Reduced Drug Sensitivity Research Articles

Hydroxy-wybutosine tRNA modifications as indicators of disease progression and therapeutic targets in leukaemia.

Therapeutic approaches for acute myeloid leukaemia (AML) and myelodysplastic syndromes (MDS) differ due to distinct diagnostic criteria and treatment strengths. However, reliable biomarkers to differentiate AML from MDS are needed. This study investigated transfer RNA (tRNA) modifications, particularly hydroxy-wybutosine (OHyW), in the transition from MDS to AML. We found a significant decrease in OHyW and its biosynthetic enzyme leucine carboxyl methyltransferase 2 (LCMT2, alias symbol is TYW4) levels in AML compared to MDS. Mass spectrometric analysis revealed distinct tRNA modification patterns, with AML showing decreased OHyW and increased precursor levels, indicating a disrupted biosynthetic pathway. Lower LCMT2 expression correlated with reduced drug sensitivity and limited differentiation potential in AML cell lines. The results highlight the pivotal role of tRNA modifications in the progression from MDS to AML and suggest that targeting LCMT2 may enhance therapeutic outcomes in AML. By understanding these molecular mechanisms, we can develop new diagnostic markers and therapeutic strategies, potentially transforming the clinical management of AML and improving patient outcomes.

Effector Functions of Dendritic Cells in Cancer: Role of Cytotoxicity and Growth Inhibition.

The tumor microenvironment plays a critical role in modulating immune responses associated with tumorigenesis, tumor progression, and metastasis. Dendritic cells (DC) play a key role in preventing and progression of metastatic neoplasia by driving and restoring dysfunctional immune systems and obliterating immunosuppression, thus obstructing tumor evasion. In this review, we will discuss the functions of tumor-infiltrating DC in anti-tumor resistance, prevention of tumor recurrence, and immunosuppression. We will also describe DC metabolism, differentiation, and plasticity, which are essential for its function. Cancers like Lymphomas may be able to corrupt immune surveillance by reducing natural killer cell numbers. Thus, interactions between lymphoma and DC with reference to cytotoxicity may be an important event, likely to be mediated via activation with interferon-γ (IFN-γ) and Toll like receptors (TLR) ligands. Mechanisms of DC-mediated cytotoxicity and the role of apoptosis and death receptors, including the role played by nitric oxide, etc., are of immense significance. We will also look into the molecular mechanisms in the tumor microenvironment, reduced drug sensitivity, and tumor relapse, as well as methods for combating drug resistance and focusing on immunosuppressive tumor networks. We will address how DC mediated cytotoxicity in combination with drugs affects tumor growth and expansion in relation to checkpoint inhibitors and regulatory T cells. Innovative approaches for therapeutic modulation of this immunosuppressive adoptive DC immunotherapy will be highlighted, which is necessary for future personalized therapeutic applications.

Matrix stiffening facilitates stemness of liver cancer stem cells by YAP activation and BMF inhibition

Matrix stiffening is one of the major risk factors for hepatocellular carcinoma (HCC) and drives tumor progression. The extracellular matrix (ECM) stiffness of HCC displays mechanical heterogeneity, with stiffness increasing from the core to the invasive frontier. The distribution of liver cancer stem cells (CSCs) is related to this mechanical property. However, it is not sufficiently understood how heterogeneous matrix stiffness regulates the stemness of CSCs. In this study, we developed an adjustable gelatin/alginate hydrogel to investigate the effect of various matrix stiffnesses on CSC stemness under three-dimensional culture conditions. Gelatin/alginate hydrogel with the stiffness of soft (5 kPa), medium (16 kPa), and stiff (81 kPa) were prepared by altering the concentration of calcium ions. It was found that a stiffer matrix promoted stemness-associated gene expression, reduced drug sensitivity, enhanced sphere-forming and clonogenic ability, and tumorigenic potential. Mechanistically, matrix stiffening facilitates CSC stemness by increasing Yes-associated protein (YAP) activity and inhibiting Bcl-2 modifying factor (BMF) expression. Knockdown of YAP or overexpression of BMF significantly attenuated matrix stiffening-induced stemness, suggesting the involvement of YAP and BMF in this process. Together, our results unravel the regulatory mechanism of heterogeneous matrix stiffness on CSC stemness and also provide a novel therapeutic strategy for eradicating CSCs and improving the efficiency of HCC treatment.

