The Johns Hopkins Physician-Scientist Training Program to Enhance Institutional Retention and Entry Into Academic Positions: An Evaluation of Program Effectiveness and Outcomes.

PURPOSE: The Johns Hopkins Physician-Scientist Training Program (PSTP) was implemented to overcome well-documented challenges in training and retaining physician-scientists by providing physician-scientist pathway training for residents and clinical fellows. The program's core tenets include monthly seminars, individualized feedback on project proposals, access to mentors, and institutional funding opportunities. This study evaluated the effectiveness and outcomes of the PTSP and provides a framework for replication. METHOD: A query of institutional demographic data and bibliometric variables of the PSTP participants (2017-2020) at a single academic medical center was conducted in 2021. In addition, a voluntary survey collected personal and program evaluation information. RESULTS: Of 145 PSTP scholars, 59 (41%) were women, and 41 (31%), 8 (6%), and 6 (5%) of scholars self-identified as Asian, Hispanic, and Black, respectively. Thirty-three (23%) scholars received PSTP research support or career development microgrants. Of 66 PSTP graduates, 29 (44%) remained at Johns Hopkins as clinical fellows or faculty. Of 48 PSTP graduates in a post-training position, 42 (88%) were in academia, with the majority, 29 (76%), holding the rank of assistant professor. Fifty-nine of 140 available participants responded to the survey (42% response rate). The top-cited reason for joining the PSTP was exposure to mentors and administration (50/58 respondents, 86%), followed by seeking scholarly opportunities (37/58 respondents, 64%). Most scholars intended to continue a career as a physician-scientist. CONCLUSIONS: The PSTP provides internal research support and institutional oversight. Although establishing close mentor-mentee relationships requires individualized approaches, the PSTP provided structured academic pathways that enhanced participating scholars' ability to apply for grants and jobs. The vast majority continued their careers as physician-scientists after training. In light of the national evidence of a "leaky physician-scientist pipeline," programs such as the PSTP can be critical to entry into early academic career positions and institutional retention.

Optimization of Advanced Molecular Genetic Testing Utilization in Hematopathology: A Goldilocks Approach to Bone Marrow Testing.

PURPOSE: This study investigated the effectiveness of algorithmic testing in hematopathology at the Brigham and Women's Hospital and Dana-Farber Cancer Institute (DFCI). The algorithm was predicated on test selection after an initial pathologic evaluation to maximize cost-effective testing, especially for expensive molecular and cytogenetic assays. MATERIALS AND METHODS: Standard ordering protocols (SOPs) for 17 disease categories were developed and encoded in a decision support application. Six months of retrospective data from application beta testing was obtained and compared with actual testing practices during that timeframe. In addition, 2 years of prospective data were also obtained from patients at one community satellite site. RESULTS: A total of 460 retrospective cases (before introduction of algorithmic testing) and 109 prospective cases (following introduction) were analyzed. In the retrospective data, 61.7% of tests (509 of 825) were concordant with the SOPs while 38.3% (316 of 825) were overordered and 30.8% (227 of 736) of SOP-recommended tests were omitted. In the prospective data, 98.8% of testing was concordant (244 of 247 total tests) with only 1.2% overordered tests (3 of 247) and 7.6% omitted tests (20 of 264 SOP-recommended tests; overall P < .001). The cost of overordered tests before implementing SOP indicates a potential annualized saving of $1,347,520 in US dollars (USD) in overordered testing at Brigham and Women's Hospital/DFCI. Only two of 316 overordered tests (0.6%) returned any additional information, both for extremely rare clinical circumstances. CONCLUSION: Implementation of SOPs dramatically improved test ordering practices, with a just right number of ancillary tests that minimizes cost and has no significant impact on acquiring key informative test results.

Structures, functions and adaptations of the human LINE-1 ORF2 protein.

