Computational Modeling of NLRP3 Identifies Enhanced ATP Binding and Multimerization in Cryopyrin-Associated Periodic Syndromes.

Cyropyrin-associated periodic syndromes (CAPS) are clinically distinct syndromes that encompass a phenotypic spectrum yet are caused by alterations in the same gene, NLRP3. Many CAPS cases and other NLRP3-autoinflammatory diseases (NLRP3-AIDs) are directly attributed to protein-coding alterations in NLRP3 and the subsequent dysregulation of the NLRP3 inflammasome leading to IL-1ß-mediated inflammatory states. Here, we used bioinformatics tools, computational modeling, and computational assessments to explore the proteomic consequences of NLRP3 mutations, which potentially drive NLRP3 inflammasome dysregulation. We analyzed 177 mutations derived from familial cold autoinflammatory syndrome (FCAS), Muckle-Wells Syndrome (MWS), and the non-hereditary chronic infantile neurologic cutaneous and articular syndrome, also known as neonatal-onset multisystem inflammatory disease (CINCA/NOMID), as well as other NLRP3-AIDs. We found an inverse relationship between clinical severity and the severity of predicted structure changes resulting from mutations in NLRP3. Bioinformatics tools and computational modeling revealed that NLRP3 mutations that are predicted to be structurally severely-disruptive localize around the ATP binding pocket and that specific proteo-structural changes to the ATP binding pocket lead to enhanced ATP binding affinity by altering hydrogen-bond and charge interactions. Furthermore, we demonstrated that NLRP3 mutations that are predicted to be structurally mildly- or moderately-disruptive affect protein-protein interactions, such as NLRP3-ASC binding and NLRP3-NLRP3 multimerization, enhancing inflammasome formation and complex stability. Taken together, we provide evidence that proteo-structural mechanisms can explain multiple mechanisms of inflammasome activation in NLRP3-AID.

Inflammasome Sensor NLRP1 Confers Acquired Drug Resistance to Temozolomide in Human Melanoma.

Cancer cells gain drug resistance through a complex mechanism, in which nuclear factor-κB (NF-κB) and interleukin-1ß (IL-1ß) are critical contributors. Because NACHT, LRR and PYD domains-containing protein (NLRP) inflammasomes mediate IL-1ß maturation and NF-κB activation, we investigated the role of inflammasome sensor NLRP1 in acquired drug resistance to temozolomide (TMZ) in melanoma. The sensitivity of melanoma cells to TMZ was negatively correlated with the expression levels of O6-methylguanine-DNA methyltransferase (MGMT), the enzyme to repair TMZ-induced DNA lesions. When MGMT-low human melanoma cells (1205Lu and HS294T) were treated with TMZ for over two months, MGMT was upregulated, and cells became resistant. However, the resistance mechanism was independent of MGMT, and the cells that acquired TMZ resistance showed increased NLRP1 expression, NLRP inflammasome activation, IL-1ß secretion, and NF-κB activity, which contributed to the acquired resistance to TMZ. Finally, blocking IL-1 receptor (IL-1R) signaling with IL-1R antagonist decreased TMZ-resistant 1205Lu tumor growth in vivo. Although inflammation has been associated with drug resistance in various cancers, our paper is the first to demonstrate the involvement of NLRP in the development of acquired drug resistance. Because drug-tolerant cancer cells become cross-tolerant to other classes of cancer drugs, NLRP1 might be a suitable therapeutic target in drug-resistant melanoma, as well as in other cancers.

NLRP1 Functions Downstream of the MAPK/ERK Signaling via ATF4 and Contributes to Acquired Targeted Therapy Resistance in Human Metastatic Melanoma.

