Vertebrate brain development is associated with prominent neuronal cell death and DNA breaks, but their causes and functions are not well understood. DNA transposable elements could contribute to somatic genome rearrangements; however, their contributions to brain development are largely unknown. PiggyBac transposable element derived 5 (PGBD5) is an evolutionarily conserved vertebrate DNA transposase-derived gene with DNA remodeling activities in human cells. Here, we show that PGBD5 contributes to normal brain development in mice and humans, and its deficiency causes disorder of intellectual disability, movement disorders, and epilepsy. In mice, Pgbd5 is required for the developmental induction of postmitotic DNA breaks and recurrent somatic brain genome rearrangements. In the cerebral cortex, loss of Pgbd5 leads to aberrant neuronal gene expression, including of specific types of glutamatergic neurons, which partly explains the features of PGBD5 deficiency in humans. Thus, PGBD5 is a transposase-derived gene required for brain development in mammals.

Cancer genome sequencing is essential for understanding tumor evolution and advancing precision medicine.1 However, reference gaps and germline variants obscure detection of small and large somatic variants and methylation in repetitive regions.1-3 It is common for tumor cells to gain or lose chromosome arms due to somatic structural changes that occur inside highly repetitive satellite DNA sequences in the centromeres.4 To identify the full spectrum of somatic variants, including complex rearrangements, we construct and curate near-complete, haplotype-resolved assemblies of the most recent common ancestor of an early-passage broadly-consented hypodiploid pancreatic cancer cell line and matched normal tissues. The tumor assembly completely recapitulates all 35 tumor chromosomes observed with karyotyping, with multiple translocation-induced hybrid chromosomes. The hybrid chromosomes contain putative functional dicentric and fused centromeres, nested foldback inversions causing 14 breakpoints with a haplotype switch in a single event, and centromeric satellite tandem duplications up to 136 kbp. Direct comparison of tumor and normal assembly haplotypes uncovers >7,000 variants altering >1 Mbp of sequence in repetitive regions that have been hidden by reference gaps and germline variants. 44 % of somatic small variants change representation because they alter germline variants on GRCh38, impacting mutational signatures and kataegis/omikli clusters. Most somatic LINE insertions originate from two hypomethylated non-reference germline LINE insertions, highlighting their impact on insertion mutation burden. These assemblies demonstrate that centromeric, acrocentric, and telomeric regions conventionally excluded from analysis harbor extensive somatic and epigenetic changes. Resolving complete tumor genomes enables a deeper understanding of cancer structural plasticity and the endpoints of breakage-fusion-bridge cycles. These assembled, curated paired normal-tumor benchmarks will serve as a critical foundation for developing future algorithms to characterize the most intractable regions of cancer genomes.

