Candidate variants in DNA replication and repair genes in early-onset renal cell carcinoma patients referred for germline testing.

BACKGROUND: Early-onset renal cell carcinoma (eoRCC) is typically associated with pathogenic germline variants (PGVs) in RCC familial syndrome genes. However, most eoRCC patients lack PGVs in familial RCC genes and their genetic risk remains undefined. METHODS: Here, we analyzed biospecimens from 22 eoRCC patients that were seen at our institution for genetic counseling and tested negative for PGVs in RCC familial syndrome genes. RESULTS: Analysis of whole-exome sequencing (WES) data found enrichment of candidate pathogenic germline variants in DNA repair and replication genes, including multiple DNA polymerases. Induction of DNA damage in peripheral blood monocytes (PBMCs) significantly elevated numbers of [Formula: see text]H2AX foci, a marker of double-stranded breaks, in PBMCs from eoRCC patients versus PBMCs from matched cancer-free controls. Knockdown of candidate variant genes in Caki RCC cells increased [Formula: see text]H2AX foci. Immortalized patient-derived B cell lines bearing the candidate variants in DNA polymerase genes (POLD1, POLH, POLE, POLK) had DNA replication defects compared to control cells. Renal tumors carrying these DNA polymerase variants were microsatellite stable but had a high mutational burden. Direct biochemical analysis of the variant Pol δ and Pol η polymerases revealed defective enzymatic activities. CONCLUSIONS: Together, these results suggest that constitutional defects in DNA repair underlie a subset of eoRCC cases. Screening patient lymphocytes to identify these defects may provide insight into mechanisms of carcinogenesis in a subset of genetically undefined eoRCCs. Evaluation of DNA repair defects may also provide insight into the cancer initiation mechanisms for subsets of eoRCCs and lay the foundation for targeting DNA repair vulnerabilities in eoRCC.

CRISPR/Cas9 genome-wide loss-of-function screening identifies druggable cellular factors involved in sunitinib resistance in renal cell carcinoma.

BACKGROUND: Multi-targeted tyrosine kinase inhibitors (TKIs) are the standard of care for patients with advanced clear cell renal cell carcinoma (ccRCC). However, a significant number of ccRCC patients are primarily refractory to targeted therapeutics, showing neither disease stabilisation nor clinical benefits. METHODS: We used CRISPR/Cas9-based high-throughput loss of function (LOF) screening to identify cellular factors involved in the resistance to sunitinib. Next, we validated druggable molecular factors that are synthetically lethal with sunitinib treatment using cell and animal models of ccRCC. RESULTS: Our screening identified farnesyltransferase among the top hits contributing to sunitinib resistance in ccRCC. Combined treatment with farnesyltransferase inhibitor lonafarnib potently augmented the anti-tumour efficacy of sunitinib both in vitro and in vivo. CONCLUSION: CRISPR/Cas9 LOF screening presents a promising approach to identify and target cellular factors involved in the resistance to anti-cancer therapeutics.

Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD).

Autosomal-dominant polycystic kidney disease (ADPKD) is associated with progressive formation of renal cysts, kidney enlargement, hypertension, and typically end-stage renal disease. In ADPKD, inherited mutations disrupt function of the polycystins (encoded by PKD1 and PKD2), thus causing loss of a cyst-repressive signal emanating from the renal cilium. Genetic studies have suggested ciliary maintenance is essential for ADPKD pathogenesis. Heat shock protein 90 (HSP90) clients include multiple proteins linked to ciliary maintenance. We determined that ganetespib, a clinical HSP90 inhibitor, inhibited proteasomal repression of NEK8 and the Aurora-A activator trichoplein, rapidly activating Aurora-A kinase and causing ciliary loss in vitro. Using conditional mouse models for ADPKD, we performed long-term (10 or 50 wk) dosing experiments that demonstrated HSP90 inhibition caused durable in vivo loss of cilia, controlled cystic growth, and ameliorated symptoms induced by loss of Pkd1 or Pkd2. Ganetespib efficacy was not increased by combination with 2-deoxy-d-glucose, a glycolysis inhibitor showing some promise for ADPKD. These studies identify a new biologic activity for HSP90 and support a cilia-based mechanism for cyst repression.-Nikonova, A. S., Deneka, A. Y., Kiseleva, A. A., Korobeynikov, V., Gaponova, A., Serebriiskii, I. G., Kopp, M. C., Hensley, H. H., Seeger-Nukpezah, T. N., Somlo, S., Proia, D. A., Golemis, E. A. Ganetespib limits ciliation and cystogenesis in autosomal-dominant polycystic kidney disease (ADPKD).

