Activation of phospholipase C-dependent inositol polyphosphate signaling pathways generates distinct messengers derived from inositol 1,4,5-trisphosphate that control gene expression and mRNA export. Here we report the regulation of telomere length by production of a diphosphorylinositol tetrakisphosphate, PP-IP 4 , synthesized by the KCS1 gene product. Loss of PP-IP 4 production results in lengthening of telomeres, whereas overproduction leads to their shortening. This effect requires the presence of Tel1, the yeast homologue of ATM, the protein mutated in the human disease ataxia telangiectasia. Our data provide in vivo evidence of a regulatory link between inositol polyphosphate signaling and the checkpoint kinase family and describe a third nuclear process modulated by phospholipase C activation.Appropriate cellular responses to environmental changes involve intracellular second messenger systems that transduce the signals from cytoplasm to nucleus, thereby initiating adaptive genetic programs. One well described intracellular messenger system works through receptor-coupled activation of phospholipase C (PLC), 1 which hydrolyzes phosphatidylinositol 4,5-bisphosphate to yield inositol 1,4,5-trisphosphate (IP 3 ) (reviewed in Refs. 1 and 2). In metazoans, cellular production of IP 3 functions as a signal for calcium release from intracellular stores through allosteric activation of an IP 3 receptor channel. Recent studies indicate that IP 3 also plays an important role as precursor to multiple inositol polyphosphates (IPs), each with potentially unique signaling capability (reviewed in Ref. 3). This signaling potential is not restricted to the cytoplasm, because IPs are known to function in nuclear processes in budding yeast (reviewed in Ref. 4). Activation of yeast phospholipase C (Plc1) produces IP 3 , which is rapidly phosphorylated by the dual specificity 6-/3-kinase Ipk2 (initially cloned as ArgRIII/Arg82 (5)) to yield IP 4 and IP 5 (6 -8). This phosphorylation step is coupled to transcriptional regulation, possibly through chromatin remodeling (7, 9 -11). IP 5 is then phosphorylated by the 2-kinase Ipk1, generating IP 6 , which is required for efficient mRNA export from the nucleus (12-14). Both IP 5 and IP 6 can be converted to the diphosphoryl inositols PP-IP 4 and PP-IP 5 through the action of Kcs1, a kinase required for normal vacuolar morphology (15-18). PP-IP 5 has recently been suggested to play a role in chemotaxis in Dictyostelium (19). A nuclear role for Kcs1 is implied by its initial cloning as a regulator of mitotic DNA recombination, and inositol kinase activity is required for this regulation, but further understanding of its nuclear activity is lacking (20,21).Recent work has provided further linking of IP production and nuclear function through an important family of protein serine/threonine kinases known as phosphatidylinositol 3-kinase related kinases (PIKKs). IP 6 stimulates DNA repair by non-homologous end joining with mammalian proteins in vitro (22). Non-homologous end joining can b...

Oncogenic EGFR mutations are found in 10-35% of lung adenocarcinomas. Such mutations, which present most commonly as small in-frame deletions in exon 19 or point mutations in exon 21 (L858R), confer sensitivity to EGFR tyrosine kinase inhibitors (TKIs). In analyzing the tumor from a 33-year-old male never smoker, we identified a novel EGFR alteration in lung cancer: EGFR exon 18-25 kinase domain duplication (EGFR-KDD). Through analysis of a larger cohort of tumor samples, we detected additional cases of EGFR-KDD in lung, brain, and other cancers. In vitro, EGFR-KDD is constitutively active, and computational modeling provides potential mechanistic support for its auto-activation. EGFR-KDD-transformed cells are sensitive to EGFR TKIs and, consistent with these in vitro findings, the index patient had a partial response to the EGFR TKI, afatinib. The patient eventually progressed, at which time, re-sequencing revealed an EGFR-dependent mechanism of acquired resistance to afatinib, thereby validating EGFR-KDD as a driver alteration and therapeutic target.