The International Myeloma Society and International Myeloma Working Group recently updated the definition of high-risk multiple myeloma and for the first time this definition includes genomic features that can only be detected by DNA sequencing. As a consequence, the working group recommended clinical testing should transition from FISH to DNA sequencing-based technologies that can detect the translocations, copy number alterations, and coding mutations used to calculate risk along with beta-2-microglobulin levels. To support this transition, we characterized the translocation breakpoints in 68 commercially or publicly available myeloma cell lines to create a gold-standard reference set for the community. This was then used to evaluate a series of different structural callers or dedicated immunoglobulin translocation calling tools. Additionally, as part of the clinical validation of a rapid whole genome sequencing platform, we established the accuracy, limit of detection and precision of our platform and analytical workflow for the detection of immunoglobulin translocations. All cell lines and patient samples used for clinical validation were sequenced on a NovaSeq X Plus using PCR-free libraries sequenced to 30-40x and >118x, respectively. To confirm complex events in the cell lines, we performed long-read sequencing using a PacBio Revio with libraries selected to have inserts exceeding 15kb. We tested the ability of Manta v1.6, IgCaller v1.3, DRAGEN v4.4.6, and SCITAV v0.6.5 to call the individual derivative chromosomes or at least any one derivative from a given translocation. Within the panel of myeloma cell lines, we detected 160 junctions from 92 distinct balanced or unbalanced translocation events in 66 cell lines between a common target gene in myeloma (NSD2, CCND3, MYC, MAFA, CCND1, CCND2, MAF, MAFB) and one of the immunoglobulin loci (IgH, IgK, IgL). Of the individual junctions, we found 9 with breakpoints proximal but outside of the immunoglobulin loci, 15 with insertions of a tertiary part of the genome ranging from 42bp-338kb and one with an immunoglobulin breakpoint containing 565bp of novel sequence. Approximately, 5.6% of derivative chromosome junctions will be undetectable by standard short-read sequencing approaches given the 8/15 events with insertions exceeding a typical WGS library or the one with unmappable sequence. Although 7/15 events with insertions under 260bp could likely be detected with some whole genome sequencing tests, we limited our comparison of bioinformatics approaches to the 135 junctions from 82 translocations that are within the immunoglobulin loci and do not contain defined insertions at the junctions. Using each calling tool in a tumor-only mode, the individual derivative call rate ranged from 66.7% (Manta) to 97.8% (SCITAV), with IgCaller detecting 76.3% while DRAGEN detected 88.2%. Since the majority of translocations are balanced events with two detectable derivatives, we assessed which percentage of translocations are detectable when at least one derivative is detected. This raised the detection rate range from 81.7% (Manta) to 100% (SCITAV), with IgCaller detecting 87.8% and DRAGEN 91.5%. A common theme in undetected breakpoints was immunoglobulin windows with low mapping quality resulting in read pairs being distributed between homologous regions, which resulted in the same event being called multiple times or these reads being ignored, leading to a missed call. For clinical validation we compared the WGS results with gold standard FISH assays from each patient and SCITAV had a sensitivity and specificity of 100%, while DRAGEN had a sensitivity of 94.4% and specificity of 100%. The limit of detection for SCITAV was established to be 6% VAF by diluting tumor DNA into matched normal DNA and additional precision replicates at this VAF resulted in a 96% recovery of calls across all replicates. We have shown that the majority of immunoglobulin translocations are detectable with WGS, but comprehensive detection requires optimized approaches to overcome mapping quality issues existing within immunoglobulin loci. These gold standards and particularly the difficult to detect events can be used for the establishment of clinical sequencing assays for multiple myeloma, which will provide more uniform and complete risk assessments and hold the promise of impacting patient care by identifying novel therapeutic options or predicting if specific immunotherapies will be effective.

Despite the potential of targeted epigenetic therapies, most cancers do not respond to current epigenetic drugs. The polycomb repressive complex EZH2 inhibitor tazemetostat was recently approved for the treatment of SMARCB1-deficient epithelioid sarcomas, based on the functional antagonism between PRC2 and SMARCB1. Through the analysis of tumors of patients treated with tazemetostat, we recently defined key principles of their response and resistance to EZH2 epigenetic therapy. Here, using transcriptomic inference from SMARCB1-deficient tumor cells, we nominate the DNA damage repair kinase ATR as a target for rational EZH2 combination epigenetic therapy. We showed that EZH2 inhibition promotes DNA damage in epithelioid and rhabdoid tumor cells, at least in part via its induction of piggyBac transposable element derived 5 (PGBD5). We leveraged this collateral synthetic lethal dependency to target PGBD5-dependent DNA damage by inhibition of ATR, but not CHK1, using the ATR inhibitor elimusertib. Consequently, combined EZH2 and ATR inhibition improved therapeutic responses in diverse patient-derived epithelioid and rhabdoid tumors in vivo. This advances a combination epigenetic therapy based on EZH2-PGBD5 synthetic lethal dependency suitable for immediate translation to clinical trials for patients.

Oligonucleotide Table: Complete list of gRNAs and primers used in this study.

Supplementary Table S1: List of patient tumor specimens used for RNA-seq and MSK-IMPACT analysis. *We note that two primary tumors in patients who responded to TAZ harbored deletions of RB1 (patient 2, sample ES_02_T_02) in one tumor and CDKN2A/B in another tumor (patient 5, sample ES_05_T_01). However, these primary tumors were fully resected prior to the initiation of TAZ treatment and did not recur at the primary sites. In the case of patient 2, a later TAZ-responsive metastasis (ES_2_T_03) did not harbor the RB1 loss. In the case of patient 5, a later TAZ-responsive metastasis (ES_05_T_09) did not harbor the CDKN2A/B loss. This suggests that these mutations were subclonal and were not present in tumors exposed to TAZ treatment. Thus, the mutations in these tumors were unlikely to have impacted their response to TAZ. Supplementary Table S2: List of mutations found in all patient tumor specimens in Supplementary table 1 for which MSK-IMPACT data is available.

