The Cytoplasmic DNA Sensor cGAS Promotes Mitotic Cell Death.

Pathogenic and other cytoplasmic DNAs activate the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway to induce inflammation via transcriptional activation by IRF3 and nuclear factor κB (NF-κB), but the functional consequences of exposing cGAS to chromosomes upon mitotic nuclear envelope breakdown are unknown. Here, we show that nucleosomes competitively inhibit DNA-dependent cGAS activation and that the cGAS-STING pathway is not effectively activated during normal mitosis. However, during mitotic arrest, low level cGAS-dependent IRF3 phosphorylation slowly accumulates without triggering inflammation. Phosphorylated IRF3, independently of its DNA-binding domain, stimulates apoptosis through alleviating Bcl-xL-dependent suppression of mitochondrial outer membrane permeabilization. We propose that slow accumulation of phosphorylated IRF3, normally not sufficient for inducing inflammation, can trigger transcription-independent induction of apoptosis upon mitotic aberrations. Accordingly, expression of cGAS and IRF3 in cancer cells makes mouse xenograft tumors responsive to the anti-mitotic agent Taxol. The Cancer Genome Atlas (TCGA) datasets for non-small cell lung cancer patients also suggest an effect of cGAS expression on taxane response.

A targetable secreted neural protein drives pancreatic cancer metastatic colonization and HIF1a nuclear retention.

Pancreatic ductal adenocarcinoma (PDAC) is an increasingly diagnosed cancer that kills 90% of afflicted patients, with most patients receiving palliative chemotherapy. We identified neuronal pentraxin 1 (NPTX1) as a cancer secreted protein that becomes over-expressed in human and murine PDAC cells during metastatic progression and identified adhesion molecule with Ig like domain 2 (AMIGO2) as its receptor. Molecular, genetic, biochemical and pharmacologic experiments revealed that secreted NPTX1 acts cell-autonomously on the AMIGO2 receptor to drive PDAC metastatic colonization of the liver-the primary site of PDAC metastasis. NPTX1-AMIGO2 signaling enhanced hypoxic growth and was critically required for hypoxia induced factor-1a (HIF1a) nuclear retention and function. NPTX1 is over-expressed in human PDAC tumors and upregulated in liver metastases. Therapeutic targeting of NPTX1 with a high-affinity monoclonal antibody substantially reduced PDAC liver metastatic colonization. We thus identify NPTX1-AMIGO2 as druggable critical upstream regulators of the HIF1a hypoxic response in PDAC.

Arginine limitation drives a directed codon-dependent DNA sequence evolution response in colorectal cancer cells.

Utilization of specific codons varies between organisms. Cancer represents a model for understanding DNA sequence evolution and could reveal causal factors underlying codon evolution. We found that across human cancer, arginine codons are frequently mutated to other codons. Moreover, arginine limitation-a feature of tumor microenvironments-is sufficient to induce arginine codon-switching mutations in human colon cancer cells. Such DNA codon switching events encode mutant proteins with arginine residue substitutions. Mechanistically, arginine limitation caused rapid reduction of arginine transfer RNAs and the stalling of ribosomes over arginine codons. Such selective pressure against arginine codon translation induced an adaptive proteomic shift toward low-arginine codon-containing genes, including specific amino acid transporters, and caused mutational evolution away from arginine codons-reducing translational bottlenecks that occurred during arginine starvation. Thus, environmental availability of a specific amino acid can influence DNA sequence evolution away from its cognate codons and generate altered proteins.

Arginine limitation causes a directed DNA sequence evolution response in colorectal cancer cells.

