Functional impact and targetability of PI3KCA, GNAS, and PTEN mutations in a spindle cell rhabdomyosarcoma with MYOD1 L122R mutation

Spindle cell/sclerosing rhabdomyosarcoma (ssRMS) is a rare subtype of rhabdomyosarcoma, commonly harboring a gain-of-function L122R mutation in the muscle-specific master transcription factor MYOD1. MYOD1-mutated ssRMS is almost invariably fatal, and development of novel therapeutic approaches based on the biology of the disease is urgently needed. MYOD1 L122R affects the DNA-binding domain and is believed to confer MYC-like properties to MYOD1, driving oncogenesis. Moreover, the majority of the MYOD1-mutated ssRMS harbor additional alterations activating the PI3K/AKT pathway. It is postulated that the PI3K/AKT pathway cooperates with MYOD1 L122R. To address this biological entity, we established and characterized a new patient-derived ssRMS cell line OHSU-SARC001, harboring MYOD1 L122R as well as alterations in PTEN, PIK3CA, and GNAS. We explored the functional impact of these aberrations on oncogenic signaling with gain-of-function experiments in C2C12 murine muscle lineage cells. These data reveal that PIK3CAI459_T462del, the novel PIK3CA variant discovered in this patient specimen, is a constitutively active kinase, albeit to a lesser extent than PI3KCAE545K, a hotspot oncogenic mutation. Furthermore, we examined the effectiveness of molecularly targeted PI3K/AKT/mTOR and RAS/MAPK inhibitors to block oncogenic signaling and suppress the growth of OHSU-SARC001 cells. Dual PI3K/mTOR (LY3023414, bimiralisib) and AKT inhibitors (ipatasertib, afuresertib) induced dose-dependent reductions in cell growth. However, mTOR-selective inhibitors (everolimus, rapamycin) alone did not exert cytotoxic effects. The MEK1/2 inhibitor trametinib did not impact proliferation even at the highest doses tested. Our data suggest that molecularly targeted strategies may be effective in PI3K/AKT/mTOR-activated ssRMS. Taken together, these data highlight the importance of utilizing patient-derived models to assess molecularly targetable treatments and their potential as future treatment options.

AP-1 transcription factor network explains diverse patterns of cellular plasticity in melanoma

SummaryCellular plasticity associated with fluctuations in transcriptional programs allows individual cells in a tumor to adopt heterogeneous differentiation states and switch phenotype during their adaptive responses to therapies. Despite increasing knowledge of such transcriptional programs, the molecular basis of cellular plasticity remains poorly understood. Here, we combine multiplexed transcriptional and protein measurements at population and single-cell levels with multivariate statistical modeling to show that the state of AP-1 transcription factor network plays a unifying role in explaining cellular plasticity in melanoma. We find that a tightly regulated balance between AP-1 factors cJUN, FRA2, FRA1 and cFOS determines the intrinsic diversity of differentiation states and adaptive responses to MAPK inhibitors in individual melanoma cells. Perturbing this balance through genetic depletion of specific AP-1 proteins, or by MAPK inhibitors, shifts cellular heterogeneity in a predictable fashion. Thus, AP-1 may serve as a critical node for manipulating cellular plasticity with potential therapeutic implications.

IL-18 Mediates Vascular Calcification Induced by High-Fat Diet in Rats With Chronic Renal Failure

Objective: Vascular calcification (VC) is an important predictor of cardiovascular morbidity and mortality in patients with chronic renal failure (CRF). It is well-known that obesity and metabolic syndrome (OB/MS) predicts poor prognosis of CRF patients. However, the influence of OB/MS on VC in CRF patients isn't clear. IL-18 mediates OB/MS-related inflammation, but whether IL-18 is involved in OB/MS -mediated VC in CRF patients hasn't been studied. In this study, it was explored that whether OB/MS caused by high-fat diet (HFD) can affect the level of serum IL-18 and aggravate the degree of VC in CRF rats. Furthermore, it was studied that whether IL-18 induces rat vascular smooth muscle cells (VSMCs) calcification by activating the MAPK pathways.Approach: The rats were randomly assigned to the sham-operated, CRF and CRF + HFD groups. CRF was induced by 5/6 nephrectomy. Serum IL-18 levels and aortic calcification indicators were compared in each group. Primary rat VSMCs calcification were induced by β-glycerophosphate and exposed to IL-18. VSMCs were also treated with MAPK inhibitors.Results: The weight, serum levels of hsCRP, TG and LDL-C in CRF + HFD group were significantly higher than those in sham-operated and CRF groups (p < 0.05). Compared with the sham-operated group, the calcium content and the expression of BMP-2 of aorta in CRF and CRF + HFD groups were significantly increased (p < 0.05). Moreover, the calcium content and the expression of BMP-2 of aorta in CRF + HFD group was significantly higher than those in CRF group (p < 0.05). And the serum IL-18 level was positively correlated with aortic calcium content. It was also found that p38 inhibitor SB203580 can suppress the VSMCs calcification and osteoblast phenotype differentiation induced by IL-18. But the JNK inhibitor SP600125 can't suppress the VSMCs calcification and osteoblast phenotype differentiation induced by IL-18.Conclusions: These findings suggest that obesity-related inflammation induced by high-fat diet could exacerbate VC in CRF rats. Furthermore, serum IL-18 level had a positive correlation with the degree of VC. It is also found that IL-18 promoted osteogenic differentiation and calcification of rat VSMCs via p38 pathway activation.