The LINE-1 (L1) retrotransposon is an ancient genetic parasite that has written around one-third of the human genome through a 'copy and paste' mechanism catalysed by its multifunctional enzyme, open reading frame 2 protein (ORF2p)1. ORF2p reverse transcriptase (RT) and endonuclease activities have been implicated in the pathophysiology of cancer2,3, autoimmunity4,5 and ageing6,7, making ORF2p a potential therapeutic target. However, a lack of structural and mechanistic knowledge has hampered efforts to rationally exploit it. We report structures of the human ORF2p 'core' (residues 238-1061, including the RT domain) by X-ray crystallography and cryo-electron microscopy in several conformational states. Our analyses identified two previously undescribed folded domains, extensive contacts to RNA templates and associated adaptations that contribute to unique aspects of the L1 replication cycle. Computed integrative structural models of full-length ORF2p show a dynamic closed-ring conformation that appears to open during retrotransposition. We characterize ORF2p RT inhibition and reveal its underlying structural basis. Imaging and biochemistry show that non-canonical cytosolic ORF2p RT activity can produce RNA:DNA hybrids, activating innate immune signalling through cGAS/STING and resulting in interferon production6-8. In contrast to retroviral RTs, L1 RT is efficiently primed by short RNAs and hairpins, which probably explains cytosolic priming. Other biochemical activities including processivity, DNA-directed polymerization, non-templated base addition and template switching together allow us to propose a revised L1 insertion model. Finally, our evolutionary analysis demonstrates structural conservation between ORF2p and other RNA- and DNA-dependent polymerases. We therefore provide key mechanistic insights into L1 polymerization and insertion, shed light on the evolutionary history of L1 and enable rational drug development targeting L1.

LINE-1 retrotransposition and its deregulation in cancers: implications for therapeutic opportunities.

Long interspersed element 1 (LINE-1) is the only protein-coding transposon that is active in humans. LINE-1 propagates in the genome using RNA intermediates via retrotransposition. This activity has resulted in LINE-1 sequences occupying approximately one-fifth of our genome. Although most copies of LINE-1 are immobile, ∼100 copies are retrotransposition-competent. Retrotransposition is normally limited via epigenetic silencing, DNA repair, and other host defense mechanisms. In contrast, LINE-1 overexpression and retrotransposition are hallmarks of cancers. Here, we review mechanisms of LINE-1 regulation and how LINE-1 may promote genetic heterogeneity in tumors. Finally, we discuss therapeutic strategies to exploit LINE-1 biology in cancers.

Transforming ovarian cancer care by targeting minimal residual disease.

Frontline treatment and resultant cure rates in patients with advanced ovarian cancer have changed little over the past several decades. Here, we outline a multidisciplinary approach aimed at gaining novel therapeutic insights by focusing on the poorly understood minimal residual disease phase of ovarian cancer that leads to eventual incurable recurrences.

Meeting report: transposable elements at the crossroads of evolution, health and disease 2023.

The conference "Transposable Elements at the Crossroads of Evolution, Health and Disease" was hosted by Keystone Symposia in Whistler, British Columbia, Canada, on September 3-6, 2023, and was organized by Kathleen Burns, Harmit Malik and Irina Arkhipova. The central theme of the meeting was the incredible diversity of ways in which transposable elements (TEs) interact with the host, from disrupting the existing genes and pathways to creating novel gene products and expression patterns, enhancing the repertoire of host functions, and ultimately driving host evolution. The meeting was organized into six plenary sessions and two afternoon workshops with a total of 50 invited and contributed talks, two poster sessions, and a career roundtable. The topics ranged from TE roles in normal and pathological processes to restricting and harnessing TE activity based on mechanistic insights gained from genetic, structural, and biochemical studies.

Proceedings of the inaugural Dark Genome Symposium: November 2022.

In November 2022 the first Dark Genome Symposium was held in Boston, USA. The meeting was hosted by Rome Therapeutics and Enara Bio, two biotechnology companies working on translating our growing understanding of this vast genetic landscape into therapies for human disease. The spirit and ambition of the meeting was one of shared knowledge, looking to strengthen the network of researchers engaged in the field. The meeting opened with a welcome from Rosana Kapeller and Kevin Pojasek followed by a first session of field defining talks from key academics in the space. A series of panels, bringing together academia and industry views, were then convened covering a wide range of pertinent topics. Finally, Richard Young and David Ting gave their views on the future direction and promise for patient impact inherent in the growing understanding of the Dark Genome.

Vitamin C activates young LINE-1 elements in mouse embryonic stem cells via H3K9me3 demethylation.

