by RUNX1.

ETP-ALL (early T-cell precursor acute lymphoblastic leukemia) is a high-risk subtype of early T-cell leukemia. The distinctive immunophenotype of ETP-ALL is characterized by CD1a-, CD8-, CD5-, or weak-positive <75%. The complex karyotype and genomic instability are well-known characteristics of ETP-ALL. Throughout all disease stages, we examined the karyotypes, protein expressions, immunophenotypes, and gene expression profiles. The relapse's karyotype analysis reveals new mutations not found during diagnosis, some of which are unprecedented in any ETP-ALL case reported in the literature. Alterations are present in chromosomes 4, 6, 16, and 18 (BCL-2 locus). Immunophenotype analysis shows drastic changes from diagnosis to relapse in ETP-ALL, including CD56 switch and unusual CD5 and CD8 switches. After comparing the patient's relapse-stage marrow with the healthy subject's control marrow and the non-ETP ALL subject at the same stage, protein analysis showed the IL23R-SGK1-RANBP1 asset is overexpressed, along with MARCH5 and BLC-2. Additionally, the expression of BIM, a negative regulator of BCL-2, decreases, as well as Mitofusin-2 (MFN2), which plays a role in mitochondrial fusion and network maintenance. A test conducted on ex vivo blasts confirms that Venetoclax is sensitive at different levels during the relapse phase but not in the early stages. This confirms that disease-related genetic changes led to blast expansion, dependent on BCL2, and is associated with the feedback regulation of transcript expression. Throughout the disease progression, we examined bone marrow and peripheral blood smears. These prove the failures of all therapeutic lines and HSCT, as well as the knockdown of leukemic blasts and the resumption of bone marrow activity (measured by neutrophil levels) with venetoclax administration. In summary, ourunderstanding of the BCL2 pathway's impact in ETP-ALL is limited, and the occasional use of venetoclax is not yet standardized in guidelines. Our results highlight the importance of assessing BCL2 expression in refractory ETP-ALL cases.

Deubiquitylating enzymes are proteases that reverse the ubiquitination of proteins, an important process for maintaining normal homeostasis.

USP16 is a deubiquitylating enzyme that belongs to the family of ubiquitin-specific proteases (USPs) and is involved in cell cycle progression and chromatin remodeling.

To elucidate the role of USP16 in lung cancer progression, we first analyzed its expression in a cohort of biopsies obtained from patients with non-small lung cancer (N=18). Real-time PCR analysis and Western blot analysis showed that USP16 is highly expressed in NSCLC tissues compared to normal tissues. To characterize the role of USP16, we used two lung cancer cell lines (NCI-H460 and A549), in which USP16 was knocked down by lentiviral RNA interference (shUSP16).

The knockdown of USP16 affects cancer cell behavior in terms of proliferation and drug sensitivity. The knockdown of USP16 reduces the proliferation of cancer cells (≈40%) compared to control cells, which is due to defects in mitotic cell phase. These effects could be attributed to the deubiquitinating effect on its substrates. Indeed, the silencing of USP16 decreased the protein stability of the transcription factor Myc and Polo like kinase-1 (PLK1). Conversely, we found that USP16 impaired the response of lung cancer cells to platinum-containing compounds. The reduction of USP16 expression impairs the cytotoxicity of cisplatin by approximately 50% compared to control cells. This could likely be due to the impaired recruitment of repair proteins in proximity of double-strand breaks (DSBs) in lung cancer cells. These results prompted us to perform further analysis to investigate the role of USP16 in DNA repair and drug sensitivity.

Introduction: Emulsions are attractive delivery systems for hydrophobic drug molecules compared to hydrophilic drug molecules. An emulsion is a biphasic liquid preparation, containing two immiscible liquids. In the delivery of therapeutically active compounds, nanoemulsions have gained considerable interest because approximately 50% of new chemical entities are hydrophobic. Nanoemulsions are a group of dispersed particles which are used as pharmaceutical biomedical aids and vehicles that show great promise for the future of cosmetics, diagnostics, drug therapies, and biotechnologies. To achieve targeted drug delivery and to reduce side effects, we have prepared a magnetic nanoemulsion.