MicroRNA-15a-5p acts as a tumor suppressor in histiocytosis by mediating CXCL10-ERK-LIN28a-let-7 axis

Erdheim–Chester disease (ECD) is characterized by excessive production and accumulation of histiocytes within multiple tissues and organs. ECD patients harbor recurrent mutations of genes associated with the RAS/RAF/MEK/ERK signaling pathway, particularly, the BRAFV600E mutation. Following our previous finding that miR-15a-5p is the most prominently downregulated microRNA in ECD patients compared to healthy individuals, we elucidated its role in ECD pathogenesis. Bioinformatics analysis followed by a luciferase assay showed that chemokine ligand 10 (CXCL10) is a target gene regulated by miRNA-15a-5p. This was confirmed in 24/34 ECD patients that had low expression of miR-15a-5p concurrent with upregulated CXCL10. Overexpression of miR-15a-5p in cell lines harboring BRAF or RAS mutations (Ba/F3, KG-1a and OCI-AML3) resulted in CXCL10 downregulation, followed by LIN28a and p-ERK signaling downregulation and let-7 family upregulation. Overexpression of miR-15a-5p inhibited cell growth and induced apoptosis by decreasing Bcl-2 and Bcl-xl levels. Analysis of sequential samples from 7 ECD patients treated with MAPK inhibitors (vemurafenib/cobimetinib) for 4 months showed miR-15a-5p upregulation and CXCL10 downregulation. Our findings suggest that miR-15a-5p is a tumor suppressor in ECD through the CXCL10-ERK-LIN28a-let7 axis, highlighting another layer of post-transcriptional regulation in this disease. Upregulation of miR-15a-5p in ECD patients may have a potential therapeutic role.

7-Epitaxol Induces Apoptosis and Autophagy in Head and Neck Squamous Cell Carcinoma through Inhibition of the ERK Pathway

As the main derivative of paclitaxel, 7-Epitaxol is known to a have higher stability and cytotoxicity. However, the anticancer effect of 7-Epitaxol is still unclear. The purpose of this study was to explore the anticancer effects of 7-Epitaxol in squamous cell carcinoma of the head and neck (HNSCC). Our study findings revealed that 7-Epitaxol potently suppressed cell viability in SCC-9 and SCC-47 cells by inducing cell cycle arrest. Flow cytometry and DAPI staining demonstrated that 7-Epitaxol treatment induced cell death, mitochondrial membrane potential and chromatin condensation in OSCC cell lines. The compound regulated the proteins of extrinsic and intrinsic pathways at the highest concentration, and also increased the activation of caspases 3, 8, 9, and PARP in OSCC cell lines. Interestingly, a 7-Epitaxol-mediated induction of LC3-I/II expression and suppression of p62 expression were observed in OSCC cells lines. Furthermore, the MAPK inhibitors indicated that 7-Epitaxol induces apoptosis and autophagy marker proteins (cleaved-PARP and LC3-I/II) by reducing the phosphorylation of ERK1/2. In conclusion, these findings indicate the involvement of 7-Epitaxol in inducing apoptosis and autophagy through ERK1/2 signaling pathway, which identify 7-Epitaxol as a potent cytotoxic agent in HNSCC.

Inflammatory cytokines TNFα, IL-1β, and IL-6 are induced in endotoxin- stimulated microglia through different signaling cascades

By using an animal model in which inflammatory cytokines are induced in lipopolysaccharide (LPS)-injected rat brain, we investigated the induction of tumor necrosis factor alpha (TNFα), interleukin-1beta (IL-1β), and IL-6. Immunoblotting and immunohistochemistry revealed that all three cytokines were transiently induced in the cerebral cortex at about 12 h after LPS injection. To clarify which glial cell type induced the cytokines, we examined the respective abilities of astrocytes and microglia in vitro. Primary microglia largely induced TNFα, IL-1β and IL-6 in response to LPS, but primary astrocytes induced only limited levels of TNFα. Thus, we used specific inhibitors to focus on microglia in surveying signaling molecules involved in the induction of TNFα, IL-1β, and IL-6. The experiments using mitogen-activated protein kinases (MAPK) inhibitors revealed that c-Jun N-terminal kinase (JNK)/p38, external signal regulated kinase (ERK)/JNK, and ERK/JNK/p38 are necessary for the induction of TNFα, IL-1β, and IL-6, respectively. The experiments using protein kinase C (PKC) inhibitor clarified that PKCα is required for the induction of all these cytokines in LPS-stimulated microglia. Furthermore, LPS-dependent IL-1β/IL-6 induction was suppressed by pretreatment with a nitric oxide (NO) scavenger, suggesting that NO is involved in the signaling cascade of IL-1β/IL-6 induction. Thus, an inducible NO synthase induced in the LPS-injected cerebral cortex might be related to the induction of IL-1β/IL-6 through the production of NO in vivo. Taken together, these results demonstrated that microglia induce different kinds of inflammatory cytokine through specific combinations of MAPKs and by the presence or absence of NO.