Musashi-2 (MSI2) supports TGF-ß signaling and inhibits claudins to promote non-small cell lung cancer (NSCLC) metastasis.

Non-small cell lung cancer (NSCLC) has a 5-y survival rate of ∼16%, with most deaths associated with uncontrolled metastasis. We screened for stem cell identity-related genes preferentially expressed in a panel of cell lines with high versus low metastatic potential, derived from NSCLC tumors of Kras(LA1/+);P53(R172HΔG/+) (KP) mice. The Musashi-2 (MSI2) protein, a regulator of mRNA translation, was consistently elevated in metastasis-competent cell lines. MSI2 was overexpressed in 123 human NSCLC tumor specimens versus normal lung, whereas higher expression was associated with disease progression in an independent set of matched normal/primary tumor/lymph node specimens. Depletion of MSI2 in multiple independent metastatic murine and human NSCLC cell lines reduced invasion and metastatic potential, independent of an effect on proliferation. MSI2 depletion significantly induced expression of proteins associated with epithelial identity, including tight junction proteins [claudin 3 (CLDN3), claudin 5 (CLDN5), and claudin 7 (CLDN7)] and down-regulated direct translational targets associated with epithelial-mesenchymal transition, including the TGF-ß receptor 1 (TGFßR1), the small mothers against decapentaplegic homolog 3 (SMAD3), and the zinc finger proteins SNAI1 (SNAIL) and SNAI2 (SLUG). Overexpression of TGFßRI reversed the loss of invasion associated with MSI2 depletion, whereas overexpression of CLDN7 inhibited MSI2-dependent invasion. Unexpectedly, MSI2 depletion reduced E-cadherin expression, reflecting a mixed epithelial-mesenchymal phenotype. Based on this work, we propose that MSI2 provides essential support for TGFßR1/SMAD3 signaling and contributes to invasive adenocarcinoma of the lung and may serve as a predictive biomarker of NSCLC aggressiveness.

Genetic Variants That Predispose to DNA Double-Strand Breaks in Lymphocytes From a Subset of Patients With Familial Colorectal Carcinomas.

BACKGROUND & AIMS: DNA structural lesions are prevalent in sporadic colorectal cancer. Therefore, we proposed that gene variants that predispose to DNA double-strand breaks (DSBs) would be found in patients with familial colorectal carcinomas of an undefined genetic basis (UFCRC). METHODS: We collected primary T cells from 25 patients with UFCRC and matched patients without colorectal cancer (controls) and assayed for DSBs. We performed exome sequence analyses of germline DNA from 20 patients with UFCRC and 5 undiagnosed patients with polyposis. The prevalence of identified variants in genes linked to DNA integrity was compared with that of individuals without a family history of cancer. The effects of representative variants found to be associated with UFCRC was confirmed in functional assays with HCT116 cells. RESULTS: Primary T cells from most patients with UFCRC had increased levels of the DSB marker γ(phosphorylated)histone2AX (γH2AX) after treatment with DNA damaging agents, compared with T cells from controls (P < .001). Exome sequence analysis identified a mean 1.4 rare variants per patient that were predicted to disrupt functions of genes relevant to DSBs. Controls (from public databases) had a much lower frequency of variants in the same genes (P < .001). Knockdown of representative variant genes in HCT116 CRC cells increased γH2AX. A detailed analysis of immortalized patient-derived B cells that contained variants in the Werner syndrome, RecQ helicase-like gene (WRN, encoding T705I), and excision repair cross-complementation group 6 (ERCC6, encoding N180Y) showed reduced levels of these proteins and increased DSBs, compared with B cells from controls. This phenotype was rescued by exogenous expression of WRN or ERCC6. Direct analysis of the recombinant variant proteins confirmed defective enzymatic activities. CONCLUSIONS: These results provide evidence that defects in suppression of DSBs underlie some cases of UFCRC; these can be identified by assays of circulating lymphocytes. We specifically associated UFCRC with variants in WRN and ERCC6 that reduce the capacity for repair of DNA DSBs. These observations could lead to a simple screening strategy for UFCRC, and provide insight into the pathogenic mechanisms of colorectal carcinogenesis.