Methods: Using both high-and low-energy methods, we will prepare the nano emulsion. Microfluidization, ultrasonication, and high-pressure homogenization are examples of high-energy methods, whereas we will also use low-energy techniques like the phase inversion emulsification method and the self-nanoemulsification method. We also may use the self-nanoemulsification method for the preparation and formulation of letrozole magnetic nanoemulsion.

Results: The pseudo ternary phase diagram explains the optimal concentration of excipients. The studies reported that the formulation was stable under the magnetic field. In vitro characterization studies have reported that the average globule size of the LMNEs was 49.63 nm, with a charge of 26.9 eV and a polydispersity index of 0.428. FT-IR results showed that citric acid successfully stabilized the magnetic nanoparticles and confirmed that interaction between the drug and liposomes. The report also found the rheological properties of LMNEs .

Conclusion: The nanoemulsion formulation parameters were evaluated to indicate that the results were obtained within an acceptable range. From in vitro data, it can be concluded that the developed magnetic nanoemulsion has great potential with better pharmaceutical and therapeutical properties. Letrozole magnetic nanoemulsion is a suitable module used for controlled and targeted drug delivery in order to combat breast cancer. The formulation of a letrozole-loaded magnetic nanoemulsion was performed and in vitro cell line studies and pre-clinical studies may be performed in the future.

Introduction. The design and development of antitumor compounds based on an isatin core led to the synthesis of 1-substituted isatin-5-sulfonamides with potent antiproliferative activity. This investigation is aimed at new 1-substituted isatin-5-sulfonamides with pro-apoptotic properties alone and in combination with AKT and Hsp90 inhibitors on hormone-sensitive and hormone-resistant breast cancer cells.

Methods. The synthesis of target 1-substituted isatin-5-sulfonamides involved 3 stages. The alkylation of isatin-5-sulfonamide via various benzyl chlorides allowed us to synthesize previously unknown series of 1-substituted isatin-5-sulfonamides. 4-Hydroxytamoxifen (HT) was used to develop a MCF7-resistant subline (MCF7/HT) via the long-term incubation of MCF7 cells with HT. MCF7 cells were transfected with the p53 luciferase reporter plasmid to obtain MCF7/p53-LUC cells.

Results. 1-(4-((trifluoromethyl)thio)benzyl)isatin-5-sulfonamide (LCTA-3344) exhibited the highest antiproliferative activity, suppressing tumor cells at low micromolar concentrations. After the development of the resistant subline, the IC₅₀ values of HT were 5.1±0.3 μМ (MCF7) and 10.2±0.4 μМ (MCF7/HT), and a resistance index (RI) of 2 was found. Compound LCTA-3344 showed higher antiproliferative activity in MCF7/HT (IC₅₀=1.4±0.1 μМ), than in MCF7 (2.6±0.3 μМ). A search for effective combinations with AKT Inhibitor IV (6-(2-benzothiazolyl)-1-ethyl-2-[2-(methylphenylamino)ethenyl]-3-phenyl-1H-benzimidazolium, monoiodide), AKT Inhibitor X (10-DEBC; 2-chloro-N,N-diethyl-10H-phenoxazine-10-butanamine), and Hsp90 Inhibitor (luminespib, NVP-AUY922; 5-[2,4-dihydroxy-5-(1-methylethyl)phenyl]-N-ethyl-4-[4-(4-morpholinylmethyl)phenyl]-3-isoxazolecarboxamide) was performed. The combinations of LCTA-3344 and AKT Inhibitor IV on MCF7 and MCF7/HT were synergetic with combination index (CI) values equal to 0.8 and 0.4 (a higher effect), correspondingly. For comparison, combinations of LCTA-3344 with 10-DEBC and luminespib did not demonstrate such a high effect with a minimal value of CI 0.9. MCF7/p53-LUC cells were used to assess p53 activity. LCTA-3344 did not increase luciferase activity in MCF7/p53-LUC cells, whereas doxorubicin has been identified as its strong inducer.