IL-33-dependent NF-κB activation inhibits apoptosis and drives chemoresistance in acute myeloid leukemia

BackgroundDespite recent advances in therapeutic regimens, the prognosis of acute myeloid leukemia (AML) remains poor. Following our previous finding that interleukin-33 (IL-33) promotes cell survival along with activated NF-κB in AML, we further investigated the role of NF-κB during leukemia development. MethodsFlow cytometry was performed to value the apoptosis and proliferation. qRT-PCR and western blot were performed to detect the expression of IL-6, active caspase 3, BIRC2, Bcl-2, and Bax, as well as activated NF-κB p65 and AKT. Finally, xenograft mouse models and AML patient samples were used to verify the findings observed in AML cell lines. ResultsIL-33-mediated NF-κB activation in AML cell lines contributes to a reduction in apoptosis, an increase in proliferation rate as well as a decrease in drug sensitivity, which were reversed by NF-κB inhibitor, Bay-117085. Moreover, IL-33 decreased the expression of active caspase-3 while increasing the levels of BIRC2, Bcl-2, and Bax, and these effects were blocked by Bay-117085. Additionally, NF-κB activation induced by IL-33 increases the production of IL-6 and autocrine activation of AKT. Co-culture of bone marrow stroma with AML cells resulted in increased IL-33 expression by leukemia cells, along with decreased apoptosis level and reduced drug sensitivity. Finally, we confirmed the in vivo pro-tumor effect mediated by IL-33/ NF-κB axis using a xenograft model of AML. ConclusionOur data indicate that IL-33/IL1RL1-dependent signaling contributes to AML cell activation of NF-κB, which in turn causes autocrine IL-6-induced activation of pAKT, supporting IL-33/NF-κB/pAKT as a potential target for AML therapy.

Molecular and functional characterization of reversible-sunitinib-tolerance state in human renal cell carcinoma.

Therapy failure with the tyrosine kinase inhibitor (TKI) sunitinib remains a great challenge in metastatic renal cell carcinoma (mRCC). Growing evidence indicates that the tumour subpopulation can enter a transient, non-mutagenic drug-tolerant state to endure the treatment underlying the minimal residual disease and tumour relapse. Drug tolerance to sunitinib remains largely unexplored in RCC. Here, we show that sunitinib-tolerant 786-O/S and Caki-2/S cells are induced by prolonged drug treatment showing reduced drug sensitivity, enhanced clonogenicity, and DNA synthesis. Sunitinib-tolerance developed via dynamic processes, including (i) engagement of c-MET and AXL pathways, (ii) alteration of stress-induced p38 kinase and pro-survival BCL-2 signalling, (iii) extensive actin remodelling, which was correlated with activation of focal adhesion proteins. Remarkably, the acute drug response in both sensitive and sunitinib-tolerant cell lines led to dramatic fine-tuning of the actin-cytoskeleton and boosted cellular migration and invasion, indicating that the drug-response might depend on cell state transition rather than pre-existing mutations. The drug-tolerant state was transiently acquired, as the cells resumed initial drug sensitivity after >10 passages under drug withdrawal, reinforcing the concept of dynamic regulation and phenotypic heterogeneity. Our study described molecular events contributing to the reversible switch into sunitinib-tolerance, providing possible novel therapeutic opportunities in RCC.

Identification and validation of an anoikis-related genes signature for prognostic implication in papillary thyroid cancer.

Thyroid cancer, notably papillary thyroid cancer (PTC), is a global health concern with increasing incidence. Anoikis, a regulator of programmed cell death, is pivotal in normal physiology and, when dysregulated, can drive cancer progression and metastasis. This study explored the impact of anoikis on PTC prognosis. Analyzing data from GEO, TCGA, and GeneCards, we identified a prognostic signature consisting of six anoikis-related genes (ARGs): EZH2, PRKCQ, CD36, INHBB, TDGF1, and MMP9. This signature independently predicted patient outcomes, with high-risk scores associated with worse prognoses. A robust predictive ability was confirmed via ROC analysis, and a nomogram achieved a C-index of 0.712. Differences in immune infiltration levels were observed between high- and low-risk groups. Importantly, the high-risk group displayed reduced drug sensitivity and poor responses to immunotherapy. This research provides insights into anoikis in PTC, offering a novel ARG signature for predicting patient prognosis and guiding personalized treatment strategies.

Reduced Anticancer Potency of Carboplatin in the Presence of Sialic Acid in SH-SY5Y Cells

Background: Neuroblastoma (NB) is the most common solid childhood tumor with aggressive behavior and a high mortality rate. Multiple therapeutic approaches have been developed and applied to treat neuroblastoma, but resistance to therapies is inevitable, leading to treatment failure and cancer relapse. Therefore, perceiving the mechanisms of reduced drug sensitivity seems appropriate and promising in NB treatment to determine synergistic effects strategies like combination therapy. Objectives: The present study aimed to investigate the modulation effect of the therapeutic efficacy of carboplatin (a chemotherapeutic agent) by co-administration of sialic acid (an alpha-keto acid sugar with a 9-carbon backbone located at the outermost end of N-linked and O-linked carbohydrate chains and in lipid-associated glycoconjugates showing aberrant expression in tumor cells caused tumor-genesis) in NB cells. Methods: In the present study, the effects of sialic acid, carboplatin, and Sia-Carbo (sialic acid in combination with carboplatin) were evaluated on viability and apoptosis using the determination of EC50 and IC50. Also, the capability of metastasis and apoptosis were assessed using real-time PCR. Moreover, the expression of MRP-1 encoded by ABCC1 (as a target for therapeutic suppression in high-risk neuroblastoma) was studied in different treatment groups. Conclusions: Regarding IC50 increasing carboplatin in the presence of sialic acid, the results showed that sialic acid treatment significantly modulated carboplatin's effect on cell apoptosis and induced ABCC1 expression. These findings show that sialic acid metabolic engineering can be a good approach to neuroblastoma therapy. It was suggested that targeting aberrant sialylation in combination with carboplatin exerts more profound apoptotic and anticancer effects on the neuroblastoma SH-SY5Y cell line than carboplatin monotherapy.