Inhibiting the HSP90 chaperone slows cyst growth in a mouse model of autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a progressive genetic syndrome with an incidence of 1:500 in the population, arising from inherited mutations in the genes for polycystic kidney disease 1 (PKD1) or polycystic kidney disease 2 (PKD2). Typical onset is in middle age, with gradual replacement of renal tissue with thousands of fluid-filled cysts, resulting in end-stage renal disease requiring dialysis or kidney transplantation. There currently are no approved therapies to slow or cure ADPKD. Mutations in the PKD1 and PKD2 genes abnormally activate multiple signaling proteins and pathways regulating cell proliferation, many of which we observe, through network construction, to be regulated by heat shock protein 90 (HSP90). Inhibiting HSP90 with a small molecule, STA-2842, induces the degradation of many ADPKD-relevant HSP90 client proteins in Pkd1(-/-) primary kidney cells and in vivo. Using a conditional Cre-mediated mouse model to inactivate Pkd1 in vivo, we find that weekly administration of STA-2842 over 10 wk significantly reduces initial formation of renal cysts and kidney growth and slows the progression of these phenotypes in mice with preexisting cysts. These improved disease phenotypes are accompanied by improved indicators of kidney function and reduced expression and activity of HSP90 clients and their effectors, with the degree of inhibition correlating with cystic expansion in individual animals. Pharmacokinetic analysis indicates that HSP90 is overexpressed and HSP90 inhibitors are selectively retained in cystic versus normal kidney tissue, analogous to the situation observed in solid tumors. These results provide an initial justification for evaluating HSP90 inhibitors as therapeutic agents for ADPKD.

Compounds identified by virtual docking to a tetrameric EGFR extracellular domain can modulate Grb2 internalization.

BACKGROUND: Overexpression or mutation of the epidermal growth factor receptor (EGFR) potently enhances the growth of many solid tumors. Tumor cells frequently display resistance to mechanistically-distinct EGFR-directed therapeutic agents, making it valuable to develop therapeutics that work by additional mechanisms. Current EGFR-targeting therapeutics include antibodies targeting the extracellular domains, and small molecules inhibiting the intracellular kinase domain. Recent studies have identified a novel prone extracellular tetrameric EGFR configuration, which we identify as a potential target for drug discovery. METHODS: Our focus is on the prone EGFR tetramer, which contains a novel protein-protein interface involving extracellular domain III. This EGFR tetramer is computationally targeted for stabilization by small molecule ligand binding. This study performed virtual screening of a Life Chemicals, Inc. small molecule library of 345,232 drug-like compounds against a molecular dynamics simulation of protein-protein interfaces distinct to the novel tetramer. One hundred nine chemically diverse candidate molecules were selected and evaluated using a cell-based high-content imaging screen that directly assessed induced internalization of the EGFR effector protein Grb2. Positive hits were further evaluated for influence on phosphorylation of EGFR and its effector ERK1/2. RESULTS: Fourteen hit compounds affected internalization of Grb2, an adaptor responsive to EGFR activation. Most hits had limited effect on cell viability, and minimally influenced EGFR and ERK1/2 phosphorylation. Docked hit compound poses generally include Arg270 or neighboring residues, which are also involved in binding the effective therapeutic cetuximab, guiding further chemical optimization. CONCLUSIONS: These data suggest that the EGFR tetrameric configuration offers a novel cancer drug target.

