LINE-1s are active human DNA parasites that are agents of genome dynamics in evolution and disease. These streamlined elements require host factors to complete their lifecycles, whereas hosts have developed mechanisms to combat retrotransposition’s mutagenic effects. As such, endogenous L1 expression levels are extremely low, creating a roadblock for detailed interactomic analyses. Here we describe a system to express and purify highly active L1 RNP complexes from human suspension cell culture and characterize the co-purified proteome, identifying 37 high-confidence candidate interactors. These datasets include known interactors PABPC1 and MOV10 and, with in-cell imaging studies, suggest existence of at least three types of compositionally and functionally distinct L1 RNPs. Among the novel findings, UPF1, a key nonsense-mediated decay factor, and PCNA, the polymerase-delta-associated sliding DNA clamp, were identified and validated. PCNA interacts with ORF2p via a PIP box motif; mechanistic studies suggest this occurs during or immediately after target-primed reverse transcription.

KRAS is the most frequently mutated human oncogene, and KRAS inhibition has been a longtime goal. Recently, inhibitors were developed that bind KRASG12C-GDP and react with Cys-12 (G12C-Is). Using new affinity reagents to monitor KRASG12C activation and inhibitor engagement, we found that an SHP2 inhibitor (SHP2-I) increases KRAS-GDP occupancy, enhancing G12C-I efficacy. The SHP2-I abrogated RTK feedback signaling and adaptive resistance to G12C-Is in vitro, in xenografts, and in syngeneic KRASG12C-mutant pancreatic ductal adenocarcinoma (PDAC) and non–small cell lung cancer (NSCLC). SHP2-I/G12C-I combination evoked favorable but tumor site–specific changes in the immune microenvironment, decreasing myeloid suppressor cells, increasing CD8+ T cells, and sensitizing tumors to PD-1 blockade. Experiments using cells expressing inhibitor-resistant SHP2 showed that SHP2 inhibition in PDAC cells is required for PDAC regression and remodeling of the immune microenvironment but revealed direct inhibitory effects on tumor angiogenesis and vascularity. Our results demonstrate that SHP2-I/G12C-I combinations confer a substantial survival benefit in PDAC and NSCLC and identify additional potential combination strategies.

LINE-1/L1 retrotransposon sequences comprise 17% of the human genome. Among the many classes of mobile genetic elements, L1 is the only autonomous retrotransposon that still drives human genomic plasticity today. Through its co-evolution with the human genome, L1 has intertwined itself with host cell biology. However, a clear understanding of L1’s lifecycle and the processes involved in restricting its insertion and intragenomic spread remains elusive. Here we identify modes of L1 proteins’ entrance into the nucleus, a necessary step for L1 proliferation. Using functional, biochemical, and imaging approaches, we also show a clear cell cycle bias for L1 retrotransposition that peaks during the S phase. Our observations provide a basis for novel interpretations about the nature of nuclear and cytoplasmic L1 ribonucleoproteins (RNPs) and the potential role of DNA replication in L1 retrotransposition.

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Retrotransposons are mutagenic units able to move within the genome. Despite many defenses deployed by the host to suppress potentially harmful activities of retrotransposons, these genetic units have found ways to meld with normal cellular functions through processes of exaptation and domestication. The same host mechanisms targeting transposon mobility allow for expansion and rewiring of gene regulatory networks on an evolutionary time scale. Recent works demonstrating retrotransposon activity during development, cell differentiation and neurogenesis shed new light on unexpected activities of transposable elements. Moreover, new technological advances illuminated subtler nuances of the complex relationship between retrotransposons and the host genome, clarifying the role of retroelements in evolution, development and impact on human disease.

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SignificanceRetrotransposons replicate through RNA intermediates that are reverse transcribed and inserted at new genomic locations. LINE-1 (L1) elements constitute ∼17% of the human genome, making them the most successful retrotransposons in the human genome by mass. The activity of L1s was shown first in the germline or during early embryogenesis. More recent studies demonstrate a wider prevalence of L1 expression in somatic cells including neurons, aging cells, and different types of cancer. In this study, we developed the MapRRCon pipeline and performed a comprehensive computational analysis of L1 transcriptional regulators using ENCODE ChIP-seq datasets. We revealed the binding of various transcription factors, including Myc and CTCF, to the 5′ UTR promoter of the youngest human L1 family (L1HS) and their potential functional impact on L1HS expression.

Long Interspersed Nuclear Element-1 (LINE-1, L1) is a mobile genetic element active in human genomes. L1-encoded ORF1 and ORF2 proteins bind L1 RNAs, forming ribonucleoproteins (RNPs). These RNPs interact with diverse host proteins, some repressive and others required for the L1 lifecycle. Using differential affinity purifications, quantitative mass spectrometry, and next generation RNA sequencing, we have characterized the proteins and nucleic acids associated with distinctive, enzymatically active L1 macromolecular complexes. Among them, we describe a cytoplasmic intermediate that we hypothesize to be the canonical ORF1p/ORF2p/L1-RNA-containing RNP, and we describe a nuclear population containing ORF2p, but lacking ORF1p, which likely contains host factors participating in target-primed reverse transcription.