Current Advances and Future Strategies for BCL-2 Inhibitors: Potent Weapons against Cancers.

Targeting the intrinsic apoptotic pathway regulated by B-cell lymphoma-2 (BCL-2) antiapoptotic proteins can overcome the evasion of apoptosis in cancer cells. BCL-2 inhibitors have evolved into an important means of treating cancers by inducing tumor cell apoptosis. As the most extensively investigated BCL-2 inhibitor, venetoclax is highly selective for BCL-2 and can effectively inhibit tumor survival. Its emergence and development have significantly influenced the therapeutic landscape of hematological malignancies, especially in chronic lymphocytic leukemia and acute myeloid leukemia, in which it has been clearly incorporated into the recommended treatment regimens. In addition, the considerable efficacy of venetoclax in combination with other agents has been demonstrated in relapsed and refractory multiple myeloma and certain lymphomas. Although venetoclax plays a prominent antitumor role in preclinical experiments and clinical trials, large individual differences in treatment outcomes have been characterized in real-world patient populations, and reduced drug sensitivity will lead to disease recurrence or progression. The therapeutic efficacy may vary widely in patients with different molecular characteristics, and key genetic mutations potentially result in differential sensitivities to venetoclax. The identification and validation of more novel biomarkers are required to accurately predict the effectiveness of BCL-2 inhibition therapy. Furthermore, we summarize the recent research progress relating to the use of BCL-2 inhibitors in solid tumor treatment and demonstrate that a wealth of preclinical models have shown promising results through combination therapies. The applications of venetoclax in solid tumors warrant further clinical investigation to define its prospects.

Tumor-Infiltrating Dendritic Cells: Decisive Roles in Cancer Immunosurveillance, Immunoediting, and Tumor T Cell Tolerance.

The tumor microenvironment plays a key role in progression of tumorigenesis, tumor progression, and metastasis. Accumulating data reveal that dendritic cells (DCs) appear to play a key role in the development and progression of metastatic neoplasia by driving immune system dysfunction and establishing immunosuppression, which is vital for tumor evasion of host immune response. Consequently, in this review, we will discuss the function of tumor-infiltrating DCs in immune cell signaling pathways that lead to treatment resistance, tumor recurrence, and immunosuppression. We will also review DC metabolism, differentiation, and plasticity, which are essential for metastasis and the development of lung tumors. Furthermore, we will take into account the interaction between myeloid cells and DCs in tumor-related immunosuppression. We will specifically look into the molecular immune-related mechanisms in the tumor microenvironment that result in reduced drug sensitivity and tumor relapse, as well as methods for combating drug resistance and focusing on immunosuppressive tumor networks. DCs play a crucial role in modulating the immune response. Especially, as cancer progresses, DCs may switch from playing an immunostimulatory to an inhibitory role. This article’s main emphasis is on tumor-infiltrating DCs. We address how they affect tumor growth and expansion, and we highlight innovative approaches for therapeutic modulation of these immunosuppressive DCs which is necessary for future personalized therapeutic approaches.

Glutathione Peroxidase 4 as a Therapeutic Target for Anti-Colorectal Cancer Drug-Tolerant Persister Cells.

BackgroundDespite the effectiveness of chemotherapy and targeted therapy for colorectal cancer, drug resistance drives therapy failure and tumor relapse. Increasing evidence has suggested that cancer cells can enter a reversible drug-tolerant persister state to survive chemotherapy or targeted agents. However, the traits and treatable vulnerabilities of anti-colorectal cancer drug-tolerant persister cells is not yet known.MethodsIn this study, we established 5-fluorouracil and AZ628-tolerant persister cell models in two colorectal cancer cell lines, namely HCT116 and SW620, and revealed the characteristics of colorectal cancer persister cells by cell viability assay and flow cytometry. We investigated the efficacy and mechanism of ferroptosis inducers RSL3 and FIN56 on persister cells, which are glutathione peroxidase 4 inhibitors. In the xenograft mouse model, we further evaluated the inhibitory effect of RSL3 on tumor regrowth.ResultsColorectal cancer persister cells, which were enriched in the residual cancer cell population, exhibited reduced drug sensitivity, were largely quiescent and expressed high levels of stem cell-related genes and mesenchymal markers but not epithelial markers. The persister cells were more sensitive and underwent ferroptosis induced by glutathione peroxidase 4 inhibitors. Mechanistically, glutathione peroxidase 4 and ferrous iron, which are pivotal ferroptosis regulators, were upregulated in residual cells or tumors, and were hence potential therapeutic targets of persister cells. In the xenograft model, we confirmed that inhibition of glutathione peroxidase 4 restrained tumor regrowth after discontinuation of anti-cancer drug treatment. Moreover, biopsies obtained from patients with colorectal cancer undergoing neoadjuvant chemoradiotherapy revealed upregulated glutathione peroxidase 4 and ferritin heavy chain 1. High glutathione peroxidase 4 expression correlates with a worse prognosis in colorectal cancer patients.ConclusionsOur work reveals that the upregulated glutathione peroxidase 4 and ferrous iron in anti-colorectal cancer drug-tolerant persister cells were potential therapeutic targets. Glutathione peroxidase 4 inhibition combined with chemotherapy or targeted therapy may be a promising therapy for colorectal cancer.