Aurora A kinase (AURKA) in normal and pathological cell division.

Temporally and spatially controlled activation of the Aurora A kinase (AURKA) regulates centrosome maturation, entry into mitosis, formation and function of the bipolar spindle, and cytokinesis. Genetic amplification and mRNA and protein overexpression of Aurora A are common in many types of solid tumor, and associated with aneuploidy, supernumerary centrosomes, defective mitotic spindles, and resistance to apoptosis. These properties have led Aurora A to be considered a high-value target for development of cancer therapeutics, with multiple agents currently in early-phase clinical trials. More recently, identification of additional, non-mitotic functions and means of activation of Aurora A during interphase neurite elongation and ciliary resorption have significantly expanded our understanding of its function, and may offer insights into the clinical performance of Aurora A inhibitors. Here we review the mitotic and non-mitotic functions of Aurora A, discuss Aurora A regulation in the context of protein structural information, and evaluate progress in understanding and inhibiting Aurora A in cancer.

Aurora kinases in head and neck cancer.

In healthy cells, controlled activation of aurora kinases regulates mitosis. Overexpression and hyperactivation of aurora kinases A and B have major roles in tumorigenesis, and can induce aneuploidy and genomic instability. In squamous-cell carcinomas of the head and neck, overexpression of aurora kinase A is associated with decreased survival, and a reduction in aurora kinase A and aurora kinase B expression inhibits cell growth and increases apoptosis. In this Review, we provide an overview of the biological functions of aurora kinases in healthy cells and in cancer cells, and we review small studies and high-throughput datasets that particularly implicate aurora kinase A in the pathogenesis of squamous-cell carcinomas of the head and neck. Early phase trials are beginning to assess the activity of small-molecule inhibitors of aurora kinases. We summarise trials of aurora kinase inhibitors in squamous-cell carcinomas of the head and neck, and discuss directions for future drug combination trials and biomarkers to use with drugs that inhibit aurora kinases.

Synergy of EGFR and AURKA Inhibitors in KRAS-mutated Non-small Cell Lung Cancers.

The most common oncogenic driver mutations for non-small cell lung cancer (NSCLC) activate EGFR or KRAS. Clinical trials exploring treatments for EGFR- or KRAS-mutated (EGFRmut or KRASmut) cancers have focused on small-molecule inhibitors targeting the driver mutations. Typically, these inhibitors perform more effectively based on combination with either chemotherapies, or other targeted therapies. For EGFRmut NSCLC, a combination of inhibitors of EGFR and Aurora-A kinase (AURKA), an oncogene commonly overexpressed in solid tumors, has shown promising activity in clinical trials. Interestingly, a number of recent studies have indicated that EGFR activity supports overall viability of tumors lacking EGFR mutations, and AURKA expression is abundant in KRASmut cell lines. In this study, we have evaluated dual inhibition of EGFR and AURKA in KRASmut NSCLC models. These data demonstrate synergy between the EGFR inhibitor erlotinib and the AURKA inhibitor alisertib in reducing cell viability and clonogenic capacity in vitro, associated with reduced activity of EGFR pathway effectors, accumulation of enhanced aneuploid cell populations, and elevated cell death. Importantly, the erlotinib-alisertib combination also synergistically reduces xenograft growth in vivo. Analysis of signaling pathways demonstrated that the combination of erlotinib and alisertib was more effective than single-agent treatments at reducing activity of EGFR and pathway effectors following either brief or extended administration of the drugs. In sum, this study indicates value of inhibiting EGFR in KRASmut NSCLC, and suggests the specific value of dual inhibition of AURKA and EGFR in these tumors. SIGNIFICANCE: The introduction of specific KRAS G12C inhibitors to the clinical practice in lung cancer has opened up opportunities that did not exist before. However, G12C alterations are only a subtype of all KRAS mutations observed. Given the high expression of AURKA in KRASmut NSCLC, our study could point to a potential therapeutic option for this subgroup of patients.

Source, co-occurrence, and prognostic value of PTEN mutations or loss in colorectal cancer.

