Characterization and clustering of kinase isoform expression in metastatic melanoma.

Mutations to the human kinome are known to play causal roles in cancer. The kinome regulates numerous cell processes including growth, proliferation, differentiation, and apoptosis. In addition to aberrant expression, aberrant alternative splicing of cancer-driver genes is receiving increased attention as it could lead to loss or gain of functional domains, altering a kinase's downstream impact. The present study quantifies changes in gene expression and isoform ratios in the kinome of metastatic melanoma cells relative to primary tumors. We contrast 538 total kinases and 3,040 known kinase isoforms between 103 primary tumor and 367 metastatic samples from The Cancer Genome Atlas (TCGA). We find strong evidence of differential expression (DE) at the gene level in 123 kinases (23%). Additionally, of the 468 kinases with alternative isoforms, 60 (13%) had significant difference in isoform ratios (DIR). Notably, DE and DIR have little correlation; for instance, although DE highlights enrichment in receptor tyrosine kinases (RTKs), DIR identifies altered splicing in non-receptor tyrosine kinases (nRTKs). Using exon junction mapping, we identify five examples of splicing events favored in metastatic samples. We demonstrate differential apoptosis and protein localization between SLK isoforms in metastatic melanoma. We cluster isoform expression data and identify subgroups that correlate with genomic subtypes and anatomic tumor locations. Notably, distinct DE and DIR patterns separate samples with BRAF hotspot mutations and (N/K/H)RAS hotspot mutations, the latter of which lacks effective kinase inhibitor treatments. DE in RAS mutants concentrates in CMGC kinases (a group including cell cycle and splicing regulators) rather than RTKs as in BRAF mutants. Furthermore, isoforms in the RAS kinase subgroup show enrichment for cancer-related processes such as angiogenesis and cell migration. Our results reveal a new approach to therapeutic target identification and demonstrate how different mutational subtypes may respond differently to treatments highlighting possible new driver events in cancer.

Recent Progress in RXLR Effector Research.

Some of the most devastating oomycete pathogens deploy effector proteins, with the signature amino acid motif RXLR, that enter plant cells to promote virulence. Research on the function and evolution of RXLR effectors has been very active over the decade that has transpired since their discovery. Comparative genomics indicate that RXLR genes play a major role in virulence for Phytophthora and downy mildew species. Importantly, gene-for-gene resistance against these oomycete lineages is based on recognition of RXLR proteins. Comparative genomics have revealed several mechanisms through which this resistance can be broken, most notably involving epigenetic control of RXLR gene expression. Structural studies have revealed a core fold that is present in the majority of RXLR proteins, providing a foundation for detailed mechanistic understanding of virulence and avirulence functions. Finally, functional studies have demonstrated that suppression of host immunity is a major function for RXLR proteins. Host protein targets are being identified in a variety of plant cell compartments. Some targets comprise hubs that are also manipulated by bacteria and fungi, thereby revealing key points of vulnerability in the plant immune network.

The Megacomplex protects ER-alpha from degradation by Fulvestrant in epithelial ovarian cancer.

Ovarian cancer, a significant contributor to cancer-related mortality, exhibits limited responsiveness to hormonal therapies targeting the estrogen receptor (ERα). This study aimed to elucidate the mechanisms behind ERα resistance to the therapeutic drug Fulvestrant (ICI182780 or ICI). Notably, compared to the cytoplasmic version, nuclear ERα was minimally degraded by ICI, suggesting a mechanism for drug resistance via the protective confines of the nuclear substructures. Of these substructures, we identified a 1.3MDa Megacomplex comprising transcription factors ERα, FOXA1, and PITX1 using size exclusion chromatography (SEC) in the ovarian cancer cell line, PEO4. ChIP-seq revealed these factors colocalized at 6,775 genomic positions representing sites of Megacomplex formation. Megacomplex ERα exhibited increased resistance to degradation by ICI compared to cytoplasmic and nuclear ERα. A small molecule inhibitor of active chromatin and super-enhancers, JQ1, in combination with ICI significantly enhanced ERα degradation from Megacomplex as revealed by SEC and ChIP-seq. Interestingly, this combination degraded both the cytoplasmic as well as nuclear ERa. Pathway enrichment analysis showed parallel results for RNA-seq gene sets following Estradiol, ICI, or ICI plus JQ1 treatments as those defined by Megacomplex binding identified through ChIP-seq. Furthermore, similar pathway enrichments were confirmed in mass-spec analysis of the Megacomplex macromolecule fractions after modulation by Estradiol or ICI. These findings implicate Megacomplex in ERα-driven ovarian cancer chromatin regulation. This combined treatment strategy exhibited superior inhibition of cell proliferation and viability. Therefore, by uncovering ERα's resistance within the Megacomplex, the combined ICI plus JQ1 treatment elucidates a novel drug treatment vulnerability.

Homologous RXLR effectors from Hyaloperonospora arabidopsidis and Phytophthora sojae suppress immunity in distantly related plants.

Diverse pathogens secrete effector proteins into plant cells to manipulate host cellular processes. Oomycete pathogens contain large complements of predicted effector genes defined by an RXLR host cell entry motif. The genome of Hyaloperonospora arabidopsidis (Hpa, downy mildew of Arabidopsis) contains at least 134 candidate RXLR effector genes. Only a small subset of these genes is conserved in related oomycetes from the Phytophthora genus. Here, we describe a comparative functional characterization of the Hpa RXLR effector gene HaRxL96 and a homologous gene, PsAvh163, from the Glycine max (soybean) pathogen Phytophthora sojae. HaRxL96 and PsAvh163 are induced during the early stages of infection and carry a functional RXLR motif that is sufficient for protein uptake into plant cells. Both effectors can suppress immune responses in soybean. HaRxL96 suppresses immunity in Nicotiana benthamiana, whereas PsAvh163 induces an HR-like cell death response in Nicotiana that is dependent on RAR1 and Hsp90.1. Transgenic Arabidopsis plants expressing HaRxL96 or PsAvh163 exhibit elevated susceptibility to virulent and avirulent Hpa, as well as decreased callose deposition in response to non-pathogenic Pseudomonas syringae. Both effectors interfere with defense marker gene induction, but do not affect salicylic acid biosynthesis. Together, these experiments demonstrate that evolutionarily conserved effectors from different oomycete species can suppress immunity in plant species that are divergent from the source pathogen's host.