Cell-Intrinsic Mechanisms of Drug Tolerance to Systemic Therapies in Cancer.

In patients with cancer with metastatic disease, the rate of complete tumor response to systemic therapies is low, and residual lesions persist in the majority of patients due to early molecular adaptation in cancer cells. A growing body of evidence suggests that a subpopulation of drug-tolerant persister cells-a reversible phenotype characterized by reduced drug sensitivity and decreased cell proliferation-maintains residual disease and may serve as a reservoir for resistant phenotypes. The survival of these residual tumor cells can be caused by reactivation of specific signaling pathways, phenotypic plasticity (i.e., transdifferentiation), epigenetic or metabolic reprogramming, downregulation of apoptosis as well as transcriptional remodeling. In this review, we discuss the molecular mechanisms that enable adaptive survival in drug-tolerant cells. We describe the main characteristics and dynamic nature of this persistent state, and highlight the current therapeutic strategies that may be used to interfere with the establishment of drug-tolerant cells, as an alternative to improve objective response to systemic therapies and delay the emergence of resistance to improve long-term survival.

A Promising Esophageal Cancer Prognostic Signature of Ferroptosis-Related LncRNA to Predict Immune Scenery and Immunotherapy Response

PurposeFerroptosis and long non-coding RNA (lncRNA) expression signatures have been associated with the clinical progression and immune-contexture of different solid tumors. The study aimed to identify a prognostic signature of ferroptosis-related lncRNAs (falncRNAs) to forecast the immune scenery and immunotherapy response in esophageal cancer (EC).Patients and MethodsGene expression profiles of EC were extracted from The Cancer Genome Atlas (TCGA) database, and ferroptosis-related genes were downloaded from the FerrDb database, which identified differentially expressed falncRNAs (DEfalncRNAs) via differential analysis. DEfalncRNA pairs associated with prognosis were identified by constructing a matrix, univariate and least absolute shrinkage and selection operator (LASSO) analysis. The prognostic signature was constructed by multivariate analysis. We appraised the forecasting capability of prognostic signature in survival, clinicopathological features, immune landscape, efficacy of immunotherapy, and drug sensitivity. The potential molecular mechanism of signature was investigated by gene set enrichment analysis (GSEA).ResultsWe obtained 18 DEfalncRNA pairs to define a novel prognostic signature that was determined on a discovery cohort of 158 tumor samples and 11 adjacent normal tissues from TCGA and internally validated, with the definition of high- vs low-risk groups based on 3 years overall survival. We demonstrated that the high- vs low-risk groups differed for clinical parameters and computationally predicted drug sensitivity and tumor immune contexture, with the high-risk group having worse survival, more aggressive disease (node involvement, metastasis), reduced drug sensitivity, higher tumor mutation load, and gene signatures of infiltration of pro-tumoral immune cell subsets. The GSEA results revealed that ferroptosis and immunoregulatory pathways were significantly enriched in the high-risk group.ConclusionThe prognostic signature based on falncRNAs has the potential to forecast the survival, immune scenery, efficacy of immunotherapy, and drug sensitivity of EC, which is helpful for clinical prediction and individualized treatment.

Elevated Expression of the S100A8/S100A9 Complex in AML Correlates with Reduced Sensitivity to the BCL2 Inhibitor Venetoclax

