Neuronal differentiation drives the antitumor activity of mitogen-activated protein kinase kinase (MEK) inhibition in glioblastoma.

Background: Epidermal growth factor receptor (EGFR) amplification is found in nearly 40%-50% of glioblastoma cases. Several EGFR inhibitors have been tested in glioblastoma but have failed to demonstrate long-term therapeutic benefit, presumably because of acquired resistance. Targeting EGFR downstream signaling with mitogen-activated protein kinase kinase 1 and 2 (MEK1/2) inhibitors would be a more effective approach to glioblastoma treatment. We tested the therapeutic potential of MEK1/2 inhibitors in glioblastoma using 3D cultures of glioma stem-like cells (GSCs) and mouse models of glioblastoma. Methods: Several MEK inhibitors were screened in an unbiased high-throughput platform using GSCs. Cell death was evaluated using flow cytometry and Western blotting (WB) analysis. RNA-seq, real-time quantitative polymerase chain reaction, immunofluorescence, and WB analysis were used to identify and validate neuronal differentiation. Results: Unbiased screening of multiple MEK inhibitors in GSCs showed antiproliferative and apoptotic cell death in sensitive cell lines. An RNA-seq analysis of cells treated with trametinib, a potent MEK inhibitor, revealed upregulation of neurogenesis and neuronal differentiation genes, such as achaete-scute homolog 1 (ASCL1), delta-like 3 (DLL3), and neurogenic differentiation 4 (NeuroD4). We validated the neuronal differentiation phenotypes in vitro and in vivo using selected differentiation markers (ß-III-tubulin, ASCL1, DLL3, and NeuroD4). Oral treatment with trametinib in an orthotopic GSC xenograft model significantly improved animal survival, with 25%-30% of mice being long-term survivors. Conclusions: Our findings demonstrated that MEK1/2 inhibition promotes neuronal differentiation in glioblastoma, a potential additional mechanism of action of MEK1/2 inhibitors. Thus, MEK inhibitors could be efficacious in glioblastoma patients with activated EGFR/MAPK signaling.

Fibroblast-derived PI16 sustains inflammatory pain via regulation of CD206+ myeloid cells.

Originally identified in fibroblasts, Protease Inhibitor (PI)16 was recently shown to be crucial for the development of neuropathic pain via effects on blood-nerve barrier permeability and leukocyte infiltration, though its impact on inflammatory pain has not been established. Using the complete Freund's Adjuvant inflammatory pain model, we show that Pi16-/- mice are protected against sustained inflammatory pain. Accordingly, intrathecal delivery of a PI16 neutralizing antibody in wild-type mice prevented sustained CFA pain. In contrast to neuropathic pain models, we did not observe any changes in blood-nerve barrier permeability due to PI16 deletion. Instead, Pi16-/- mice display reduced macrophage density in the CFA-injected hindpaw. Furthermore, there was a significant bias toward CD206hi (anti-inflammatory) macrophages in the hindpaw and associated dorsal root ganglia. Following CFA, intrathecal depletion of CD206+ macrophages using mannosylated clodronate liposomes promoted sustained pain in Pi16-/- mice. Similarly, an IL-10 neutralizing antibody also promoted sustained CFA pain in the Pi16-/ when administered intrathecally. Collectively, our results point to fibroblast-derived PI16 mediating substantial differences in macrophage phenotype in the pain neuroaxis under conditions of inflammation. The co-expression of PI16 alongside fibroblast markers in human DRG raise the likelihood that a similar mechanism operates in human inflammatory pain states. Collectively, our findings may have implications for targeting fibroblast-immune cell crosstalk for the treatment of chronic pain.

Sensory neuron dysfunction in orthotopic mouse models of colon cancer.

Reports of neurological sequelae related to colon cancer are largely restricted to rare instances of paraneoplastic syndromes, due to autoimmune reactions. Systemic inflammation associated with tumor development influences sensory neuron function in other disease models, though the extent to which this occurs in colorectal cancer is unknown. We induced orthotopic colorectal cancer via orthotopic injection of two colorectal cancer cell lines (MC38 and CT26) in two different mouse strains (C57BL/6 and Balb/c, respectively). Behavioral tests of pain sensitivity and activity did not detect significant alterations in sensory sensitivity or diminished well-being throughout tumor development. However, immunohistochemistry revealed widespread reductions in intraepidermal nerve fiber density in the skin of tumor-bearing mice. Though loss of nerve fiber density was not associated with increased expression of cell injury markers in dorsal root ganglia, lumbar dorsal root ganglia neurons of tumor-bearing animals showed deficits in mitochondrial function. These neurons also had reduced cytosolic calcium levels in live-cell imaging and reduced spontaneous activity in multi-electrode array analysis. Bulk RNA sequencing of DRGs from tumor-bearing mice detected activation of gene expression pathways associated with elevated cytokine and chemokine signaling, including CXCL10. This is consistent with the detection of CXCL10 (and numerous other cytokines, chemokines and growth factors) in MC38 and CT26 cell-conditioned media, and the serum of tumor-bearing mice. Our study demonstrates in a pre-clinical setting that colon cancer is associated with latent sensory neuron dysfunction and implicates cytokine/chemokine signaling in this process. These findings may have implications for determining risk factors and treatment responsiveness related to neuropathy in colorectal cancer.