Utilization of specific codons varies significantly across organisms. Cancer represents a model for understanding DNA sequence evolution and could reveal causal factors underlying codon evolution. We found that across human cancer, arginine codons are frequently mutated to other codons. Moreover, arginine restriction-a feature of tumor microenvironments-is sufficient to induce arginine codon-switching mutations in human colon cancer cells. Such DNA codon switching events encode mutant proteins with arginine residue substitutions. Mechanistically, arginine limitation caused rapid reduction of arginine transfer RNAs and the stalling of ribosomes over arginine codons. Such selective pressure against arginine codon translation induced a proteomic shift towards low arginine codon containing genes, including specific amino acid transporters, and caused mutational evolution away from arginine codons-reducing translational bottlenecks that occurred during arginine starvation. Thus, environmental availability of a specific amino acid can influence DNA sequence evolution away from its cognate codons and generate altered proteins.

Functional genetic screen identifies ITPR3/calcium/RELB axis as a driver of colorectal cancer metastatic liver colonization.

Metastatic colonization is the primary cause of death from colorectal cancer (CRC). We employed genome-scale in vivo short hairpin RNA (shRNA) screening and validation to identify 26 promoters of CRC liver colonization. Among these genes, we identified a cluster that contains multiple targetable genes, including ITPR3, which promoted liver-metastatic colonization and elicited similar downstream gene expression programs. ITPR3 is a caffeine-sensitive inositol 1,4,5-triphosphate (IP3) receptor that releases calcium from the endoplasmic reticulum and enhanced metastatic colonization by inducing expression of RELB, a transcription factor that is associated with non-canonical NF-κB signaling. Genetic, cell biological, pharmacologic, and clinical association studies revealed that ITPR3 and RELB drive CRC colony formation by promoting cell survival upon substratum detachment or hypoxic exposure. RELB was sufficient to drive colonization downstream of ITPR3. Our findings implicate the ITPR3/calcium/RELB axis in CRC metastatic colony formation and uncover multiple clinico-pathologically associated targetable proteins as drivers of CRC metastatic colonization.

Adaptive stimulation of macropinocytosis overcomes aspartate limitation in cancer cells under hypoxia.

Stress-adaptive mechanisms enable tumour cells to overcome metabolic constraints under nutrient and oxygen shortage. Aspartate is an endogenous metabolic limitation under hypoxic conditions, but the nature of the adaptive mechanisms that contribute to aspartate availability and hypoxic tumour growth are poorly understood. Here we identify GOT2-catalysed mitochondrial aspartate synthesis as an essential metabolic dependency for the proliferation of pancreatic tumour cells under hypoxic culture conditions. In contrast, GOT2-catalysed aspartate synthesis is dispensable for pancreatic tumour formation in vivo. The dependence of pancreatic tumour cells on aspartate synthesis is bypassed in part by a hypoxia-induced potentiation of extracellular protein scavenging via macropinocytosis. This effect is mutant KRAS dependent, and is mediated by hypoxia-inducible factor 1 (HIF1A) and its canonical target carbonic anhydrase-9 (CA9). Our findings reveal high plasticity of aspartate metabolism and define an adaptive regulatory role for macropinocytosis by which mutant KRAS tumours can overcome nutrient deprivation under hypoxic conditions.

Advances in Oncology in US and Japan: Focusing on Cancer and Infectious Diseases.

This is a review article based on the international symposium report of the "US-Japan Conference on Advances in Oncology: Cancer and Infectious Diseases" held online on June 25, 2021, which provided an update on the association between oncology and infectious disease research from cutting-edge basic science to high-impact clinical trials.

Functional Genomics In Vivo Reveal Metabolic Dependencies of Pancreatic Cancer Cells.

Pancreatic ductal adenocarcinoma (PDAC) cells require substantial metabolic rewiring to overcome nutrient limitations and immune surveillance. However, the metabolic pathways necessary for pancreatic tumor growth in vivo are poorly understood. To address this, we performed metabolism-focused CRISPR screens in PDAC cells grown in culture or engrafted in immunocompetent mice. While most metabolic gene essentialities are unexpectedly similar under these conditions, a small fraction of metabolic genes are differentially required for tumor progression. Among these, loss of heme synthesis reduces tumor growth due to a limiting role of heme in vivo, an effect independent of tissue origin or immune system. Our screens also identify autophagy as a metabolic requirement for pancreatic tumor immune evasion. Mechanistically, autophagy protects cancer cells from CD8+ T cell killing through TNFα-induced cell death in vitro. Altogether, this resource provides metabolic dependencies arising from microenvironmental limitations and the immune system, nominating potential anti-cancer targets.