BackgroundDespite recent drug development efforts, multi-drug resistance remains a major challenge in the treatment of AML. Understanding resistance mechanisms could help to design combinatorial therapies beneficial for AML patients. Earlier studies have shown that sensitivity of AML cells to the B-cell lymphoma-2 (BCL2) inhibitor venetoclax is associated with mutations in genes encoding chromatin modifiers, WT1 and IDH1/IDH2 (Konopleva et al, Cancer Disc. 2016). We have also observed that overexpression of a subset of HOXA and HOXB genes is linked to venetoclax sensitivity (Kontro et al, Leukemia 2016). Besides, induction of alternate cell survival mechanisms (e.g. BCLXL and MCL1), there is little knowledge of molecular indicators of venetoclax resistance. In this study, we used RNA sequence and ex vivo drug sensitivity data from a cohort of AML patients to identify gene expression profiles linked to venetoclax resistance.MethodsRNA sequencing was performed on bone marrow or peripheral blood mononuclear cells (MNCs) collected from 112 AML patients and 4 healthy individuals. Sensitivity of MNCs from 18 AML patient samples was assessed to venetoclax by measuring cell viability after 72 h incubation with 6 different concentrations (1-1000 nM) using the CellTiter-Glo (CTG) assay. Free cytosolic calcium was measured in venetoclax-resistant (OCI-AML3, SHI-1) and sensitive cell lines (MOLM-13, Kasumi-1, all from DSMZ) using a calcium assay kit (BD Biosciences) after exposure to venetoclax (100 nM) for 2, 4 and 6 h. Statistical dependence between two variables was assessed by Pearson's correlation coefficient modeling.ResultsThe S100 family is comprised of 21 different low molecular weight, calcium-binding proteins. Interestingly, nearly half of the S100 family members had elevated expression in AML patient samples as compared with healthy individuals, with S100A8 and S100A9 being the most highly expressed genes. Based on this observation and a previous analysis of overexpressed genes in venetoclax resistant samples (Kontro et al, Leukemia 2016), we studied correlation of S100 gene expression with resistance to venetoclax. Wetested sensitivity of AML patient samples with the highest versus lowest expression of the genes (n = 9, each group) to venetoclax and confirmed correlation of high gene expression with reduced drug sensitivity (Figure 1A). High expression of S100A8, S100A9 and S100A12 negatively correlated with sensitivity to venetoclax (Figure 1B-D). Over-expression of S100A8 and S100A9 on venetoclax-resistant cell lines (OCI-AML3, SHI-1) was also associated with failure to release free-cytosolic Ca2+ compared with venetoclax-sensitive cell lines (MOLM-13, Kasumi-1). The S100A8 and S100A9 proteins form a complex and bind free Ca2+, which can inhibit mitochondrial membrane depololarization and the initiation of apoptosis. Over expression of these genes in AML could therefore potentially contribute to the development of venetoclax resistance.ConclusionsBCL2 inhibitors such as venetoclax are effective novel therapies for several malignancies, including AML. However, identifying responding patients and understanding mechanisms of resistance are important. Our results showing high expression of the S100A8 and S100A9 calcium-binding proteins correlates with AML cell resistance to venetoclax warrant further validation in a clinical setting. However, these data provide evidence of a novel mechanism of resistance to this class of drug.AcknowledgmentRK and ML equal contribution. [Display omitted] DisclosuresPorkka:Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding. Heckman:Celgene: Research Funding; Novartis: Research Funding; Orion Pharma: Research Funding; Pfizer: Research Funding; IMI2 project HARMONY: Research Funding.

Coexisting Oncogenic Signaling Pathway Mutations in Cnl/Acml/CMML/MPN-U

Purpose: Chronic neutrophilic leukemia (CNL), chronic myelomonocytic leukemia (CMML), atypical myeloid leukemia (aCML), and unclassified myeloproliferative neoplasms (unMPN) are rare entities of myeloproliferative or myelodysplastic/myeloproliferative (MDS/MPN) syndromes. Due to poorly defined molecular mechanisms, these diseases have been, until now, diagnosed based on morphology and exclusionary criteria and treated empirically, resulting in dismal outcomes. Recent application of next generation sequencing has shed light on the genetic mechanisms of CNL/aCML/CMML/MPN-U. In particular, Maxson et al. and others have shown that CSF3R membrane proximal and transmembrane mutations are present in 83-89% of CNL and 3.3-44% of aCML patients, and expression of CSF3R membrane proximal mutants is sufficient to recapitulate the clinical features (mature granulocyte accumulation) of CNL in a mouse model. This has led to revision of the WHO diagnostic criteria for CNL, enabling a more accurate diagnosis and facilitating the development of targeted treatment for this disease. Aside from the CSF3R driver mutations, few recurrent signaling pathway mutations have been characterized in this group of diseases that could serve as the basis for such targeted therapeutic strategies. The aim of the current study is to characterize the frequency and distribution of signaling pathway driver mutations in a large cohort of CNL/aCML/CMML/MPN-U patients with the hope of informing diagnosis, prognosis and treatment.Methods and results: We performed whole exome sequencing on a large cohort of patients (n=160) with plausible diagnosis of CNL/aCML/MPN-U/CMML. We screened hotspot variants in genes representing kinases, non-kinase growth factor and cytokine receptors. We observed that 66.9% of patients harbor at least one signaling pathway driver mutations (CSF3R 20.6%, NRAS 17.5%, JAK2 9.4%, CBL 6.5%, PTPTN11 5.6%, ETNK1 5.6, KRAS 4.4%, NF1 3.1%, FLT3 3.1%, SH3B2 3.1%, etc.), largely involving JAK/STAT and RAS signaling pathways. Interestingly, MPL mutations and CALR indels were rare in our cohort. The variants of signaling pathway driver mutations were observed with high allele frequencies: CSF3R (52.1%), JAK2 (48.9%), NRAS (46.3%), PTPN11 (40.7%), CBL (34.4%) and KRAS (29.5%), indicating the majority of these driver mutations are present in the dominant leukemic clones.Interestingly, 21.5% and 3.7% patients harbor two and three different active signaling pathway mutations, respectively. Clonality analysis demonstrated a co-occurrence pattern in more than half of the cases where more than two signaling pathway mutations were identified, underscoring a complex genetic pathogenesis. CSF3R was shown to co-occur with NRAS, SH2B3, PTPN11, NTRK2, CBL, ETNK1 and ABL1 mutations; NRAS mutations were present in combination with CSF3R, KRAS, JAK2, CBL, STAT3, NTRK2, FLT3, and GNB1 mutation. Since CSF3R and RAS mutation co-occur at high frequencies (n=5, 22%) in this cohort. We performed drug sensitivity assays, and we observed reduced drug sensitivity to either JAK or MEK inhibitors in cells transformed by co-existing mutations, whereas drug combinations targeting the co-existing mutations demonstrated synergistic cytotoxicity .Conclusions: In sum, CNL/aCML/MPN-U/CMML is a group of heterogeneous diseases associated with high frequencies of signaling pathway mutations in the major clone. More than 25% of patients demonstrate multiple different pathway mutations in a co-existing pattern, which warrants comprehensive screening of molecular targets and high-efficacy drug combination strategies. DisclosuresTyner:AstraZeneca: Research Funding; Incyte Corporation: Research Funding; Leap Oncology: Consultancy; Syros: Research Funding; Seattle Genetics: Research Funding; Agios Pharmaceuticals: Research Funding; Takeda Pharmaceutical Company: Research Funding; Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding; Janssen Pharmaceutica: Research Funding; Gilead: Research Funding; Genentech: Research Funding; Array Biopharma: Research Funding.

