Acquired resistance to KRAS G12C small-molecule inhibitors via genetic/nongenetic mechanisms in lung cancer.

Inherent or acquired resistance to sotorasib poses a substantialt challenge for NSCLC treatment. Here, we demonstrate that acquired resistance to sotorasib in isogenic cells correlated with increased expression of integrin ß4 (ITGB4), a component of the focal adhesion complex. Silencing ITGB4 in tolerant cells improved sotorasib sensitivity, while overexpressing ITGB4 enhanced tolerance to sotorasib by supporting AKT-mTOR bypass signaling. Chronic treatment with sotorasib induced WNT expression and activated the WNT/ß-catenin signaling pathway. Thus, silencing both ITGB4 and ß-catenin significantly improved sotorasib sensitivity in tolerant, acquired, and inherently resistant cells. In addition, the proteasome inhibitor carfilzomib (CFZ) exhibited synergism with sotorasib by down-regulating ITGB4 and ß-catenin expression. Furthermore, adagrasib phenocopies the combination effect of sotorasib and CFZ by suppressing KRAS activity and inhibiting cell cycle progression in inherently resistant cells. Overall, our findings unveil previously unrecognized nongenetic mechanisms underlying resistance to sotorasib and propose a promising treatment strategy to overcome resistance.

Intrinsically Disordered Proteins: Critical Components of the Wetware.

Despite the wealth of knowledge gained about intrinsically disordered proteins (IDPs) since their discovery, there are several aspects that remain unexplored and, hence, poorly understood. A living cell is a complex adaptive system that can be described as a wetware─a metaphor used to describe the cell as a computer comprising both hardware and software and attuned to logic gates─capable of "making" decisions. In this focused Review, we discuss how IDPs, as critical components of the wetware, influence cell-fate decisions by wiring protein interaction networks to keep them minimally frustrated. Because IDPs lie between order and chaos, we explore the possibility that they can be modeled as attractors. Further, we discuss how the conformational dynamics of IDPs manifests itself as conformational noise, which can potentially amplify transcriptional noise to stochastically switch cellular phenotypes. Finally, we explore the potential role of IDPs in prebiotic evolution, in forming proteinaceous membrane-less organelles, in the origin of multicellularity, and in protein conformation-based transgenerational inheritance of acquired characteristics. Together, these ideas provide a new conceptual framework to discern how IDPs may perform critical biological functions despite their lack of structure.

Co-opting disorder into order: Intrinsically disordered proteins and the early evolution of complex multicellularity.

Intrinsically disordered proteins (IDPs) are proteins that lack rigid structures yet play important roles in myriad biological phenomena. A distinguishing feature of IDPs is that they often mediate specific biological outcomes via multivalent weak cooperative interactions with multiple partners. Here, we show that several proteins specifically associated with processes that were key in the evolution of complex multicellularity in the lineage leading to the multicellular green alga Volvox carteri are IDPs. We suggest that, by rewiring cellular protein interaction networks, IDPs facilitated the co-option of ancestral pathways for specialized multicellular functions, underscoring the importance of IDPs in the early evolution of complex multicellularity.

Protein Phosphatase 2A as a Therapeutic Target in Small Cell Lung Cancer.

Protein phosphatase 2A (PP2A), a serine/threonine phosphatase involved in the regulation of apoptosis, proliferation, and DNA-damage response, is overexpressed in many cancers, including small cell lung cancer (SCLC). Here we report that LB100, a small molecule inhibitor of PP2A, when combined with platinum-based chemotherapy, synergistically elicited an antitumor response both in vitro and in vivo with no apparent toxicity. Using inductively coupled plasma mass spectrometry, we determined quantitatively that sensitization via LB100 was mediated by increased uptake of carboplatin in SCLC cells. Treatment with LB100 alone or in combination resulted in inhibition of cell viability in two-dimensional culture and three-dimensional spheroid models of SCLC, reduced glucose uptake, and attenuated mitochondrial and glycolytic ATP production. Combining LB100 with atezolizumab increased the capacity of T cells to infiltrate and kill tumor spheroids, and combining LB100 with carboplatin caused hyperphosphorylation of the DNA repair marker γH2AX and enhanced apoptosis while attenuating MET signaling and invasion through an endothelial cell monolayer. Taken together, these data highlight the translational potential of inhibiting PP2A with LB100 in combination with platinum-based chemotherapy and immunotherapy in SCLC.

Activation of EPHA2-ROBO1 Heterodimer by SLIT2 Attenuates Non-canonical Signaling and Proliferation in Squamous Cell Carcinomas.