Therapeutic targeting of SLC6A8 creatine transporter suppresses colon cancer progression and modulates human creatine levels.

Colorectal cancer (CRC) is a leading cause of cancer mortality. Creatine metabolism was previously shown to critically regulate colon cancer progression. We report that RGX-202, an oral small-molecule SLC6A8 transporter inhibitor, robustly inhibits creatine import in vitro and in vivo, reduces intracellular phosphocreatine and ATP levels, and induces tumor apoptosis. RGX-202 suppressed CRC growth across KRAS wild-type and KRAS mutant xenograft, syngeneic, and patient-derived xenograft (PDX) tumors. Antitumor efficacy correlated with tumoral expression of creatine kinase B. Combining RGX-202 with 5-fluorouracil or the DHODH inhibitor leflunomide caused regressions of multiple colorectal xenograft and PDX tumors of distinct mutational backgrounds. RGX-202 also perturbed creatine metabolism in patients with metastatic CRC in a phase 1 trial, mirroring pharmacodynamic effects on creatine metabolism observed in mice. This is, to our knowledge, the first demonstration of preclinical and human pharmacodynamic activity for creatine metabolism targeting in oncology, thus revealing a critical therapeutic target.

PCK1 and DHODH drive colorectal cancer liver metastatic colonization and hypoxic growth by promoting nucleotide synthesis.

Colorectal cancer (CRC) is a major cause of human death. Mortality is primarily due to metastatic organ colonization, with the liver being the main organ affected. We modeled metastatic CRC (mCRC) liver colonization using patient-derived primary and metastatic tumor xenografts (PDX). Such PDX modeling predicted patient survival outcomes. In vivo selection of multiple PDXs for enhanced metastatic colonization capacity upregulated the gluconeogenic enzyme PCK1, which enhanced liver metastatic growth by driving pyrimidine nucleotide biosynthesis under hypoxia. Consistently, highly metastatic tumors upregulated multiple pyrimidine biosynthesis intermediary metabolites. Therapeutic inhibition of the pyrimidine biosynthetic enzyme DHODH with leflunomide substantially impaired CRC liver metastatic colonization and hypoxic growth. Our findings provide a potential mechanistic basis for the epidemiologic association of anti-gluconeogenic drugs with improved CRC metastasis outcomes, reveal the exploitation of a gluconeogenesis enzyme for pyrimidine biosynthesis under hypoxia, and implicate DHODH and PCK1 as metabolic therapeutic targets in CRC metastatic progression.

Amine-Controlled Divergent Reaction: Iminolactonization and Olefination in the Presence of a Cu(I) Catalyst.

α-Bromoamides and styrenes underwent iminolactonization reactions (carbooxygenation), in which simultaneous C-C and C-O formation occurred in the presence of a copper catalyst with triethylamine as the base. Conversely, olefination reactions occurred in the presence of a Cu catalyst with piperidine as the base. The selectivities in those reactions were very high.

Pulmonary Extranodal Marginal Zone Lymphoma with Macroglobulinemia and Mixed Cryoglobulinemia Developed in a Patient with Chronic Hepatitis C.

We report a 65-year-old woman with a chronic hepatitis C virus infection who developed pulmonary extranodal marginal zone lymphoma (EMZL) of mucosa-associated lymphoid tissues complicated with macroglobulinemia and mixed cryoglobulinemia. She was treated with immunochemotherapy which resulted in the reduction of both the tumors and the serum immunoglobulin (Ig) M level. This case exemplifies an extensive stimulation upon immune system with derangement in the production of immunoglobulines associated with EMZL, and suggests that it is necessary to consider the possibility of B-cell lymphoma when IgM paraprotein is detected.

Comparison of cardiac events associated with liposomal doxorubicin, epirubicin and doxorubicin in breast cancer: a Bayesian network meta-analysis.

