Type II mode of JAK2 inhibition and destabilization are potential therapeutic approaches against the ruxolitinib resistance driven myeloproliferative neoplasms.

Background: Ruxolitinib has been approved by the US FDA for the treatment of myeloproliferative neoplasms such as polycythemia vera and primary myelofibrosis. Ruxolitinib will remain a main stay in the treatment of MPN patients due to its effective therapeutic benefits. However, there have been instances of ruxolitinib resistance in MPN patients. As JAK2 is a direct target of ruxolitinib, we generated ruxolitinib-resistant clones to find out the mechanism of resistance. Methods: Cell-based screening strategy was used to detect the ruxolitinib-resistant mutations in JAK2. The Sanger sequencing method was used to detect the point mutations in JAK2. Mutations were re-introduced using the site-directed mutagenesis method and stably expressed in Ba/F3 cells. Drug sensitivities against the JAK2 inhibitors were measured using an MTS-based assay. JAK2 and STAT5 activation levels and total proteins were measured using immunoblotting. Computational docking studies were performed using the Glide module of Schrodinger Maestro software. Results: In this study, we have recovered seven residues in the kinase domain of JAK2 that affect ruxolitinib sensitivity. All these mutations confer cross-resistance across the panel of JAK2 kinase inhibitors except JAK2-L983F. JAK2-L983F reduces the sensitivity towards ruxolitinib. However, it is sensitive towards fedratinib indicating that our screen identifies the drug-specific resistance profiles. All the ruxolitinib-resistant JAK2 variants displayed sensitivity towards type II JAK2 inhibitor CHZ-868. In this study, we also found that JAK1-L1010F (homologous JAK2-L983F) is highly resistant towards ruxolitinib suggesting the possibility of JAK1 escape mutations in JAK2-driven MPNs and JAK1 mutated ALL. Finally, our study also shows that HSP90 inhibitors are potent against ruxolitinib-resistant variants through the JAK2 degradation and provides the rationale for clinical evaluation of potent HSP90 inhibitors in genetic resistance driven by JAK2 inhibitors. Conclusion: Our study identifies JAK1 and JAK2 resistance variants against the type I JAK2 inhibitors ruxolitinib, fedratinib, and lestaurtinib. The sensitivity of these resistant variants towards the type II JAK2 inhibitor CHZ-868 indicates that this mode of type II JAK2 inhibition is a potential therapeutic approach against ruxolitinib refractory leukemia. This also proposes the development of potent and specific type II JAK2 inhibitors using ruxolitinib-resistance variants as a prototype.

Generation of induced pluripotent stem cell line NIMHi010-A from dermal fibroblast cells of a healthy individual.

In this study, we have established human induced pluripotent stem cell (hiPSC) line, NIMHi010-A of a 42-year-old healthy donor. The iPSC line was generated from human dermal fibroblasts using Sendai viruses carrying reprogramming factors c-MYC, SOX2, KLF4, and OCT4 under a feeder-free culture system. The generated hiPSC line expressed typical pluripotency markers, displayed a normal karyotype, and demonstrated the potential to differentiate into the three germ layers. This hiPSC line will serve as a healthy control model for physiological processes and drug screening of Asian origin from Indian population.

Targeting cancer-derived extracellular vesicles by combining CD147 inhibition with tissue factor pathway inhibitor for the management of urothelial cancer cells.

BACKGROUND: Extracellular vesicles (EVs), including microvesicles, hold promise for the management of bladder urothelial carcinoma (BLCA), particularly because of their utility in identifying therapeutic targets and their diagnostic potential using easily accessible urine samples. Among the transmembrane glycoproteins highly enriched in cancer-derived EVs, tissue factor (TF) and CD147 have been implicated in promoting tumor progression. In this in vitro study, we explored a novel approach to impede cancer cell migration and metastasis by simultaneously targeting these molecules on urothelial cancer-derived EVs. METHODS: Cell culture supernatants from invasive and non-invasive bladder cancer cell lines and urine samples from patients with BLCA were collected. Large, microvesicle-like EVs were isolated using sequential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and flow cytometry. The impact of urinary or cell supernatant-derived EVs on cellular phenotypes was evaluated using cell-based assays following combined treatment with a specific CD147 inhibitor alone or in combination with a tissue factor pathway inhibitor (TFPI), an endogenous anticoagulant protein that can be released by low-molecular-weight heparins. RESULTS: We observed that EVs obtained from the urine samples of patients with muscle-invasive BLCA and from the aggressive bladder cancer cell line J82 exhibited higher TF activity and CD147 expression levels than did their non-invasive counterparts. The shedding of GFP-tagged CD147 into isolated vesicles demonstrated that the vesicles originated from plasma cell membranes. EVs originating from invasive cancer cells were found to trigger migration, secretion of matrix metalloproteinases (MMPs), and invasion. The same induction of MMP activity was replicated using EVs obtained from urine samples of patients with invasive BLCA. EVs derived from cancer cell clones overexpressing TF and CD147 were produced in higher quantities and exhibited a higher invasive potential than those from control cancer cells. TFPI interfered with the effect when used in conjunction with the CD147 inhibitor, further suppressing homotypic EV-induced migration, MMP production, and invasion. CONCLUSIONS: Our findings suggest that combining a CD147 inhibitor with low molecular weight heparins to induce TFPI release may be a promising therapeutic approach for urothelial cancer management. This combination can potentially suppress the tumor-promoting actions of cancer-derived microvesicle-like EVs, including collective matrix invasion.