Somatic PTEN mutations are common and have driver function in some cancer types. However, in colorectal cancers (CRCs), somatic PTEN-inactivating mutations occur at a low frequency (~8-9%), and whether these mutations are actively selected and promote tumor aggressiveness has been controversial. Analysis of genomic data from ~53,000 CRCs indicates that hotspot mutation patterns in PTEN partially reflect DNA-dependent selection pressures, but also suggests a strong selection pressure based on protein function. In microsatellite stable (MSS) tumors, PTEN alterations co-occur with mutations activating BRAF or PI3K, or with TP53 deletions, but not in CRC with microsatellite instability (MSI). Unexpectedly, PTEN deletions are associated with poor survival in MSS CRC, whereas PTEN mutations are associated with improved survival in MSI CRC. These and other data suggest use of PTEN as a prognostic marker is valid in CRC, but such use must consider driver mutation landscape, tumor subtype, and category of PTEN alteration.

Protein-intrinsic and signaling network-based sources of resistance to EGFR- and ErbB family-targeted therapies in head and neck cancer.

Agents targeting EGFR and related ErbB family proteins are valuable therapies for the treatment of many cancers. For some tumor types, including squamous cell carcinomas of the head and neck (SCCHN), antibodies targeting EGFR were the first protein-directed agents to show clinical benefit, and remain a standard component of clinical strategies for management of the disease. Nevertheless, many patients display either intrinsic or acquired resistance to these drugs; hence, major research goals are to better understand the underlying causes of resistance, and to develop new therapeutic strategies that boost the impact of EGFR/ErbB inhibitors. In this review, we first summarize current standard use of EGFR inhibitors in the context of SCCHN, and described new agents targeting EGFR currently moving through pre-clinical and clinical development. We then discuss how changes in other transmembrane receptors, including IGF1R, c-Met, and TGF-ß, can confer resistance to EGFR-targeted inhibitors, and discuss new agents targeting these proteins. Moving downstream, we discuss critical EGFR-dependent effectors, including PLC-γ; PI3K and PTEN; SHC, GRB2, and RAS and the STAT proteins, as factors in resistance to EGFR-directed inhibitors and as alternative targets of therapeutic inhibition. We summarize alternative sources of resistance among cellular changes that target EGFR itself, through regulation of ligand availability, post-translational modification of EGFR, availability of EGFR partners for hetero-dimerization and control of EGFR intracellular trafficking for recycling versus degradation. Finally, we discuss new strategies to identify effective therapeutic combinations involving EGFR-targeted inhibitors, in the context of new system level data becoming available for analysis of individual tumors.

Association of TP53 and CDKN2A Mutation Profile with Tumor Mutation Burden in Head and Neck Cancer.

PURPOSE: Head and neck squamous cell carcinoma (HNSCC) is a frequently devastating cancer that affects more than a half million people annually worldwide. Although some cases arise from infection with human papillomavirus (HPV), HPV-negative HNSCC is more common, and associated with worse outcome. Advanced HPV-negative HNSCC may be treated with surgery, chemoradiation, targeted therapy, or immune checkpoint inhibition (ICI). There is considerable need for predictive biomarkers for these treatments. Defects in DNA repair capacity and loss of cell-cycle checkpoints sensitize tumors to cytotoxic therapies, and can contribute to phenotypes such as elevated tumor mutation burden (TMB), associated with response to ICI. Mutation of the tumor suppressors and checkpoint mediators TP53 and CDKN2A is common in HPV-negative HNSCC. EXPERIMENTAL DESIGN: To gain insight into the relation of the interaction of TP53 and CDKN2A mutations with TMB in HNSCC, we have analyzed genomic data from 1,669 HPV-negative HNSCC tumors with multiple criteria proposed for assessing the damaging effect of TP53 mutations. RESULTS: Data analysis established the TP53 and CDKN2A mutation profiles in specific anatomic subsites and suggested that specific categories of TP53 mutations are more likely to associate with CDKN2A mutation or high TMB based on tumor subsite. Intriguingly, the pattern of hotspot mutations in TP53 differed depending on the presence or absence of a cooccurring CDKN2A mutation. CONCLUSIONS: These data emphasize the role of tumor subsite in evaluation of mutational profiles in HNSCC, and link defects in TP53 and CDKN2A to elevated TMB levels in some tumor subgroups.