Immunomodulatory Properties of BRAF and MEK Inhibitors Used for Melanoma Therapy—Paradoxical ERK Activation and Beyond

The advent of mitogen–activated protein kinase (MAPK) inhibitors that directly inhibit tumor growth and of immune checkpoint inhibitors (ICI) that boost effector T cell responses have strongly improved the treatment of metastatic melanoma. In about half of all melanoma patients, tumor growth is driven by gain–of–function mutations of BRAF (v–rat fibrosarcoma (Raf) murine sarcoma viral oncogene homolog B), which results in constitutive ERK activation. Patients with a BRAF mutation are regularly treated with a combination of BRAF and MEK (MAPK/ERK kinase) inhibitors. Next to the antiproliferative effects of BRAF/MEKi, accumulating preclinical evidence suggests that BRAF/MEKi exert immunomodulatory functions such as paradoxical ERK activation as well as additional effects in non–tumor cells. In this review, we present the current knowledge on the immunomodulatory functions of BRAF/MEKi as well as the non–intended effects of ICI and discuss the potential synergistic effects of ICI and MAPK inhibitors in melanoma treatment.

Lymphatic Abnormalities in Noonan Syndrome Spectrum Disorders: A Systematic Review

Noonan syndrome spectrum disorders are a group of phenotypically related conditions, resembling Noonan syndrome, caused by germline pathogenic variants in genes within the Ras/mitogen-activated protein kinase (Ras/MAPK) signalling pathway. Lymphatic dysplasia with a clinical lymphatic abnormality is one of the major features. We performed a systematic review to get more insight in (1) the prevalence of clinically lymphatic abnormalities in patients with a genetically proven Noonan syndrome spectrum disorder, (2) if a genotype-lymphatic phenotype relation can be found and describe the clinical presentation and course of the lymphatic abnormality. Most studies report patients with Noonan syndrome. Prenatally, the prevalence of increased nuchal translucency differs from 7% in patients with pathogenic <i>PTPN11</i> variant<i>s</i> to 38% in patients with pathogenic <i>RIT1</i> variants, and the prevalence of pleural effusions differed from 7% in patients with pathogenic <i>SOS1</i> to 29% in patients with pathogenic <i>RIT1</i> variants. Postnatally, the prevalence of lymphedema differs from 16% in patients with pathogenic <i>PTPN11</i> variants to 44% in patients with pathogenic <i>SOS1</i> variants, and the prevalence of acquired chylothorax is 4% in patients with pathogenic <i>RIT1</i> variants. Lymphatic abnormalities do occur in patients with cardiofaciocutaneous syndrome and Costello syndrome. In conclusion, Noonan syndrome spectrum disorders, Noonan syndrome in particular, are associated with lymphatic abnormalities. Combining the available published literature about genetically proven Noonan syndrome spectrum disorders, it appears likely that the lifetime prevalence of these abnormalities in Noonan syndrome is higher than the 20% that were generally accepted so far. This is increasingly important, because the activation of the RAS/MAPK pathway can be inhibited by RAS/MAPK inhibitors, and clinically severe lymphatic abnormalities may improve.

miR-143-3p Inhibits the Differentiation of Osteoclast Induced by Synovial Fibroblast and Monocyte Coculture in Adjuvant-Induced Arthritic Rats

Osteoclast, which mediates overactive bone resorption, is one of the key factors for bone destruction in rheumatoid arthritis (RA). Existing studies have shown that abnormal miR-143-3p expression was observed in both RA patients and arthritis animals, which can participate in osteoclast differentiation, and mitogen-activated protein kinase (MAPK) signaling pathway was closely related to osteoclast differentiation. The primary objective of the current study was to determine the role of miR-143-3p in the progression of osteoclast differentiation and its relationship with MAPK signaling pathways. The results showed that miR-143-3p inhibited osteoclast differentiation and decreased the levels of M-CSF and RANKL during osteoclast differentiation. miR-143-3p inhibited the expression of MAPK signaling proteins, which is ERK1/2 in the early stage and JNK in the later stage of osteoclast differentiation. It was also observed that MAPK inhibitors upregulated miR-143-3p expression in osteoclast differentiation. Taken together, our results suggested that miR-143-3p could inhibit the differentiation of osteoclast, which was related to inhibiting MAPK signaling pathways. This may provide a novel strategy for curing RA.