Conclusions. A leading 1-substituted isatin-5-sulfonamide LCTA-3344 was 1.9 times more effective against MCF7/HT, than parental cells. The most effective drug combination was LCTA-3344 with AKT Inhibitor IV on the MCF7/HT subline, CI 0.4. The isatin-5-sulfonamide LCTA-3344 induced apoptosis with the p53-independent mechanism, which may provide a basis for novel therapeutic strategies in the treatment of hormone-resistant breast cancers.

Funding. This research was partly funded by the Russian Science Foundation (agreement 20-13-00402, https://rscf.ru/project/23-13-45035/, accessed on November 24, 2023).

Invasive ductal carcinoma (IDC) constitutes approximately 80% of breast cancer cases. After treatment, there is a 3-15% chance that IDC will recur. The aim of this study was to accurately predict IDC recurrence with a low false-negative rate and to identify mutated genes associated with IDC recurrence. We hypothesize that IDC recurrence can be predicted using genomic, clinical, and phenotypic patient data and that certain genes, when mutated, are highly influential in the recurrence of IDC. To attain an understanding for the underlying causes of IDC recurrence, genomic data such as gene expression and mutation variant data, in addition to clinical and phenotypic data, were aggregated. An XGBoost framework was utilized for the classification task of predicting a patient’s status as disease-free or cancer-recurrent. The XGBoost framework and the XGBClassifier algorithm employ sequential learning and an ensemble of weaker learning models, such as individual decision trees, both of which allow for the continuous correction of errors made by previous models and the identification of complex relationships between features. The XGBClassifier algorithm predicted IDC recurrence with over 99% accuracy and 0% false-negative rates across nearly 2200 IDC patient samples, approximately half of which were recurrent cases. These results were obtained after model fine tuning upon the examination of generated ROC curves, precision—recall curves, and confusion matrices. Feature importance scores, in this case representing the significance of mutated genes in IDC recurrence, were calculated using the XGBoost model for the 40 most commonly mutated genes among IDC patients. GATA3, CDH1, and BRCA1 genes were given the highest feature importance scores despite relatively low mutation occurrences, indicating that our model did not simply associate gene importance with mutation frequency. Our results prompt further inspection, both of genes given high importance scores with low occurrences and of genes given low importance scores with high occurrences using ontological tools. The feature importance scores can aid in biomarker identification, cancer diagnoses, and drug development. Our study can be generalized to other cancers to determine common biomarkers and deepen our understanding of general cancer recurrence.

Glioblastoma multiforme (GBM) is the most common aggressive malignant primary brain tumor, afflicting approximately 3 in every 100,000 persons in the United States, with an incidence rate that is 1.6 times higher in males compared to females. Arising from the neural
and/or glial progenitor/stem cells, GBM is commonly located in the supratentorial cortical region affecting the frontal lobes. The rapid local growth and spread of GBM leads to a dismal prognosis with a 5-year survival rate of 6.9%, despite multiple therapeutic interventions, including surgery, radiotherapy, and chemotherapy. To overcome the challenge to develop curative treatments of GBM, several promising experimental methods (immune therapy, gene therapy, simulated microgravity therapy, and oncolytic viral therapy) are under investigation. Potentially beneficial effects of microgravity on GBM include (A) the repression of survival signaling pathways, (B) the induction of the apoptosis of cancerous cells, and (C) the blocking of spherical colony formation and cellular proliferation via the downregulation of ATM/ATR and CDK1/2 proteins to block cellular phase 2 to progress to G2. We combined oncolytic viral therapy using an autonomous rat parvovirus H1 with simulated microgravity, in turn utilizing the potentially beneficial effects of microgravity on tumor cells (decreased cell proliferation, disrupted mitochondrial functions, and the induction of apoptosis) (Elshourbagy T, Brašić JR. Amelioration of glioblastoma multiforme via the combination of simulated microgravity and oncolytic viral therapy. Med. Sci. Forum. 2023; 20: 9. https://doi.org/10.3390/IECC2023-14219 ) and was limited by the use of simulated microgravity on earth. We now propose to conduct similar investigations on space stations where gravity approaches zero. We hypothesize that oncolytic viral therapy in space without gravity will (A) lyse tumor cells through the induction of apoptosis, decreased cell proliferation, and the induction of an immune response, and constitute the foundation of potentially curative treatments of GBM.