GLI1 reduces drug sensitivity by regulating cell cycle through PI3K/AKT/GSK3/CDK pathway in acute myeloid leukemia

Acute myeloid leukemia (AML) is a hematological malignancy with high incidence and recurrence rates. Gene expression profiling has revealed that transcriptional overexpression of glioma‐associated oncogene 1 (GLI1), a vital gene in the Hedgehog (Hh) signaling pathway, occurs in poor-prognosis AML, and high levels of phosphoinositide-3-kinase, regulatory subunit 1 (PIK3R1) and AKT3 predict shorter overall survival in AML patients. In this study, we discovered that GLI1 overexpression promotes cell proliferation and reduces chemotherapy sensitivity in AML cells while knocking down GLI1 has the opposite effect. Moreover, GLI1 promoted cell cycle progression and led to elevated protein levels of cyclins and cyclin-dependent kinases (CDKs) in AML cells. By luciferase assays and co-immunoprecipitation, we demonstrated that the PI3K/AKT pathway is directly activated by GLI1. GLI1 overexpression significantly accelerates tumor growth and upregulated p-AKT, CDK4, and cyclinD3 in vivo. Notably, the GLI1 inhibitor GANT61 and the CDK4/6 inhibitor PD 0332991 had synergistic effects in promoting Ara-c sensitivity in AML cell lines and patient samples. Collectively, our data demonstrate that GLI1 reduces drug sensitivity by regulating cell cycle through the PI3K/AKT/GSK3/CDK pathway, providing a new perspective for involving GLI1 and CDK4/6 inhibitors in relapsed/refractory (RR) patient treatment.