Introduction Patients with small cell lung cancer (SCLC) have a poor prognosis and limited treatment options. Since access to longitudinal tumor samples is very limited in patients with this disease, we chose to focus our studies on the characterization of plasma cell-free DNA (cfDNA) for rapid, noninvasive monitoring of disease burden. Methods We developed a liquid biopsy assay that quantifies somatic variants in cfDNA. The assay detects single nucleotide variants, copy number alterations, and insertions or deletions in 14 genes that are frequently mutated in SCLC, including TP53, RB1, BRAF, KIT, NOTCH1–4, PIK3CA, PTEN, FGFR1, MYC, MYCL1, and MYCN. Results Over 26 months of peripheral blood collection, we examined 140 plasma samples from 27 patients. We detected disease-associated mutations in 85% of patient samples with mutant allele frequencies (AFs) ranging from ≤0.1% to 84%. In our cohort, 59% of the patients had extensive stage disease, and the most common mutations occurred in TP53 (70%) and RB1 (52%). In addition to mutations in TP53 and RB1, we detected alterations in 10 additional genes in our patient population (PTEN, NOTCH1–4, MYC, MYCL1, PIK3CA, KIT, and BRAF). Observed AFs and CNAs tracked closely with treatment responses. Notably, in several cases analysis of cfDNA provided evidence of disease relapse before conventional imaging. Conclusions These results suggest that liquid biopsies are readily applicable in patients with SCLC and can potentially provide improved monitoring of disease burden, depth of responses to treatment, and timely warning of disease relapse in patients with this disease.

The cation-independent mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) undergoes constitutive endocytosis, mediating the internalization of two unrelated classes of ligands, mannose 6-phosphate (Man-6-P)-containing acid hydrolases and insulin-like growth factor II (IGF-II). To determine the role of ligand valency in M6P/IGF-II receptor-mediated endocytosis, we measured the internalization rates of two ligands, ␤-glucuronidase (a homotetramer bearing multiple Man-6-P moieties) and IGF-II. We found that ␤-glucuronidase entered the cell ϳ3-4-fold faster than IGF-II. Unlabeled ␤-glucuronidase stimulated the rate of internalization of 125 I-IGF-II to equal that of 125 I-␤-glucuronidase, but a bivalent synthetic tripeptide capable of occupying both Man-6-P-binding sites on the M6P/IGF-II receptor simultaneously did not. A mutant receptor with one of the two Man-6-P-binding sites inactivated retained the ability to internalize ␤-glucuronidase faster than IGF-II. Thus, the increased rate of internalization required a multivalent ligand and a single Man-6-P-binding site on the receptor. M6P/IGF-II receptor solubilized and purified in Triton X-100 was present as a monomer, but association with ␤-glucuronidase generated a complex composed of two receptors and one ␤-glucuronidase. Neither IGF-II nor the synthetic peptide induced receptor dimerization. These results indicate that intermolecular cross-linking of the M6P/IGF-II receptor occurs upon binding of a multivalent ligand, resulting in an increased rate of internalization.The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF-II receptor) 1 is a type I transmembrane glycoprotein that cycles through the Golgi, endosomes, and the plasma membrane to carry out its role in the biogenesis of lysosomes and in the clearance of the polypeptide insulin-like growth factor II (IGF-II) (1, 2). In the Golgi, the receptor binds newly synthesized acid hydrolases modified with mannose 6-phosphate (Man-6-P) residues on their asparagine-linked oligosaccharides and transports them to endosomes via clathrincoated vesicles (3-5). The acid hydrolases are released in the acidified endosome and then packaged into lysosomes while the receptor either returns to the Golgi to bind another ligand or moves to the plasma membrane (6, 7). At the plasma membrane, the M6P/IGF-II receptor mediates internalization of Man-6-P-containing ligands and IGF-II (3,5,8).The interactions of IGF-II and Man-6-P-containing ligands with the M6P/IGF-II receptor have been characterized in several studies (8 -12). The extracellular portion of the M6P/ IGF-II receptor contains 15 homologous repeating domains of ϳ147 amino acids each (13). Domains 3 and 9 (numbering from the amino terminus) each bind 1 mol of Man-6-P, and the single IGF-II-binding site has been mapped to domain 11 in the extracellular region (14 -16). Man-6-P residues do not inhibit binding of IGF-II to the receptor, verifying that the two ligandbinding sites are distinct. However, proteins containing Man-6...