Targeting the Meningeal Compartment to Resolve Chemobrain and Neuropathy via Nasal Delivery of Functionalized Mitochondria.

Cognitive deficits (chemobrain) and peripheral neuropathy occur in ∼75% of patients treated for cancer with chemotherapy and persist long-term in >30% of survivors. Without preventive or curative interventions and with increasing survivorship rates, the population debilitated by these neurotoxicities is rising. Platinum-based chemotherapeutics, including cisplatin, induce neuronal mitochondrial defects leading to chemobrain and neuropathic pain. This study investigates the capacity of nasally administered mesenchymal stem cell-derived mitochondria coated with dextran-triphenylphosphonium polymer (coated mitochondria) to reverse these neurotoxicities. Nasally administered coated mitochondria are rapidly detectable in macrophages in the brain meninges but do not reach the brain parenchyma. The coated mitochondria change expression of >2400 genes regulating immune, neuronal, endocrine and vascular pathways in the meninges of mice treated with cisplatin. Nasal administration of coated mitochondria reverses cisplatin-induced cognitive deficits and resolves neuropathic pain at a >55-times lower dose compared to uncoated mitochondria. Reversal of these neuropathologies is associated with resolution of cisplatin-induced deficits in myelination, synaptosomal mitochondrial integrity and neurogenesis. These findings demonstrate that nasally administered coated mitochondria promote resolution of chemobrain and peripheral neuropathy, thereby identifying a novel facile strategy for clinical application of mitochondrial donation and treating central and peripheral nervous system pathologies by targeting the brain meninges.

Targeting the A3 adenosine receptor to prevent and reverse chemotherapy-induced neurotoxicities in mice.

Cisplatin is used to combat solid tumors. However, patients treated with cisplatin often develop cognitive impairments, sensorimotor deficits, and peripheral neuropathy. There is no FDA-approved treatment for these neurotoxicities. We investigated the capacity of a highly selective A3 adenosine receptor (AR) subtype (A3AR) agonist, MRS5980, to prevent and reverse cisplatin-induced neurotoxicities. MRS5980 prevented cisplatin-induced cognitive impairment (decreased executive function and impaired spatial and working memory), sensorimotor deficits, and neuropathic pain (mechanical allodynia and spontaneous pain) in both sexes. At the structural level, MRS5980 prevented the cisplatin-induced reduction in markers of synaptic integrity. In-situ hybridization detected Adora3 mRNA in neurons, microglia, astrocytes and oligodendrocytes. RNAseq analysis identified 164 genes, including genes related to mitochondrial function, of which expression was changed by cisplatin and normalized by MRS5980. Consistently, MRS5980 prevented cisplatin-induced mitochondrial dysfunction and decreased signs of oxidative stress. Transcriptomic analysis showed that the A3AR agonist upregulates genes related to repair pathways including NOTCH1 signaling and chromatin modification in the cortex of cisplatin-treated mice. Importantly, A3AR agonist administration after completion of cisplatin treatment resolved cognitive impairment, neuropathy and sensorimotor deficits. Our results highlight the efficacy of a selective A3AR agonist to prevent and reverse cisplatin-induced neurotoxicities via preventing brain mitochondrial damage and activating repair pathways. An A3AR agonist is already in cancer, clinical trials and our results demonstrate management of neurotoxic side effects of chemotherapy as an additional therapeutic benefit.

HDAC6 inhibition reverses long-term doxorubicin-induced cognitive dysfunction by restoring microglia homeostasis and synaptic integrity.