BACKGROUND: Anthracyclines play a broad and important role in the care of patients with either operable or metastatic breast cancer. However cardiotoxicity narrows the therapeutic index of this drug class leading to potentially clinically meaningful treatment delays or discontinuations. We conducted a Bayesian network meta-analysis, a validated statistical methodology, allowing direct and indirect comparison of cardiotoxicity of different anthracycline and non-anthracycline regimens. METHODS: We conducted a systematic review of prospective randomised controlled trials through MEDLINE, EMBASE, Cochrane Central Register of Controlled Trials and Google Scholar comparing non-anthracycline based regimens (NON), doxorubicin (DOX), epirubicin (EPI) and liposomal doxorubicin (LD). We included studies published up to 1st January 2014 in both adjuvant and metastatic contexts. Notably, HER2/neu-targeted regimens were excluded. We assessed the studies' eligibility criteria and data collection with consensus of two independent authors. Our primary outcome measure was cardiac events grade 3 or greater (CE3) in accordance with Common Terminology Criteria for Adverse Events (CTCAE) Version 4.0. A Bayesian pairwise and network meta-analysis was conducted to estimate pooled Odds Ratio (OR). FINDINGS: Nineteen randomised controlled trials met eligibility criteria and were included in this analysis. We found a trend showing that LD is less cardiotoxic than DOX with an OR of 0.60 (95% confidence interval (CI) 0.34-1.07) There was no difference between Epi and LD with an OR of 0.95 (95%CI 0.39-2.33). DOX is more cardiotoxic than Non with an OR of 1.57 (95%CI 0.90-2.72). INTERPRETATION: DOX has higher CE3 rates than NON does. LD statistically trended to lower cardiac event rates than DOX. Non-statistical significance among EPI, LD and DOX with regard to cardiac toxicity indicates that avoidance of CE3 should not motivate selection of a particular anthracycline in otherwise healthy women in whom total lifetime anthracycline exposure will likely be limited. Overall low incidence of CE3 with any type of anthracycline indicates that we can safely use any anthracycline if cumulative dose limits are not exceeded. While CE3 does not limit our choice of anthracycline LD appears to be the least cardiotoxic. FUNDING: Takeo Fujii is supported by the grant named Young Investigator Award for Study Abroad in Clinical Epidemiology from St. Luke's Life Science Institution.

FOXD1 expression is associated with poor prognosis in non-small cell lung cancer.

AIM: Clinical microarray datasets were analyzed to search for new therapeutic targets and prognostic markers of non-small cell lung cancer (NSCLC). MATERIALS AND METHODS: Microarray datasets from 90 lung cancer specimens, were analyzed with focus on the FOXD1 gene. Levels of FOXD1 mRNA were assessed in lung cancer cell lines and these levels were correlated with survival. RESULTS: FOXD1-knockdown led to suppression of cell proliferation. Moreover, patients with high FOXD1 expression survived for a significantly shorter time than those with low FOXD1 expression. CONCLUSION: The expression status of FOXD1 is a novel prognostic factor and may lead to new treatment strategies for NSCLC.

Brain metastases in patients with EGFR-mutated or ALK-rearranged non-small-cell lung cancers.