The BRAF V600E mutation leads to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) pathway and its downstream effector responses. Uncovering the hidden downstream effectors can aid in understanding melanoma biology and improve targeted therapy efficacy. The inflammasome sensor, NACHT, LRR, and PYD domains-containing protein 1 (NLRP1), is responsible for IL-1ß maturation and itself is a melanoma tumor promoter. Here, we report that NLRP1 is a downstream effector of MAPK/ERK signaling through the activating transcription factor 4 (ATF4), creating regulation in metastatic melanoma cells. We confirmed that the NLRP1 gene is a target of ATF4. Interestingly, ATF4/NLRP1 regulation by the MAPK/ERK pathway uses distinct mechanisms in melanoma cells before and after the acquired resistance to targeted therapy. In parental cells, ATF4/NLRP1 is regulated by the MAPK/ERK pathway through the ribosomal S6 kinase 2 (RSK2). However, vemurafenib (VEM) and trametinib (TRA)-resistant cells lose the signaling via RSK2 and activate the cAMP/protein kinase A (PKA) pathway to redirect ATF4/NLRP1. Therefore, NLRP1 expression and IL-1ß secretion were downregulated in response to VEM and TRA in parental cells but enhanced in drug-resistant cells. Lastly, silencing NLRP1 in drug-resistant cells reduced their cell growth and inhibited colony formation. In summary, we demonstrated that NLRP1 functions downstream of the MAPK/ERK signaling via ATF4 and is a player of targeted therapy resistance in melanoma. Targeting NLRP1 may improve the therapeutic efficacy of targeted therapy in melanoma.

Clinical implications of ALDH1A1 and ALDH1A3 mRNA expression in melanoma subtypes.

Aldehyde dehydrogenase (ALDH) activity is not only a valuable marker for cancer cells with stem-like features, but also plays a vital role in drug resistance and disease progression in many tumors including melanoma. However, the precise role of ALDH activity in patient prognosis remains unclear. In this study, using the Cancer Genome Atlas (TCGA) RNA-sequencing expression data, we analyzed gene expression of ALDH isozymes in melanoma tumors to define the expression patterns and the prognostic and predictive values of these enzymes. We found that ALDH1A1 and ALDH1A3 had both higher and broader expression ranges in melanoma patients, and that ALDH1A3 expression correlated with better overall survival in metastatic melanoma. Further, stratification of the TCGA cohorts by the mutational subtypes of melanoma specifically revealed that expression of ALDH1A3 correlated with better prognosis in metastatic BRAF-mutant melanoma while expression of ALDH1A1 correlated with better prognosis in BRAF wild-type melanoma. Gene set enrichment analysis (GSEA) of these cohorts identified upregulation in oxidative phosphorylation, adipogenesis, and fatty acid metabolism signaling in ALDH1Alo patients, suggesting BRAF/MEK inhibitor resistance in that subset of patients. On the other hand, GSEA of ALDH1A3hi cohorts revealed upregulation in glycolysis, hypoxia and angiogenesis, suggesting BRAF/MEK inhibitor sensitivity in that subset of patients. Gene expression analysis using pre-treatment tumor samples supports high ALDH1A3 expression before BRAF/MEK inhibitor treatment as predictive of better treatment response in BRAF-mutant melanoma patients. Our study provides evidence that high ALDH1A3 mRNA expression is not only a prognostic marker but also a predictive marker for BRAF/MEK inhibitor treatment response in BRAF-mutant metastatic melanoma patients.

CDK1 Interacts with Sox2 and Promotes Tumor Initiation in Human Melanoma.