BACKGROUND: Vitamin C (vitC) enhances the activity of 2-oxoglutarate-dependent dioxygenases, including TET enzymes, which catalyse DNA demethylation, and Jumonji-domain histone demethylases. The epigenetic remodelling promoted by vitC improves the efficiency of induced pluripotent stem cell derivation, and is required to attain a ground-state of pluripotency in embryonic stem cells (ESCs) that closely mimics the inner cell mass of the early blastocyst. However, genome-wide DNA and histone demethylation can lead to upregulation of transposable elements (TEs), and it is not known how vitC addition in culture media affects TE expression in pluripotent stem cells. RESULTS: Here we show that vitC increases the expression of several TE families, including evolutionarily young LINE-1 (L1) elements, in mouse ESCs. We find that TET activity is dispensable for L1 upregulation, and that instead it occurs largely as a result of H3K9me3 loss mediated by KDM4A/C histone demethylases. Despite increased L1 levels, we did not detect increased somatic insertion rates in vitC-treated cells. Notably, treatment of human ESCs with vitC also increases L1 protein levels, albeit through a distinct, post-transcriptional mechanism. CONCLUSION: VitC directly modulates the expression of mouse L1s and other TEs through epigenetic mechanisms, with potential for downstream effects related to the multiple emerging roles of L1s in cellular function.

The landscape of human SVA retrotransposons.

SINE-VNTR-Alu (SVA) retrotransposons are evolutionarily young and still-active transposable elements (TEs) in the human genome. Several pathogenic SVA insertions have been identified that directly mutate host genes to cause neurodegenerative and other types of diseases. However, due to their sequence heterogeneity and complex structures as well as limitations in sequencing techniques and analysis, SVA insertions have been less well studied compared to other mobile element insertions. Here, we identified polymorphic SVA insertions from 3646 whole-genome sequencing (WGS) samples of >150 diverse populations and constructed a polymorphic SVA insertion reference catalog. Using 20 long-read samples, we also assembled reference and polymorphic SVA sequences and characterized the internal hexamer/variable-number-tandem-repeat (VNTR) expansions as well as differing SVA activity for SVA subfamilies and human populations. In addition, we developed a module to annotate both reference and polymorphic SVA copies. By characterizing the landscape of both reference and polymorphic SVA retrotransposons, our study enables more accurate genotyping of these elements and facilitate the discovery of pathogenic SVA insertions.

Oncogenic Transformation Drives DNA Methylation Loss and Transcriptional Activation at Transposable Element Loci.

Transposable elements (TE) are typically silenced by DNA methylation and repressive histone modifications in differentiated healthy human tissues. However, TE expression increases in a wide range of cancers and is correlated with global hypomethylation of cancer genomes. We assessed expression and DNA methylation of TEs in fibroblast cells that were serially transduced with hTERT, SV40, and HRASR24C to immortalize and then transform them, modeling the different steps of the tumorigenesis process. RNA sequencing and whole-genome bisulfite sequencing were performed at each stage of transformation. TE expression significantly increased as cells progressed through transformation, with the largest increase in expression after the final stage of transformation, consistent with data from human tumors. The upregulated TEs were dominated by endogenous retroviruses [long terminal repeats (LTR)]. Most differentially methylated regions (DMR) in all stages were hypomethylated, with the greatest hypomethylation in the final stage of transformation. A majority of the DMRs overlapped TEs from the RepeatMasker database, indicating that TEs are preferentially demethylated. Many hypomethylated TEs displayed a concordant increase in expression. Demethylation began during immortalization and continued into transformation, while upregulation of TE transcription occurred in transformation. Numerous LTR elements upregulated in the model were also identified in The Cancer Genome Atlas datasets of breast, colon, and prostate cancer. Overall, these findings indicate that TEs, specifically endogenous retroviruses, are demethylated and transcribed during transformation. SIGNIFICANCE: Analysis of epigenetic and transcriptional changes in a transformation model reveals that transposable element expression and methylation are dysregulated during oncogenic transformation.

LINE-1 ORF1p as a candidate biomarker in high grade serous ovarian carcinoma.

Long interspersed element 1 (LINE-1) open reading frame 1 protein (ORF1p) expression is a common feature of many cancer types, including high-grade serous ovarian carcinoma (HGSOC). Here, we report that ORF1p is not only expressed but also released by ovarian cancer and primary tumor cells. Immuno-multiple reaction monitoring-mass spectrometry assays showed that released ORF1p is confidently detectable in conditioned media, ascites, and patients' plasma, implicating ORF1p as a potential biomarker. Interestingly, ORF1p expression is detectable in fallopian tube (FT) epithelial precursors of HGSOC but not in benign FT, suggesting that ORF1p expression in an early event in HGSOC development. Finally, treatment of FT cells with DNA methyltransferase inhibitors led to robust expression and release of ORF1p, validating the regulatory role of DNA methylation in LINE-1 repression in non-tumorigenic tissue.