Infusion therapy in the internal diseases’ propaedeutics

Background. Infusion therapy (IT) is one of the main methods of drug therapy optimization. The essence of IT is to correct homeostasis disorders in order to detoxify, to restore the disrupted microcirculation and tissue perfusion, to eliminate the disorders of rheological and coagulation blood properties, to eliminate metabolic disorders, to improve drug delivery to the pathological focus, to restore circulating blood volume, to normalize fluid and electrolyte and acid-base balance.
 Objective. To describe modern IT.
 Materials and methods. Analysis of the literature sources on this topic.
 Results and discussion. Requirements for modern plasma substitutes include safety, sufficient and long-lasting volemic effect, rapid renal excretion, lack of accumulation and effect on the coagulation system, maximum similarity to blood plasma, and availability. There are several classes of plasma substitutes, and each of them has its own indications. For example, crystalloids are prescribed for dehydration, and colloids – for hypovolemia. The infusion volume is calculated based on the physiological needs of the organism, taking into account pathological fluid loss (fever, shortness of breath, postoperative wound drainage, vomiting, polyuria). Endogenous intoxication (EI) – a pathological condition that occurs as a result of exposure to toxic substances of exogenous or endogenous nature, which cause dysfunction and the development of extreme conditions – is an important field of IT application. EI can accompany chronic heart failure, peripheral vessels atherosclerosis, autoimmune and allergic diseases. Toxins have a direct (direct destruction of proteins and lipids, blocking of synthetic and oxidative processes in the cell) and indirect (microcirculation system and vascular tone disorders, changes of blood rheological properties) adverse effects. Clinical manifestations of EI include fever, malaise, and the dysfunction of various internal organs. As EI depletes the natural mechanisms of detoxification, worsens the clinical course of the disease, reduces drug sensitivity, suppresses immunity, it is an indication to detoxification via infusion. The tasks of the latter are to improve tissue perfusion, to provide hemodilution with a decrease in the toxins’ concentration, to stimulate diuresis, to eliminate acidosis, and to maintain the functional state of hepatocytes. For this purpose, solutions of polyatomic alcohols (Reosorbilact, Xylate, “Yuria-Pharm”) can be used. Reosorbilact increases the circulating blood volume, improves microcirculation and rheological blood properties, increases tissue perfusion, promotes “wash-out” of toxins, corrects acidosis and fluid and electrolyte balance, normalizes hepatocyte function, improving the own detoxification mechanisms. In case of microcirculation disturbances, it is reasonable to use the combined IT with the inclusion of Reosorbilact, Latren and Tivortin (“Yuria-Pharm”). Latren increases the elasticity of erythrocytes, reduces the aggregation of erythrocytes and platelets, normalizes the electrolyte composition of blood plasma, and Tivortin acts as a substrate for the formation of nitric oxide – the main signaling molecule of the endothelium. In addition to EI, IT usage is often prescribed for diabetic ketoacidosis. In such cases, it is advisable to use Xylate (“Yuria-Pharm”), which has antiketogenic properties, improves hemodynamics, corrects acidosis, and does not increase blood glucose levels. Xylate is recommended to be used only after preliminary rehydration with the help of isotonic saline solutions.
 Conclusions. 1. IT is an important method of treating a number of diseases. 2. EI accompanies not only diseases that involve intoxication syndrome, but also almost all internal diseases. 3. Reosorbilact and Xylate are the optimal solutions for detoxification. 4. It is reasonable to use combined IT, for example, the combination of Reosorbilact with Latren and Tivortin.

Epidemiological serosurvey and molecular characterization of sexually transmitted infections among 1890 sheltered homeless people in Marseille: Cross-sectional one day-surveys (2000–2015)

ObjectivesWe observed the prevalence and distribution of potential risk factors for sexually transmitted infections (STIs) among Marseille homeless population. MethodsOver the 2000-2015 period, we enrolled 1890 sheltered homeless adults and collected serum samples. Markers of hepatitis B and C viruses (HBV, HCV) and Treponema pallidum were searched using the CMIA testing. Positive HBsAg or anti-HCV samples underwent sequencing; positive anti-T. pallidum sera were subjected to the RPR test. ResultsThe overall prevalence of HBsAg, anti-HBs, anti-HBc, anti-HCV and anti-T. pallidum (by CMIA and RPR) was 4.1%, 22.9%, 35.5%, 5.3% and (6.8%, 1.0%), respectively. We found a significantly higher prevalence of HBsAg and anti-T. pallidum among individuals born in sub-Saharan Africa (or Asia) compared to those born in Europe. Being older (>42 years), toxicomania status, cannabis use and underweight status (compared to normal status) were independent factors associated with HCV seropositivity. Using sequencing, we obtained a substantial diversity of HBV and HCV genotypes. One HCV sequence harbouring a L31M substitution in the NS5a protein may be associated with reduced drug sensitivity. ConclusionsThe positive relationship between toxicomania and HCV suggests the need for effective prevention programmes including health education activities and addiction treatment.

Excessive E2F Transcription in Single Cancer Cells Precludes Transient Cell-Cycle Exit after DNA Damage

E2F transcription factors control the expression of cell-cycle genes. Cancers often demonstrate enhanced E2F target gene expression, which can be explained by increased percentages of replicating cells. However, we demonstrate in human cancer biopsy specimens that individual neoplastic cells display abnormally high levels of E2F-dependent transcription. To mimic this situation, we delete the atypical E2F repressors (E2F7/8) or overexpress the E2F3 activator in untransformed cells. Cells with elevated E2F activity during S/G2 phase fail to exit the cell cycle after DNA damage and undergo mitosis. In contrast, wild-type cells complete S phase and then exit the cell cycle by activating the APC/CCdh1 via repression of the E2F target Emi1. Many arrested wild-type cells eventually inactivate APC/CCdh1 to execute a second round of DNA replication and mitosis, thereby becoming tetraploid. Cells with elevated E2F transcription fail to exit the cell cycle after DNA damage, which potentially causes genomic instability, promotes malignant progression, and reduces drug sensitivity.