Uncropped images of all western blots used in this study.

Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

Supplementary Table S6: List of endogenous transposable elements whose expression is up- or downregulated by TAZ treatment. Related to Supplementary Figure S12.

Supplementary Table S3: List of Hallmark gene sets identified by Gene Set Enrichment Analysis (GSEA) up- and down-regulated by TAZ treatment. Related to Supplementary Figure S2C.

Research and medical genomics require comprehensive, scalable methods for the discovery of novel disease targets, evolutionary drivers and genetic markers with clinical significance. This necessitates a framework to identify all types of variants independent of their size or location. Here we present DRAGEN, which uses multigenome mapping with pangenome references, hardware acceleration and machine learning-based variant detection to provide insights into individual genomes, with similar to 30 min of computation time from raw reads to variant detection. DRAGEN outperforms current state-of-the-art methods in speed and accuracy across all variant types (single-nucleotide variations, insertions or deletions, short tandem repeats, structural variations and copy number variations) and incorporates specialized methods for analysis of medically relevant genes. We demonstrate the performance of DRAGEN across 3,202 whole-genome sequencing datasets by generating fully genotyped multisample variant call format files and demonstrate its scalability, accuracy and innovation to further advance the integration of comprehensive genomics. Overall, DRAGEN marks a major milestone in sequencing data analysis and will provide insights across various diseases, including Mendelian and rare diseases, with a highly comprehensive and scalable platform.

Figure S1: Validation of tumor resistance mutations of EZH2. Figure S2: RB1del cells show morphological and transcriptional responses to TAZ. Figure S3: TAZ-treated RB1del cells show evidence of differentiation at the transcript, but not protein level. Figure S4: RB1del cells show increased expression of E2F targets. Figure S5: Characterization of MRT and ES cell lines. Figure S6: Generating and testing CDKN1Adel and CDKN2Adel G401 cells. Figure S7: TAZ-resistant patient tumors show upregulation of cell cycle genes. Figure S8: Transcriptomic analysis of patient tumors nominates putative biomarkers of TAZ sensitivity and resistance. Figure S9: Downstream cell cycle inhibitors overcome resistance to TAZ. Figure S10: TAZ + barasertib increases cell cycle arrest without inducing apoptosis. Figure S11: p16 induction correlates with TAZ response in vivo. Figure S12: Induction of immune-related genes and endogenous transposable elements by TAZ. Figure S13: TAZ may remodel BAF and PRC2 composition by transcriptional regulation of their subunits.

Essential epigenetic dependencies have become evident in many cancers. Based on the functional antagonism between BAF/SWI/SNF and PRC2 in SMARCB1-deficient sarcomas, we and colleagues recently completed the clinical trial of the EZH2 inhibitor tazemetostat. However, the principles of tumor response to epigenetic therapy in general, and tazemetostat in particular, remain unknown. Using functional genomics of patient tumors and diverse experimental models, we sought to define molecular mechanisms of tazemetostat resistance in SMARCB1-deficient sarcomas and rhabdoid tumors. We found distinct classes of acquired mutations that converge on the RB1/E2F axis and decouple EZH2-dependent differentiation and cell cycle control. This allows tumor cells to escape tazemetostat-induced G1 arrest despite EZH2 inhibition, and suggests a general mechanism for effective EZH2 therapy. This also enables us to develop combination strategies to circumvent tazemetostat resistance using cell cycle bypass targeting via AURKB, and synthetic lethal targeting of PGBD5-dependent DNA damage repair via ATR. This reveals prospective biomarkers for therapy stratification, including PRICKLE1 associated with tazemetostat resistance. In all, this work offers a paradigm for rational epigenetic combination therapy suitable for immediate translation to clinical trials for epithelioid sarcomas, rhabdoid tumors, and other epigenetically dysregulated cancers.

Supplementary Table S4: List of mutations found in all MRT and ES cell lines used in this study as determined by targeted MSK-IMPACT sequencing. Related to Figure 3A. Supplementary Table S5: List of PDX models used in this study, with clinical characteristics of the original tumor specimens, followed by a list of mutations found in all PDX models, as determined by targeted MSK-IMPACT sequencing.