Small particles or vesicles released by cancer cells into their surroundings have the potential to stimulate the spread and growth of cancer cells. In this study, we focused on two specific molecules presented by these cancer cell-derived vesicles that could play a role in promoting the dissemination of cancer cells: a protein related to blood clotting and a protein on the cell surface.We found that large vesicles from bladder cancer cells that have the ability to spread had higher levels of these proteins than vesicles from nonspreading cancer cells. We also found that the former could make cancer cells move about more, produce more of a substance that helps cancer cells spread, and invade other tissues.To counteract the cancer-promoting actions of these vesicles, we examined the impact of combining a naturally occurring anticlotting protein that can be released by medications derived from heparin with an inhibitor targeting the cancer cell surface protein. We found that this combination stopped the vesicles from helping cancer cells move about more, produce more of the spreading substance, and invade other tissues.This approach of simultaneously targeting the two protein molecules present on cancer cell-derived vesicles might be a new way to treat bladder cancer.

How does "locus of control" affect persons with epilepsy?

PURPOSE: Locus of control (LOC) is the degree to which people believe that they have control over the outcome of events in their lives. A person's locus can be internal, external, or chance. A person with internal locus of control believes that one can control one's own life. A person with external locus of control believes that his life is controlled by external factors or people over which he has no influence. A person with chance locus of control believes that fate, chance, or luck controls his own life. The aim of the current study was to determine the health locus of control, anxiety, and depression levels in persons with epilepsy (PWE) and to assess whether locus of control has relation to anxiety, depression, and seizure control. METHODS: Patients aged 18 years or older with a history of epilepsy for at least 1 year were recruited from the outpatient epilepsy clinic or from the inpatient epilepsy monitoring unit at SCTIMST, Trivandrum from January 2019 to May 2020. Patients filled the questionnaire form consisting of demographic data, age of onset of seizures, present seizure control, and the current antiepileptic drugs. The Hospital Anxiety and Depression (HAD) scale was used to estimate the level of anxiety and depression in these patients. The Form-C of the Multidimensional Health Locus of Control (MHLC) scale was used to evaluate the health locus of control. Healthy controls aged 18 years or older and free of any chronic disease or psychiatric illness were also recruited. They were asked to fill the questionnaire forms with basic demographic data. HAD scale was used to estimate the level of anxiety and depression and form-C of MHLC was used to evaluate the health locus of control in the healthy controls. The mean scores of anxiety, depression, and locus of control were compared between the two groups. RESULTS: A total of 170 participants were recruited which consisted of 100 PWE and 70 healthy controls. The mean anxiety and depression scores were 8.13(SD = 4.23) and 5.85(SD = 3.66) in the PWE group and 6.75(SD = 3.39) and 4.14(SD = 2.96) in the control group, respectively. The mean internal, external, and chance LOC scores were 24.95(SD = 10.92), 26.94(SD = 4.96), and 24.41(SD = 6.46) in the PWE group; and 29.44(SD = 5.62), 26.53(SD = 5.79), and 19.9(SD = 7.13) in the control group, respectively. Persons with epilepsy had higher chance LOC scores and lower internal LOC scores compared to controls (p = 0.00003, p < 0.00001 respectively). There were no differences in the external LOC scores between the two groups (p = 0.620). Persons with epilepsy with some level of anxiety had lower internal LOC scores compared to patients with no anxiety (p = 0.04). PWE with poor seizure control had higher external LOC score and lower internal LOC scores which however did not reach statistical significance. Persons with epilepsy with poor seizure control had higher anxiety and depression scores. CONCLUSIONS: Persons with epilepsy had low perceptions of internal and strong perceptions of chance health locus of control. This means that PWE feel that luck plays an important role in their disease control. This information is important in the counseling of persons with epilepsy.