Retrotransposon insertions associated with risk of neurologic and psychiatric diseases.

Transposable elements (TEs) are active in neuronal cells raising the question whether TE insertions contribute to risk of neuropsychiatric disease. While genome-wide association studies (GWAS) serve as a tool to discover genetic loci associated with neuropsychiatric diseases, unfortunately GWAS do not directly detect structural variants such as TEs. To examine the role of TEs in psychiatric and neurologic disease, we evaluated 17,000 polymorphic TEs and find 76 are in linkage disequilibrium with disease haplotypes (P < 10-6 ) defined by GWAS. From these 76 polymorphic TEs, we identify potentially causal candidates based on having insertions in genomic regions of regulatory chromatin and on having associations with altered gene expression in brain tissues. We show that lead candidate insertions have regulatory effects on gene expression in human neural stem cells altering the activity of a minimal promoter. Taken together, we identify 10 polymorphic TE insertions that are potential candidates on par with other variants for having a causal role in neurologic and psychiatric disorders.

Frequency and mechanisms of LINE-1 retrotransposon insertions at CRISPR/Cas9 sites.

CRISPR/Cas9-based genome editing has revolutionized experimental molecular biology and entered the clinical world for targeted gene therapy. Identifying DNA modifications occurring at CRISPR/Cas9 target sites is critical to determine efficiency and safety of editing tools. Here we show that insertions of LINE-1 (L1) retrotransposons can occur frequently at CRISPR/Cas9 editing sites. Together with PolyA-seq and an improved amplicon sequencing, we characterize more than 2500 de novo L1 insertions at multiple CRISPR/Cas9 editing sites in HEK293T, HeLa and U2OS cells. These L1 retrotransposition events exploit CRISPR/Cas9-induced DSB formation and require L1 RT activity. Importantly, de novo L1 insertions are rare during genome editing by prime editors (PE), cytidine or adenine base editors (CBE or ABE), consistent with their reduced DSB formation. These data demonstrate that insertions of retrotransposons might be a potential outcome of CRISPR/Cas9 genome editing and provide further evidence on the safety of different CRISPR-based editing tools.

Repetitive DNA in disease.

Transposons become a focus of speculation and scrutiny in biomedical research.

Reverse Transcriptase Inhibition Disrupts Repeat Element Life Cycle in Colorectal Cancer.

Altered RNA expression of repetitive sequences and retrotransposition are frequently seen in colorectal cancer, implicating a functional importance of repeat activity in cancer progression. We show the nucleoside reverse transcriptase inhibitor 3TC targets activities of these repeat elements in colorectal cancer preclinical models with a preferential effect in p53-mutant cell lines linked with direct binding of p53 to repeat elements. We translate these findings to a human phase II trial of single-agent 3TC treatment in metastatic colorectal cancer with demonstration of clinical benefit in 9 of 32 patients. Analysis of 3TC effects on colorectal cancer tumorspheres demonstrates accumulation of immunogenic RNA:DNA hybrids linked with induction of interferon response genes and DNA damage response. Epigenetic and DNA-damaging agents induce repeat RNAs and have enhanced cytotoxicity with 3TC. These findings identify a vulnerability in colorectal cancer by targeting the viral mimicry of repeat elements. SIGNIFICANCE: Colorectal cancers express abundant repeat elements that have a viral-like life cycle that can be therapeutically targeted with nucleoside reverse transcriptase inhibitors (NRTI) commonly used for viral diseases. NRTIs induce DNA damage and interferon response that provide a new anticancer therapeutic strategy. This article is highlighted in the In This Issue feature, p. 1397.

Visualizing surface marker expression and intratumoral heterogeneity with SERRS-NPs imaging.