Modulation of SIRT3 expression through CDK4/6 enhances the anti-cancer effect of sorafenib in hepatocellular carcinoma cells

BackgroundHepatocellular carcinoma (HCC) is the leading cause of cancer-related deaths worldwide. The only drug currently approved for clinical use in the treatment of advanced HCC is sorafenib. However, many patients with HCC show reduced sensitivity to sorafenib during treatment. SIRT3, a member of the mammalian sirtuin family, is a tumor suppressor in certain tumor types. However, only few studies have investigated the effects of SIRT3 on tumor prognosis and sorafenib sensitivity in patients with HCC. Here, we aimed to investigate the correlation between SIRT3 expression and glucose metabolism and proliferation in HCC and discover effective compounds that increase endogenous SIRT3 modulation effect of sorafenib.MethodsTo determine the correlation between SIRT3 and glucose related proteins, immunostaining was performed with liver cancer tissue using various antibodies. To investigate whether the expression of SIRT3 in HCC is related to the resistance to sorafenib, we treated sorafenib after the modulation of SIRT3 levels in HCC cell lines (overexpression in Huh7, knockdown in HepG2). We also employed PD0332991 to modulate the SIRT3 expression in HCC cell and conducted functional assays.ResultsSIRT3 expression was downregulated in high glycolytic and proliferative HCC cells of human patients, xenograft model and HCC cell lines. Moreover, SIRT3 expression was downregulated after sorafenib treatment, resulting in reduced drug sensitivity in HCC cell lines. To enhance the anti-tumor effect of sorafenib, we employed PD0332991 (CDK4/6-Rb inhibitor) based on the correlation between SIRT3 and phosphorylated retinoblastoma protein in HCC. Notably, combined treatment with sorafenib and PD0332991 showed an enhancement of the anti-tumor effect in HCC cells.ConclusionsOur data suggest that the modulation of SIRT3 by CDK4/6 inhibition might be useful for HCC therapy together with sorafenib, which, unfortunately, has limited efficacy and whose use is often associated with drug resistance.

Genome-Wide CRISPR Screening Identifies MAPK and Mtorc Pathways As Regulators of Sorafenib Resistance in Acute Myeloid Leukemia

Acute myeloid leukemia (AML) is a fast progressing blood malignancy with impaired differentiation and proliferation of myeloid precursors. It is one of the most common leukemias in adults and is known for its molecular and biological heterogeneity, with a variety of genetic lesions implicated in the disease. Among these variants, internal tandem duplication (ITD) or point mutations in the tyrosine kinase domain (TKD) of FLT3 tyrosine kinase are found in around 30% of AML patients. Sorafenib, a multi-kinase inhibitor that targets FLT3, RAF, VEGFR, FGFR, KIT and RET, is approved for use in hepatocarcinoma, renal cell carcinoma, and thyroid carcinoma treatments. Addition of different FLT3 inhibitors such as sorafenib to standard-of-care chemotherapy treatment prolongs AML patient survival with or without FLT3 mutations, although relapse caused by drug resistance remains a clinical challenge. Understanding the mechanisms of resistance to FLT3-targeted drugs, therefore, is necessary to improve treatment options and patient outcomes in AML. We aimed to elucidate resistance mechanisms to sorafenib by subjecting MOLM13 AML cells to genome-wide CRISPR screening to identify genes whose loss-of-function contributes to reduced drug sensitivity. Using Mageck along with an internally developed tiering system for screen hit prioritization, we identified negative regulators of MAPK as well as mTOR pathways as main players in sorafenib resistance. We validated prioritized hit genes using individual sgRNAs to generate single gene deficient cell models for LZTR1, NF1, TSC1 or TSC2. Drug sensitivity assays confirmed an increase in sorafenib resistance in these knockout cells. LZTR1-, TSC1- or TSC2-deficient cells also exhibited reduced sensitivity to a panel of additional FLT3 inhibitors. RNA sequencing results from 271 AML patient peripheral blood or bone marrow samples revealed a correlation between sorafenib sensitivity and lower expression of LZTR1, NF1, TSC1, and TSC2. MOLM13 cell lines resistant to crenolanib, quizartinib, and sorafenib were independently generated by incremental increase in concentration of each drug in cell culture media. Similarly, western blot analysis demonstrated up-regulation of MAPK and/or mTORC1 activity in these resistant cell lines. In addition, these cells were sensitive to MEK inhibitors, and the combination of FLT3 + MEK inhibitors showed synergistic efficacy over single agents in both resistant and parental cells. Taken together, our work identifies the contribution of the MAPK and PI3K/mTOR pathways to FLT3 inhibitor resistance in AML and suggests the combination of FLT3 + MEK inhibitors may be effective for AML patients with FLT3 mutations and those with resistance to FLT3 inhibitors. Disclosures Tyner: Aptose: Research Funding; Array: Research Funding; Agios: Research Funding; Genentech: Research Funding; Janssen: Research Funding; Syros: Research Funding; Janssen: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Array: Research Funding; Constellation: Research Funding; Genentech: Research Funding; Seattle Genetics: Research Funding; Gilead: Research Funding; AstraZeneca: Research Funding; Gilead: Research Funding; Incyte: Research Funding; Takeda: Research Funding; Syros: Research Funding; Aptose: Research Funding; Petra: Research Funding; Seattle Genetics: Research Funding; Petra: Research Funding; Constellation: Research Funding; AstraZeneca: Research Funding; Agios: Research Funding.