The tyrosine kinase receptor ephrin receptor A2 (EPHA2) is overexpressed in lung (LSCC) and head and neck (HNSCC) squamous cell carcinomas. Although EPHA2 can inhibit tumorigenesis in a ligand-dependent fashion via phosphorylation of Y588 and Y772, it can promote tumorigenesis in a ligand-independent manner via phosphorylation of S897. Here, we show that EPHA2 and Roundabout Guidance Receptor 1 (ROBO1) interact to form a functional heterodimer. Furthermore, we show that the ROBO1 ligand Slit Guidance Ligand 2 (SLIT2) and ensartinib, an inhibitor of EPHA2, can attenuate growth of HNSCC cells and act synergistically in LSCC cells. Our results suggest that patients with LSCC and HNSCC may be stratified and treated based on their EPHA2 and ROBO1 expression patterns. Although ~73% of patients with LSCC could benefit from SLIT2+ensartinib treatment, ~41% of patients with HNSCC could be treated with either SLIT2 or ensartinib. Thus, EPHA2 and ROBO1 represent potential LSCC and HNSCC theranostics.

Small Cell Lung Cancer from Traditional to Innovative Therapeutics: Building a Comprehensive Network to Optimize Clinical and Translational Research.

Small cell lung cancer (SCLC) is an aggressive, complex disease with a distinct biology that contributes to its poor prognosis. Management of SCLC is still widely limited to chemotherapy and radiation therapy, and research recruitment still poses a considerable challenge. Here, we review the current standard of care for SCLC and advances made in utilizing immunotherapy. We also highlight research in the development of targeted therapies and emphasize the importance of a team-based approach to make clinical advances. Building an integrative network between an academic site and community practice sites optimizes biomarker and drug target discovery for managing and treating a difficult disease like SCLC.

Expressed sequence tag analysis of human RPE/choroid for the NEIBank Project: over 6000 non-redundant transcripts, novel genes and splice variants.

PURPOSE: The retinal pigment epithelium (RPE) and choroid comprise a functional unit of the eye that is essential to normal retinal health and function. Here we describe expressed sequence tag (EST) analysis of human RPE/choroid as part of a project for ocular bioinformatics. METHODS: A cDNA library (cs) was made from human RPE/choroid and sequenced. Data were analyzed and assembled using the program GRIST (GRouping and Identification of Sequence Tags). Complete sequencing, Northern and Western blots, RH mapping, peptide antibody synthesis and immunofluorescence (IF) have been used to examine expression patterns and genome location for selected transcripts and proteins. RESULTS: Ten thousand individual sequence reads yield over 6300 unique gene clusters of which almost half have no matches with named genes. One of the most abundant transcripts is from a gene (named "alpha") that maps to the BBS1 region of chromosome 11. A number of tissue preferred transcripts are common to both RPE/choroid and iris. These include oculoglycan/opticin, for which an alternative splice form is detected in RPE/choroid, and "oculospanin" (Ocsp), a novel tetraspanin that maps to chromosome 17q. Antiserum to Ocsp detects expression in RPE, iris, ciliary body, and retinal ganglion cells by IF. A newly identified gene for a zinc-finger protein (TIRC) maps to 19q13.4. Variant transcripts of several genes were also detected. Most notably, the predominant form of Bestrophin represented in cs contains a longer open reading frame as a result of splice junction skipping. CONCLUSIONS: The unamplified cs library gives a view of the transcriptional repertoire of the adult RPE/choroid. A large number of potentially novel genes and splice forms and candidates for genetic diseases are revealed. Clones from this collection are being included in a large, nonredundant set for cDNA microarray construction.

Expressed sequence tag analysis of human retina for the NEIBank Project: retbindin, an abundant, novel retinal cDNA and alternative splicing of other retina-preferred gene transcripts.

PURPOSE: Expressed sequence tag (EST) analysis was performed on un-normalized, unamplified cDNA libraries constructed from adult human retina to examine the expression profile of the tissue and to contribute resources for functional genomics studies. METHODS: Two size fractionated cDNA libraries (designated hd and he) were constructed from human retina RNA. Clones were randomly selected for sequencing and analyzed using the bioinformatics program GRIST (GRouping and Identification of Sequence Tags). PCR, Northern blotting and other techniques have been used to examine selected novel transcripts. RESULTS: After informatics analysis, 2200 retina cDNAs yield 1254 unique clusters, potentially representing individual genes. Opsin is the most abundant transcript and other retina transcripts are prominently represented. One abundant cluster of cDNAs encodes retbindin, a novel, retina preferred transcript which has sequence similarity to riboflavin binding proteins and whose gene is on chromosome 19. Variant transcripts of known retina genes are also observed, including an alternative exon in the coding sequence of the transcription factor NRL and a skipped coding sequence exon in the phosphodiesterase gammasubunit (PDE6G). CONCLUSIONS: The new retina cDNA libraries compare favorably in quality with those already represented in public databases. They are rich in retina specific sequences and include abundant cDNAs for a novel protein, retbindin. The function of retbindin remains to be determined, but it is a candidate for flavinoid or carotenoid binding. Analysis of multiple clones for highly expressed retina genes reveals several alternative splice variants in both coding and noncoding sequences which may have functional significance. The validated set of retina cDNAs will contribute to a nonredundant set for microarray construction.