Comprehensive characterization of PTEN mutational profile in a series of 34,129 colorectal cancers.

Loss of expression or activity of the tumor suppressor PTEN acts similarly to an activating mutation in the oncogene PIK3CA in elevating intracellular levels of phosphatidylinositol (3,4,5)-trisphosphate (PIP3), inducing signaling by AKT and other pro-tumorigenic signaling proteins. Here, we analyze sequence data for 34,129 colorectal cancer (CRC) patients, capturing 3,434 PTEN mutations. We identify specific patterns of PTEN mutation associated with microsatellite stability/instability (MSS/MSI), tumor mutational burden (TMB), patient age, and tumor location. Within groups separated by MSS/MSI status, this identifies distinct profiles of nucleotide hotspots, and suggests differing profiles of protein-damaging effects of mutations. Moreover, discrete categories of PTEN mutations display non-identical patterns of co-occurrence with mutations in other genes important in CRC pathogenesis, including KRAS, APC, TP53, and PIK3CA. These data provide context for clinical targeting of proteins upstream and downstream of PTEN in distinct CRC cohorts.

Nedd9 Restrains Autophagy to Limit Growth of Early Stage Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) is the most common cancer worldwide. With overall 5-year survival estimated at <17%, it is critical to identify factors that regulate NSCLC disease prognosis. NSCLC is commonly driven by mutations in KRAS and TP53, with activation of additional kinases such as SRC promoting tumor invasion. In this study, we investigated the role of NEDD9, a SRC activator and scaffolding protein, in NSCLC tumorigenesis. In an inducible model of NSCLC dependent on Kras mutation and Trp53 loss (KP mice), deletion of Nedd9 (KPN mice) led to the emergence of larger tumors characterized by accelerated rates of tumor growth and elevated proliferation. Orthotopic injection of KP and KPN tumors into the lungs of Nedd9-wild-type and -null mice indicated the effect of Nedd9 loss was cell-autonomous. Tumors in KPN mice displayed reduced activation of SRC and AKT, indicating that activation of these pathways did not mediate enhanced growth of KPN tumors. NSCLC tumor growth has been shown to require active autophagy, a process dependent on activation of the kinases LKB1 and AMPK. KPN tumors contained high levels of active LKB1 and AMPK and increased autophagy compared with KP tumors. Treatment with the autophagy inhibitor chloroquine completely eliminated the growth advantage of KPN tumors. These data for the first time identify NEDD9 as a negative regulator of LKB1/AMPK-dependent autophagy during early NSCLC tumor growth. SIGNIFICANCE: This study demonstrates a novel role for the scaffolding protein NEDD9 in regulating LKB1-AMPK signaling in early stage non-small cell lung cancer, suppressing autophagy and tumor growth.

Design, synthesis and biological evaluation of 2-quinolyl-1,3-tropolone derivatives as new anti-cancer agents.

Tropolones are promising organic compounds that can have important biologic effects. We developed a series of new 2-quinolyl-1,3-tropolones derivatives that were prepared by the acid-catalyzed reaction of 4,7-dichloro-2-methylquinolines with 1,2-benzoquinones. 2-Quinolyl-1,3-tropolones have been synthesized and tested for their anti-proliferative activity against several human cancer cell lines. Two compounds (3d and mixture B of 3i-k) showed excellent activity against six cancer cell lines of different tissue of origin. The promising compounds 3d and mixture B of 3i-k also demonstrated induction of apoptotic cell death of ovarian cancer (OVCAR-3, OVCAR-8) and colon cancer (HCT 116) cell lines and affected ERK signaling. In summary, 2-quinolyl-1,3-tropolones are promising compounds for development of effective anticancer agents.