: Cancers are composed of heterogeneous subpopulations with various tumor-initiating capacities, yet key stem cell genes associated with enhanced tumor-initiating capacities and their regulatory mechanisms remain elusive. Here, we analyzed patient-derived xenografts from melanoma, colon, and pancreatic cancer tissues and identified enrichment of tumor-initiating cells in MHC class I-hi cells, where CDK1, a master regulator of the cell cycle, was upregulated. Overexpression of CDK1, but not its kinase-dead variant, in melanoma cells increased their spheroid forming ability, tumorigenic potential, and tumor-initiating capacity; inhibition of CDK1 with pharmacologic agents reduced these characteristics, which was unexplained by the role of CDK1 in regulating the cell cycle. Proteomic analysis revealed an interaction between CDK1 and the pluripotent stem cell transcription factor Sox2. Blockade or knockdown of CDK1 resulted in reduced phosphorylation, nuclear localization, and transcriptional activity of Sox2. Knockout of Sox2 in CDK1-overexpressing cells reduced CDK1-driven tumor-initiating capacity substantially. Furthermore, GSEA analysis of CDK1hi tumor cells identified a pathway signature common in all three cancer types, including E2F, G2M, MYC, and spermatogenesis, confirming a stem-like nature of CDK1hi tumor cells. These findings reveal a previously unrecognized role for CDK1 in regulating tumor-initiating capacity in melanoma and suggest a novel treatment strategy in cancer via interruption of CDK1 function and its protein-protein interactions. SIGNIFICANCE: These findings uncover CDK1 as a new regulator of Sox2 during tumor initiation and implicate the CDK1-Sox2 interaction as a potential therapeutic target in cancer.

A streptococcal lipid toxin induces membrane permeabilization and pyroptosis leading to fetal injury.

Group B streptococci (GBS) are Gram-positive bacteria that cause infections in utero and in newborns. We recently showed that the GBS pigment is hemolytic and increased pigment production promotes bacterial penetration of human placenta. However, mechanisms utilized by the hemolytic pigment to induce host cell lysis and the consequence on fetal injury are not known. Here, we show that the GBS pigment induces membrane permeability in artificial lipid bilayers and host cells. Membrane defects induced by the GBS pigment trigger K(+) efflux leading to osmotic lysis of red blood cells or pyroptosis in human macrophages. Macrophages lacking the NLRP3 inflammasome recovered from pigment-induced cell damage. In a murine model of in utero infection, hyperpigmented GBS strains induced fetal injury in both an NLRP3 inflammasome-dependent and NLRP3 inflammasome-independent manner. These results demonstrate that the dual mechanism of action of the bacterial pigment/lipid toxin leading to hemolysis or pyroptosis exacerbates fetal injury and suggest that preventing both activities of the hemolytic lipid is likely critical to reduce GBS fetal injury and preterm birth.

Decoding long nanopore sequencing reads of natural DNA.

Nanopore sequencing of DNA is a single-molecule technique that may achieve long reads, low cost and high speed with minimal sample preparation and instrumentation. Here, we build on recent progress with respect to nanopore resolution and DNA control to interpret the procession of ion current levels observed during the translocation of DNA through the pore MspA. As approximately four nucleotides affect the ion current of each level, we measured the ion current corresponding to all 256 four-nucleotide combinations (quadromers). This quadromer map is highly predictive of ion current levels of previously unmeasured sequences derived from the bacteriophage phi X 174 genome. Furthermore, we show nanopore sequencing reads of phi X 174 up to 4,500 bases in length, which can be unambiguously aligned to the phi X 174 reference genome, and demonstrate proof-of-concept utility with respect to hybrid genome assembly and polymorphism detection. This work provides a foundation for nanopore sequencing of long, natural DNA strands.

Detection and mapping of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore MspA.

Precise and efficient mapping of epigenetic markers on DNA may become an important clinical tool for prediction and identification of ailments. Methylated CpG sites are involved in gene expression and are biomarkers for diseases such as cancer. Here, we use the engineered biological protein pore Mycobacterium smegmatis porin A (MspA) to detect and map 5-methylcytosine and 5-hydroxymethylcytosine within single strands of DNA. In this unique single-molecule tool, a phi29 DNA polymerase draws ssDNA through the pore in single-nucleotide steps, and the ion current through the pore is recorded. Comparing current levels generated with DNA containing methylated CpG sites to current levels obtained with unmethylated copies of the DNA reveals the precise location of methylated CpG sites. Hydroxymethylation is distinct from methylation and can also be mapped. With a single read, the detection efficiency in a quasirandom DNA strand is 97.5 ± 0.7% for methylation and 97 ± 0.9% for hydroxymethylation.