INTRODUCTION: Brain metastases (BM) are common in non-small-cell lung cancer (NSCLC). However, the baseline incidence and evolution of BM over time in oncogene-driven NSCLCs are seldom reported. In this study, we evaluated the frequency of BM in patients with epidermal growth factor receptor (EGFR)-mutated or anaplastic lymphoma kinase (ALK)-rearranged NSCLC. METHODS: The presence of BM, clinicopathologic data, and tumor genotype were retrospectively compiled and analyzed from a cohort of 381 patients. RESULTS: We identified 86 EGFR-mutated (90.7% with metastatic disease; 85.9% received an EGFR inhibitor) and 23 ALK-rearranged (91.3% with metastatic disease; 85.7% received an ALK inhibitor) NSCLCs. BM were present in 24.4% of EGFR-mutated and 23.8% of ALK-rearranged NSCLCs at the time of diagnosis of advanced disease. This study did not demonstrate a difference in the cumulative incidence of BM over time between the two cohorts (EGFR/ALK cohort competing risk regression [CRR] coefficient of 0.78 [95% CI 0.44-1.39], p=0.41). In still living patients with advanced EGFR-mutated NSCLC, 34.2% had BM at 1 year, 38.4% at 2 years, 46.7% at 3 years, 48.7% at 4 years, and 52.9% at 5 years. In still living patients with advanced ALK-rearranged NSCLC, 23.8% had BM at 1 year, 45.5% at 2 years, and 58.4% at 3 years. CONCLUSIONS: BM are frequent in advanced EGFR-mutated or ALK-rearranged NSCLCs, with an estimated >45% of patients with CNS involvement by three years of survival with the use of targeted therapies. These data point toward the CNS as an important unmet clinical need in the evolving schema for personalized care in NSCLC.

Dual ALK and EGFR inhibition targets a mechanism of acquired resistance to the tyrosine kinase inhibitor crizotinib in ALK rearranged lung cancer.

INTRODUCTION: The multitargeted tyrosine kinase inhibitor (TKI) crizotinib is active against ALK translocated non-small-cell lung cancer (NSCLC); however acquired resistance invariably develops over time. ALK mutations have previously been implicated in only a third of resistant tumors. We sought to evaluate alternative mechanisms of resistance and preclinical strategies to overcome these in a cell line driven by EML4-ALK. METHODS: We selected the NSCLC cell line NCI-H3122 (H3122: EML4-ALK E13;A20) and derived resistant variants that were able to grow in the presence of 1 µM crizotinib. These were analyzed for ALK mutations, sensitivity to crizotinib in combination with other TKIs, and for activation of alternative tyrosine kinases. RESULTS: All H3122 crizotinib resistant (CR) clones lacked amplification or mutations in the kinase domain of ALK. To evaluate if possible alternative kinases functioned as "bypass" tracks for downstream signaling activation in these resistance cells, we performed of phosho-receptor tyrosine kinase array that demonstrated that CR clones had higher phospho-EGFR signals than H3122 cells before and after exposure to crizotinib. A functional approach of dual ALK TKI (with crizotinib) with combinatory TKI inhibition was used as a secondary screen for possible targets. Crizotinib+erlotinib (reversible EGFR TKI) and crizotinib+afatinib (irreversible EGFR/ERBB2 TKI) were able to inhibit the growth of H3122 CR clones, confirming EGFR activation as a mechanism of resistance. The removal of crizotinib from the culture media re-sensitized CR cells to crizotinib. CONCLUSIONS: We identified activation of EGFR as a mechanism of resistance to crizotinib in preclinical models of ALK translocated NSCLC. If EGFR activation is confirmed as a predominant mechanism of ALK TKI-induced resistance in patient-derived tumors, the use of ALK plus EGFR TKIs could be explored for this important cohort of NSCLCs.

Success and failure rates of tumor genotyping techniques in routine pathological samples with non-small-cell lung cancer.