Breast cancer is the most common female malignancy in both the developed and developing world. Doxorubicin is one of the most commonly used chemotherapies for breast cancer. Unfortunately, up to 60% of survivors report long-term chemotherapy-induced cognitive dysfunction (CICD) characterized by deficits in working memory, processing speed and executive function. Currently, no therapeutic standard for treating CICD exists. Here, we hypothesized that treatment with a blood-brain barrier permeable histone deacetylase 6 (HDAC6) inhibitor can successfully reverse long-term doxorubicin-induced cognitive dysfunction. Methods: The puzzle box test and novel object/place recognition test were used to assess cognitive function following a therapeutic doxorubicin dosing schedule in female mice. Mitochondrial function and morphology in neuronal synaptosomes were evaluated using the Seahorse XF24 extracellular flux analyzer and transmission electron microscopy, respectively. Hippocampal postsynaptic integrity was evaluated using immunofluorescence. Hippocampal microglia phenotype was determined using advanced imaging techniques and single-nucleus RNA sequencing. Results: A 14-day treatment with a blood-brain barrier permeable HDAC6 inhibitor successfully reversed long-term CICD in the domains of executive function, working and spatial memory. No significant changes in mitochondrial function or morphology in neuronal synaptosomes were detected. Long-term CICD was associated with a decreased expression of postsynaptic PSD95 in the hippocampus. These changes were associated with decreased microglial ramification and alterations in the microglia transcriptome that suggest a stage 1 disease-associated microglia (DAM) phenotype. HDAC6 inhibition completely reversed these doxorubicin-induced alterations, indicating a restoration of microglial homeostasis. Conclusion: Our results show that decreased postsynaptic integrity and a neurodegenerative microglia phenotype closely resembling stage 1 DAM microglia contribute to long-term CICD. Moreover, HDAC6 inhibition shows promise as an efficacious pharmaceutical intervention to alleviate CICD and improve quality of life of breast cancer survivors.

Emerging roles of alternative cleavage and polyadenylation (APA) in human disease.

In the messenger RNA (mRNA) maturation process, the 3'-end of pre-mRNA is cleaved and a poly(A) sequence is added, this is an important determinant of mRNA stability and its cellular functions. More than 60%-70% of human genes have three or more polyadenylation (APA) sites and can be cleaved at different sites, generating mRNA transcripts of varying lengths. This phenomenon is termed as alternative cleavage and polyadenylation (APA) and it plays role in key biological processes like gene regulation, cell proliferation, senescence, and also in various human diseases. Loss of regulatory microRNA binding sites and interactions with RNA-binding proteins leading to APA are largely investigated in human diseases. However, the functions of the core APA machinery and related factors during disease conditions remain largely unknown. In this review, we discuss the roles of polyadenylation machinery in relation to brain disease, cardiac failure, pulmonary fibrosis, cancer, infectious conditions, and other human diseases. Collectively, we believe this review will be a useful avenue for understanding the emerging role of APA in the pathobiology of various human diseases.

Intrinsic Interferon Signaling Regulates the Cell Death and Mesenchymal Phenotype of Glioblastoma Stem Cells.

Interferon (IFN) signaling contributes to stemness, cell proliferation, cell death, and cytokine signaling in cancer and immune cells; however, the role of IFN signaling in glioblastoma (GBM) and GBM stem-like cells (GSCs) is unclear. Here, we investigated the role of cancer-cell-intrinsic IFN signaling in tumorigenesis in GBM. We report here that GSCs and GBM tumors exhibited differential cell-intrinsic type I and type II IFN signaling, and high IFN/STAT1 signaling was associated with mesenchymal phenotype and poor survival outcomes. In addition, chronic inhibition of IFN/STAT1 signaling decreased cell proliferation and mesenchymal signatures in GSCs with intrinsically high IFN/STAT1 signaling. IFN-ß exposure induced apoptosis in GSCs with intrinsically high IFN/STAT1 signaling, and this effect was abolished by the pharmacological inhibitor ruxolitinib and STAT1 knockdown. We provide evidence for targeting IFN signaling in a specific sub-group of GBM patients. IFN-ß may be a promising candidate for adjuvant GBM therapy.

Bexarotene normalizes chemotherapy-induced myelin decompaction and reverses cognitive and sensorimotor deficits in mice.

Frequently reported neurotoxic sequelae of cancer treatment include cognitive deficits and sensorimotor abnormalities that have long-lasting negative effects on the quality of life of an increasing number of cancer survivors. The underlying mechanisms are not fully understood and there is no effective treatment. We show here that cisplatin treatment of mice not only caused cognitive dysfunction but also impaired sensorimotor function. These functional deficits are associated with reduced myelin density and complexity in the cingulate and sensorimotor cortex. At the ultrastructural level, myelin abnormalities were characterized by decompaction. We used this model to examine the effect of bexarotene, an agonist of the RXR-family of nuclear receptors. Administration of only five daily doses of bexarotene after completion of cisplatin treatment was sufficient to normalize myelin density and fiber coherency and to restore myelin compaction in cingulate and sensorimotor cortex. Functionally, bexarotene normalized performance of cisplatin-treated mice in tests for cognitive and sensorimotor function. RNAseq analysis identified the TR/RXR pathway as one of the top canonical pathways activated by administration of bexarotene to cisplatin-treated mice. Bexarotene also activated neuregulin and netrin pathways that are implicated in myelin formation/maintenance, synaptic function and axonal guidance. In conclusion, short term treatment with bexarotene is sufficient to reverse the adverse effects of cisplatin on white matter structure, cognitive function, and sensorimotor performance. These encouraging findings warrant further studies into potential clinical translation and the underlying mechanisms of bexarotene for chemobrain.