The response of mammalian cells to S n 1 DNA methylators depends on functional MutS␣ and MutL␣. Cells deficient in either of these activities are resistant to the cytotoxic effects of this class of chemotherapeutic drug. Because killing by S n 1 methylators has been attributed to O 6 -methylguanine (MeG), we have constructed nicked circular heteroduplexes that contain a single MeG-T mispair, and we have examined processing of these molecules by mismatch repair in nuclear extracts of human cells. Excision provoked by MeG-T is restricted to the incised heteroduplex strand, leading to removal of the MeG when it resides on this strand. However, when the MeG is located on the continuous strand, the heteroduplex is irreparable. MeG-T-dependent repair DNA synthesis is observed on both reparable and irreparable 3 and 5 heteroduplexes as judged by [ P]dAMP incorporation. Labeling with [␣-32 P]dATP followed by a cold dATP chase has demonstrated that newly synthesized DNA on irreparable molecules is subject to re-excision in a reaction that is MutL␣-dependent, an effect attributable to the presence of MeG on the template strand. Processing of the irreparable 3 heteroduplex is also associated with incision of the discontinuous strand of a few percent of molecules near the thymidylate of the MeG-T base pair. These results provide the first direct evidence for mismatch repairmediated iterative processing of DNA methylator damage, an effect that may be relevant to damage signaling events triggered by this class of chemotherapeutic agent.

9084 Background: Immune checkpoint inhibitors are active for patients with stage IV NSCLC who have progressed following platinum-based chemotherapy. We evaluated responses to chemotherapy in patients who had progressed on a checkpoint inhibitor. Methods: Eligible patients were adults with NSCLC who received salvage chemotherapy following PD-1/PD-L1 inhibitors (cases) versus no PD-1/PD-L1 inhibitors (controls). CT-imaging was done within 4 weeks of initiation of salvage chemotherapy and every 6 weeks thereafter. Revised RECIST guidelines were used to define response. Clinical and imaging data were abstracted from review of electronic medical records. Multivariate logistic regression analysis was used to calculate probability of response. Results: Three-hundred and fifty patients’ charts were reviewed and 82 patients met eligibility criteria. Among evaluable patients, 46 were males. Sixty-seven patients were cases versus 15 controls. Fifty-six patients received nivolumab, 7 pembrolizumab and 4 atezolizumab. Sixty-three (77%) had adenocarcinoma, 18 (22%) squamous cell carcinoma and 1 (1%) large cell carcinoma. The mean number of chemotherapy regimens prior to salvage chemotherapy was 2.37 (95% C.I. 2.10-2.64)) in cases versus 1.93 (95% C.I: 1.32-2.54) in controls. Salvage drugs included docetaxel (62%), pemetrexed (20%), gemcitabine (12%), paclitaxel (6%). Eighteen (27%) cases had partial response to chemotherapy versus 1(7%) controls. Fifteen (22%) cases had progressive disease versus 6 (40%) controls. Thirty-four (51%) cases had stable disease versus 8 (53%) controls. The odd ratio for achieving a partial response was 0.30 (95% CI: 0.18 to 0.50, P = 0.000). In multiple logistic regression model, age, gender, number of prior chemotherapy regimens, tumor histology, smoking status, different salvage chemotherapy regimens were not associated with the likelihood of achieving a partial response. Conclusions: The odds of achieving a partial response to salvage chemotherapy were more than 3 times higher inpatients with prior exposure to PD-1/PD-L1 inhibitors. Ongoing investigations include the duration of response as well as evaluation of toxicity.

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Introduction: Most patients (70%) with limited-stage SCLC (LS-SCLC) who are treated with curative-intent therapy suffer disease relapse and cancer-related death. We evaluated circulating tumor DNA (ctDNA) as a predictor of disease relapse and death after definitive therapy in patients with LS-SCLC. Methods: In our previous work, we developed a plasmabased ctDNA assay to sequence 14 genes (TP53, RB1, BRAF, KIT, NOTCH1-4, PIK3CA, PTEN, FGFR1, MYC, MYCL1, and MYCN) that are frequently mutated in SCLC. In this work, we evaluated 177 plasma samples from 23 patients with LS-SCLC who completed definitive chemoradiation (n ¼ 21) or surgical resection (n ¼ 2) and had an end-of-treatment blood collection (median 4 d, range 0-40 d from treatment completion) plus monthly surveillance blood sampling. Median overall survival (OS) and progression-free survival (PFS) were compared using a Wilcoxon test. Results: The median OS among patients in whom we ever detected ctDNA after definitive treatment (n ¼ 15) was 18.2 months compared with a median OS of greater than 48 months among patients in whom we never detected ctDNA after definitive treatment (n ¼ 8; p ¼ 0.081). The median PFS among patients in whom we ever detected ctDNA after definitive treatment was 9.1 months compared with a median PFS of greater than 48 months among patients in whom we never detected ctDNA after definitive treatment (p < 0.001). Conclusions: Detection of ctDNA in patients with LS-SCLC after curative-intent therapy predicts disease relapse and death. Prospective trials using ctDNA as an integral biomarker for therapeutic selection should be considered in SCLC.