INTRODUCTION: Identification of some somatic molecular alterations in non-small-cell lung cancer (NSCLC) has become evidence-based practice. The success and failure rate of using commercially available tumor genotyping techniques in routine day-to-day NSCLC pathology samples is not well described. We sought to evaluate the success and failure rate of EGFR mutation, KRAS mutation, and ALK FISH in a cohort of lung cancers subjected to routine clinical tumor genotype. METHODS: Clinicopathologic data, tumor genotype success and failure rates were retrospectively compiled and analyzed from 381 patient-tumor samples. RESULTS: From these 381 patients with lung cancer, the mean age was 65 years, 61.2% were women, 75.9% were white, 27.8% were never smokers, 73.8% had advanced NSCLC and 86.1% had adenocarcinoma histology. The tumor tissue was obtained from surgical specimens in 48.8%, core needle biopsies in 17.9%, and as cell blocks from aspirates or fluid in 33.3% of cases. Anatomic sites for tissue collection included lung (49.3%), lymph nodes (22.3%), pleura (11.8%), bone (6.0%), brain (6.0%), among others. The overall success rate for EGFR mutation analysis was 94.2%, for KRAS mutation 91.6% and for ALK FISH 91.6%. The highest failure rates were observed when the tissue was obtained from image-guided percutaneous transthoracic core-needle biopsies (31.8%, 27.3%, and 35.3% for EGFR, KRAS, and ALK tests, respectively) and bone specimens (23.1%, 15.4%, and 23.1%, respectively). In specimens obtained from bone, the failure rates were significantly higher for biopsies than resection specimens (40% vs. 0%, p=0.024 for EGFR) and for decalcified compared to non-decalcified samples (60% vs. 5.5%, p=0.021 for EGFR). CONCLUSIONS: Tumor genotype techniques are feasible in most samples, outside small image-guided percutaneous transthoracic core-needle biopsies and bone samples from core biopsies with decalcification, and therefore expansion of routine tumor genotype into the care of patients with NSCLC may not require special tissue acquisition or manipulation.

ß-catenin contributes to lung tumor development induced by EGFR mutations.

The discovery of somatic mutations in EGFR and development of EGFR tyrosine kinase inhibitors (TKI) have revolutionized treatment for lung cancer. However, resistance to TKIs emerges in almost all patients and currently no effective treatment is available. Here, we show that ß-catenin is essential for development of EGFR-mutated lung cancers. ß-Catenin was upregulated and activated in EGFR-mutated cells. Mutant EGFR preferentially bound to and tyrosine phosphorylated ß-catenin, leading to an increase in ß-catenin-mediated transactivation, particularly in cells harboring the gefitinib/erlotinib-resistant gatekeeper EGFR-T790M mutation. Pharmacologic inhibition of ß-catenin suppressed EGFR-L858R-T790M mutated lung tumor growth, and genetic deletion of the ß-catenin gene dramatically reduced lung tumor formation in EGFR-L858R-T790M transgenic mice. These data suggest that ß-catenin plays an essential role in lung tumorigenesis and that targeting the ß-catenin pathway may provide novel strategies to prevent lung cancer development or overcome resistance to EGFR TKIs.

Identification and characterization of ALK kinase splicing isoforms in non-small-cell lung cancer.

INTRODUCTION: Anaplastic lymphoma kinase (ALK) rearrangements are present in an important subset of non-small-cell lung cancer (NSCLC) and predict for response to the tyrosine kinase inhibitor crizotinib. In this study, we evaluated the yet unknown frequency and functional role of ALK splicing isoforms in NSCLC. METHODS: We analyzed 270 cases of NSCLC for ALK kinase domain splicing aberrations and in addition generated constructs with full-length echinoderm microtubule-associated protein-like 4 (EML4)-ALK (E13;A20) and a splicing isoform. RESULTS: Splicing isoforms of the kinase domain of ALK-including complete skipping of exon 23 (ALKdel23, ALK p.I1171fs*42) and exon 27 (ALKdel27, ALK p.T1312fs*0)-were identified in 11.1% (30 of 270 cases) of NSCLC, and these changes coexisted with ALK rearrangements, KRAS mutations, and EGFR mutations. ALK splicing isoforms were observed with full-length EML4-ALK in crizotinib-naive and treated NSCLCs. ALK T1312fs*0 was unable to render cells solely dependent on ALK signaling. Unlike EML4-ALK and EML4-ALK p.L1196M, EML4-ALK T1312fs*0 did not autophosphorylate ALK or other phosphotyrosine sites. Coexpression of equal amounts of EML4-ALK T1312fs*0 and EML4-ALK did not result in resistance to crizotinib, whereas coexpression of EML4-ALK L1196M with EML4-ALK resulted in resistance to inhibition of ALK by crizotinib. CONCLUSIONS: ALK kinase splicing isoforms were present in NSCLC and even if translated seemed to be nonfunctional variants of ALK.