Regenerative Metaplastic Clones in COPD Lung Drive Inflammation and Fibrosis.

Chronic obstructive pulmonary disease (COPD) is a progressive condition of chronic bronchitis, small airway obstruction, and emphysema that represents a leading cause of death worldwide. While inflammation, fibrosis, mucus hypersecretion, and metaplastic epithelial lesions are hallmarks of this disease, their origins and dependent relationships remain unclear. Here we apply single-cell cloning technologies to lung tissue of patients with and without COPD. Unlike control lungs, which were dominated by normal distal airway progenitor cells, COPD lungs were inundated by three variant progenitors epigenetically committed to distinct metaplastic lesions. When transplanted to immunodeficient mice, these variant clones induced pathology akin to the mucous and squamous metaplasia, neutrophilic inflammation, and fibrosis seen in COPD. Remarkably, similar variants pre-exist as minor constituents of control and fetal lung and conceivably act in normal processes of immune surveillance. However, these same variants likely catalyze the pathologic and progressive features of COPD when expanded to high numbers.

Cloning of ground-state intestinal stem cells from endoscopic biopsy samples.

'Adult' or 'somatic' stem cells harbor an intrinsic ability to regenerate tissues. Heterogeneity of such stem cells along the gastrointestinal tract yields the known segmental specificity of this organ and may contribute to the pathology of certain enteric conditions. Here we detail technology for the generation of 'libraries' of clonogenic cells from 1-mm-diamter endoscopic biopsy samples from the human gastrointestinal tract. Each of the 150-300 independent clones in a typical stem cell library can be clonally expanded to billions of cells in a few weeks while maintaining genomic stability and the ability to undergo multipotent differentiation to the specific epithelia from which the sample originated. The key to this methodology is the intrinsic immortality of normal intestinal stem cells (ISCs) and culture systems that maintain them as highly immature, ground-state ISCs marked by a single-cell clonogenicity of 70% and a corresponding 250-fold proliferative advantage over spheroid technologies. Clonal approaches such as this enhance the resolution of molecular genetics, make genome editing easier, and may be useful in regenerative medicine, unravelling heterogeneity in disease, and facilitating drug discovery.

Unlimited expansion of intestinal stem cells from a wide range of ages.

The recent technical advance in cloning and culturing ground-state intestinal stem cells (ISC) provides us an opportunity of accurate assessment of age-related impact on the function of highly proliferative intestinal stem cells. Our ability of indefinitely and robustly expanding single-stem-cell derived pedigrees in vitro allows us to study intestinal stem cells at the clonal level. Interestingly, comparable number of ISC clones was yielded from 1mm endoscopic biopsy of all donors despite the age. They were passaged in vitro as pedigrees and expanded to 1 billion cells in approximately sixty days without changes in stemness demonstrated by clonogenicity and multipotency. Therefore, our study shows that ISCs from a wide range of ages can be cloned and expanded to unlimited number in vitro with similar efficiency and stability. These patient-derived ISCs harbor intrinsic immortality and are ideal for autologous transplantation, supporting the promise of adult-stem-cell based personalized medicine.

Ground-State Stem Cells: A Novel Approach for Adult Stem Cell Research.

A robust and reliable culture system of adult stem cells is essential for applying the cutting-edge technologies of drug screening, gene editing, and genomics to stem cell research necessary for breakthroughs in this field. In addition, personalized regenerative medicine based on autologous transplantation requires our ability to clone and expand the numbers of these stem cells in vitro. In comparison to the 3D "organoid" culture system that shows limited ability to propagate stem cells as the majority of cells are differentiated or transit amplifying cells, ground-state stem cell culture system is a novel technology that permits long-lived adult stem cells to maintain immaturity, self-renewal capacity, multi-potency and genomic stability despite long-term culturing in a 2D system. The robustness, reliability and easy-to-use features of this new technology bypass the deficiencies of 3D organoid culture systems and provided unlimited stem cell sources for research, therapeutic use, and drug discovery.