Expressed sequence tag analysis of adult human iris for the NEIBank Project: steroid-response factors and similarities with retinal pigment epithelium.

PURPOSE: The iris is a specialized tissue with important roles in the development and function of the eye. It is involved in diseases, including glaucoma and ocular melanoma, and its pigmented cells share an origin with the retinal pigment epithelium (RPE). Expressed sequence tag (EST) analysis of human iris has been performed to explore the repertoire of genes expressed in this tissue. METHODS: An unamplified, un-normalized cDNA library (designated bx) was constructed from pooled (4-80 years old) human iris tissue. Over 2000 clones were picked and sequenced. Sequences were analyzed and clustered using GRIST (GRouping and Identification of Sequence Tags). The library was then normalized (and designated fg) and a further 2200 clones were sequenced for deeper examination of rarer sequence. Some sequences of interest were investigated further by standard methods. RESULTS: From bx and fg libraries respectively, 1263 and 1604 clusters of expressed genes have been identified, giving a combined total of almost 2700 potentially unique genes. The most abundant novel transcript in bx is oculoglycan/opticin. Others include glucocorticoid induced leucine zipper protein (GILZ), Ris, a novel member of the Ras family, Iris Ring Finger (IRF), a member of the midline family, melastatin 2 (MLSN2), a member of the transient receptor potential calcium channel family, and iris expressed growth factor (IEGF), a member of the VEGF/PDGF family. Several factors involved in steroid responses are also represented. CONCLUSIONS: The iris libraries are a rich source of novel as well as known genes, including molecular markers for pigmented cells that are also shared with RPE. A number of transcripts code for proteins involved in steroid response, with interesting implications for control of intraocular pressure. These sequence verified clones provide a nonredundant set for micro-array construction.

Expressed sequence tag analysis of adult human lens for the NEIBank Project: over 2000 non-redundant transcripts, novel genes and splice variants.

PURPOSE: To explore the expression profile of the human lens and to provide a resource for microarray studies, expressed sequence tag (EST) analysis has been performed on cDNA libraries from adult lenses. METHODS: A cDNA library was constructed from two adult (40 year old) human lenses. Over two thousand clones were sequenced from the unamplified, un-normalized library. The library was then normalized and a further 2200 sequences were obtained. All the data were analyzed using GRIST (GRouping and Identification of Sequence Tags), a procedure for gene identification and clustering. RESULTS: The lens library (by) contains a low percentage of non-mRNA contaminants and a high fraction (over 75%) of apparently full length cDNA clones. Approximately 2000 reads from the unamplified library yields 810 clusters, potentially representing individual genes expressed in the lens. After normalization, the content of crystallins and other abundant cDNAs is markedly reduced and a similar number of reads from this library (fs) yields 1455 unique groups of which only two thirds correspond to named genes in GenBank. Among the most abundant cDNAs is one for a novel gene related to glutamine synthetase, which was designated "lengsin" (LGS). Analyses of ESTs also reveal examples of alternative transcripts, including a major alternative splice form for the lens specific membrane protein MP19. Variant forms for other transcripts, including those encoding the apoptosis inhibitor Livin and the armadillo repeat protein ARVCF, are also described. CONCLUSIONS: The lens cDNA libraries are a resource for gene discovery, full length cDNAs for functional studies and microarrays. The discovery of an abundant, novel transcript, lengsin, and a major novel splice form of MP19 reflect the utility of unamplified libraries constructed from dissected tissue. Many novel transcripts and splice forms are represented, some of which may be candidates for genetic diseases.

Grouping and identification of sequence tags (GRIST): bioinformatics tools for the NEIBank database.

NEIBank is a project to develop and organize genomics and bioinformatics resources for the eye. As part of this effort, tools have been developed for bioinformatics analysis and web based display of data from expressed sequence tag (EST) analyses. EST sequences are identified and formed into groups or clusters representing related transcripts from the same gene. This is carried out by a rules-based procedure called GRIST (GRouping and Identification of Sequence Tags) that uses sequence match parameters derived from BLAST programs. Linked procedures are used to eliminate non-mRNA contaminants. All data are assembled in a relational database and assembled for display as web pages with annotations and links to other informatics resources. Genome projects generate huge amounts of data that need to be classified and organized to become easily accessible to the research community. GRIST provides a useful tool for assembling and displaying the results of EST analyses. The NEIBank web site contains a growing set of pages cataloging the known transcriptional repertoire of eye tissues, derived from new NEIBank cDNA libraries and from eye-related data deposited in the dbEST section of GenBank.