Cell surface marker expression in tumors dictates the selection of therapeutics, therapy response, and survival. However, biopsies are invasive, sample only a small area of the tumor landscape and may miss significant areas of heterogeneous expression. Here, we investigated the potential of antibody-conjugated surface-enhanced resonance Raman scattering nanoparticles (SERRS-NPs) to depict and quantify high and low tumoral surface marker expression, focusing on the surface markers epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in an intracerebral and peripheral setting with an inter- and intratumoral comparison of Raman signal intensities. Methods: ICR-Prkdc mice were injected with glioblastoma, epidermoid carcinoma, or breast tumor cell lines intracerebrally and peripherally. SERRS-NPs were functionalized with cetuximab or trastuzumab and administered via tail vein injection. Raman imaging was performed 18 hours post-injection in excised tumors and in vivo through the skull. Tumors were then fixed and processed for immunohistochemical evaluation. Results: Confirmed by MRI and immunohistochemistry for EGFR and HER2, our results demonstrate that antibody-conjugated SERRS-NPs go beyond the delineation of a tumor and offer clear and distinct Raman spectra that reflect the distribution of the targeted surface marker. The intensity of the SERRS-NP signal accurately discriminated high- versus low-expressing surface markers between tumors, and between different areas within tumors. Conclusion: Biopsies can be highly invasive procedures and provide a limited sample of molecular expression within a tumor. Our nanoparticle-based Raman imaging approach offers the potential to provide non-invasive and more comprehensive molecular imaging and an alternative to the current clinical gold standard of immunohistochemistry.

LINE-1 expression in cancer correlates with p53 mutation, copy number alteration, and S phase checkpoint.

Retrotransposons are genomic DNA sequences that copy themselves to new genomic locations via RNA intermediates; LINE-1 is the only active and autonomous retrotransposon in the human genome. The mobility of LINE-1 is largely repressed in somatic tissues but is derepressed in many cancers, where LINE-1 retrotransposition is correlated with p53 mutation and copy number alteration (CNA). In cell lines, inducing LINE-1 expression can cause double-strand breaks (DSBs) and replication stress. Reanalyzing multiomic data from breast, ovarian, endometrial, and colon cancers, we confirmed correlations between LINE-1 expression, p53 mutation status, and CNA. We observed a consistent correlation between LINE-1 expression and the abundance of DNA replication complex components, indicating that LINE-1 may also induce replication stress in human tumors. In endometrial cancer, high-quality phosphoproteomic data allowed us to identify the DSB-induced ATM-MRN-SMC S phase checkpoint pathway as the primary DNA damage response (DDR) pathway associated with LINE-1 expression. Induction of LINE-1 expression in an in vitro model led to increased phosphorylation of MRN complex member RAD50, suggesting that LINE-1 directly activates this pathway.

Autoantibodies targeting LINE-1-encoded ORF1p are associated with systemic lupus erythematosus diagnosis but not disease activity.

OBJECTIVES: Long Interspersed Element 1 (LINE-1) is an endogenous retroelement that constitutes a significant portion of the human genome and has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). The LINE-1 RNA chaperone protein ORF1p was recently identified as an SLE autoantigen. Here we analyse ORF1p for qualities underlying SLE autoantigen status, compared anti-ORF1p antibodies to markers of SLE disease activity, and performed screening for antibodies against LINE-1 reverse transcriptase ORF2p. METHODS: ORF1p was examined in epithelial cell lines treated with cytotoxic lymphocyte granules and UV irradiation. Anti-ORF1p and anti-ORF2p antibodies were assayed by ELISA and analysed in two SLE cohorts. RESULTS: We found that ORF1p localises to cytoplasmic RNA-containing blebs in apoptotic cells, and is a substrate of the cytotoxic protease granzyme B (GrB). Anti-ORF1p antibodies were present in 4.2% of healthy controls, compared to 15.8% (p=0.0157) and 15.5% (p=0.036) of subjects in the two SLE cohorts. Anti-ORF1p antibodies were not associated with SLE disease activity nor peripheral blood markers of interferon (IFN) activation. Anti-ORF1p titres demonstrated stability over serial time points. Anti-ORF1p antibodies were not associated with anti-DNA, anti-RNP, or other SLE autoantibodies. There was no difference in anti-ORF2p ELISA results in controls versus SLE patients. CONCLUSIONS: LINE-1 ORF1p is a component of apoptotic blebs and a substrate for GrB. Anti-ORF1p antibodies are enriched in SLE subjects but are not associated with dynamic markers of disease activity. These data support a potential role for LINE-1 dysregulation in SLE pathogenesis.