Musashi-2 (MSI2) regulates epidermal growth factor receptor (EGFR) expression and response to EGFR inhibitors in EGFR-mutated non-small cell lung cancer (NSCLC).

Non-small cell lung cancer (NSCLC) has limited treatment options. Expression of the RNA-binding protein (RBP) Musashi-2 (MSI2) is elevated in a subset of non-small cell lung cancer (NSCLC) tumors upon progression, and drives NSCLC metastasis. We evaluated the mechanism of MSI2 action in NSCLC to gain therapeutically useful insights. Reverse phase protein array (RPPA) analysis of MSI2-depleted versus control KrasLA1/+; Trp53R172HΔG/+ NSCLC cell lines identified EGFR as a MSI2-regulated protein. MSI2 control of EGFR expression and activity in an NSCLC cell line panel was studied using RT-PCR, Western blots, and RNA immunoprecipitation. Functional consequences of MSI2 depletion were explored for cell growth and response to EGFR-targeting drugs, in vitro and in vivo. Expression relationships were validated using human tissue microarrays. MSI2 depletion significantly reduced EGFR protein expression, phosphorylation, or both. Comparison of protein and mRNA expression indicated a post-transcriptional activity of MSI2 in control of steady state levels of EGFR. RNA immunoprecipitation analysis demonstrated that MSI2 directly binds to EGFR mRNA, and sequence analysis predicted MSI2 binding sites in the murine and human EGFR mRNAs. MSI2 depletion selectively impaired cell proliferation in NSCLC cell lines with activating mutations of EGFR (EGFRmut). Further, depletion of MSI2 in combination with EGFR inhibitors such as erlotinib, afatinib, and osimertinib selectively reduced the growth of EGFRmut NSCLC cells and xenografts. EGFR and MSI2 were significantly co-expressed in EGFRmut human NSCLCs. These results define MSI2 as a direct regulator of EGFR protein expression, and suggest inhibition of MSI2 could be of clinical value in EGFRmut NSCLC.

Formation of MacroH2A-containing senescence-associated heterochromatin foci and senescence driven by ASF1a and HIRA.

In senescent cells, specialized domains of transcriptionally silent senescence-associated heterochromatic foci (SAHF), containing heterochromatin proteins such as HP1, are thought to repress expression of proliferation-promoting genes. We have investigated the composition and mode of assembly of SAHF and its contribution to cell cycle exit. SAHF is enriched in a transcription-silencing histone H2A variant, macroH2A. As cells approach senescence, a known chromatin regulator, HIRA, enters PML nuclear bodies, where it transiently colocalizes with HP1 proteins, prior to incorporation of HP1 proteins into SAHF. A physical complex containing HIRA and another chromatin regulator, ASF1a, is rate limiting for formation of SAHF and onset of senescence, and ASF1a is required for formation of SAHF and efficient senescence-associated cell cycle exit. These data indicate that HIRA and ASF1a drive formation of macroH2A-containing SAHF and senescence-associated cell cycle exit, via a pathway that appears to depend on flux of heterochromatic proteins through PML bodies.

Identification of the KRIT1 Protein by LexA-Based Yeast Two-Hybrid System.

Cerebral cavernous malformation (CCM) is a vascular malformation of the central nervous system that is associated with leaky capillaries, and a predisposition to serious clinical conditions including intracerebral hemorrhage and seizures. Germline or sporadic mutations in the CCM1/KRIT1 gene are responsible for the majority of cases of CCM. In this article, we describe the original characterization of the CCM1/KRIT1 gene. This cloning was done through the use of a variant of the yeast two-hybrid screen known as the interaction trap, using the RAS-family GTPase KREV1/RAP1A as a bait. The partial clone of KRIT1 (Krev1 Interaction Trapped) initially identified was extended through 5'RACE and computational analysis to obtain a full-length cDNA, then used in a sequential screen to define the integrin-associated ICAP1 protein as a KRIT1 partner protein. We discuss how these interactions are relevant to the current understanding of KRIT1/CCM1 biology, and provide a protocol for library screening with the Interaction Trap.