Research and medical genomics require comprehensive and scalable solutions to drive the discovery of novel disease targets, evolutionary drivers, and genetic markers with clinical significance. This necessitates a framework to identify all types of variants independent of their size (e.g., SNV/SV) or location (e.g., repeats). Here we present DRAGEN that utilizes novel methods based on multigenomes, hardware acceleration, and machine learning based variant detection to provide novel insights into individual genomes with ~30min computation time (from raw reads to variant detection). DRAGEN outperforms all other state-of-the-art methods in speed and accuracy across all variant types (SNV, indel, STR, SV, CNV) and further incorporates specialized methods to obtain key insights in medically relevant genes (e.g., HLA, SMN, GBA). We showcase DRAGEN across 3,202 genomes and demonstrate its scalability, accuracy, and innovations to further advance the integration of comprehensive genomics for research and medical applications.

DNA transposable elements and transposase-derived genes are present in most living organisms, including vertebrates, but their function is largely unknown. PiggyBac Transposable Element Derived 5 (PGBD5) is the oldest evolutionarily conserved DNA transposase-derived gene with retained nuclease activity in vertebrates. Vertebrate brain development is known to be associated with prominent neuronal cell death and DNA breaks, but their causes and functions are not well understood. Here, we show that PGBD5 contributes to normal brain development in mice and humans, where its deficiency causes disorder of intellectual disability, movement and seizures. In mice, Pgbd5 is required for the developmental induction of post-mitotic DNA breaks and recurrent somatic genome rearrangements in neurons. Together, these studies nominate PGBD5 as the long-hypothesized neuronal DNA nuclease required for brain function in mammals.

Complex somatic genomic rearrangements and copy number alterations are hallmarks of nearly all cancers. We have developed an algorithm, LINX, to aid interpretation of structural variant and copy number data derived from short-read, whole-genome sequencing. LINX classifies raw structural variant calls into distinct events and predicts their effect on the local structure of the derivative chromosome and the functional impact on affected genes. Visualizations facilitate further investigation of complex rearrangements. LINX allows insights into a diverse range of structural variation events and can reliably detect pathogenic rearrangements, including gene fusions, immunoglobulin enhancer rearrangements, intragenic deletions, and duplications. Uniquely, LINX also predicts chained fusions that we demonstrate account for 13% of clinically relevant oncogenic fusions. LINX also reports a class of inactivation events that we term homozygous disruptions that may be a driver mutation in up to 9% of tumors and may frequently affect PTEN, TP53, and RB1.

e21545 Background: Nearly 1.5 million cases of cutaneous squamous cell cancer (cSCC) are diagnosed annually in the United States. Approximately 1% of cases are in the advanced unresectable stage. The standard of care in unresectable advanced disease is immunotherapy with anti-PD-1 agents. Cemiplimab, an anti-PD-1 antibody, was approved by the Food and Drug Administration in 2018. We present real-world clinical data from a single institution on outcomes and safety for patients treated with cemiplimab for metastatic and unresectable cSCC. Methods: We conducted a retrospective cohort analysis of patients treated with cemiplimab (350 mg every 3 weeks) for advanced cSCC from September 2018 until December 2021. Data included patient demographics, tumor characteristics, and treatment course. Patients who received at least 1 dose of cemiplimab and one clinic follow-up were included for analysis. Clinical response and adverse events (AE) were evaluated. Results: Of the 26 patients treated, 84% were male (n = 22), and 16% were female (n = 4) with a median age of 74 years (39-94). Twenty-three (23) patients were included in the final analysis as 3 patients transferred care to an outside facility and were not available for follow-up. Nineteen (19) patients (73%) had locally advanced disease and 7 (27%) had metastatic disease. The most common site of the primary tumor was head/neck (85%, n = 22) with all remaining cases on the trunk/extremities (15%, n = 4). Patients received a median of 7 doses of cemiplimab (range 1- 46). The median time to first response was 1.7 months which required a median of 2.5 doses. An objective clinical response defined as complete response (CR) or partial response (PR) was seen in 18/23 (78%) patients, of whom 8/23(35%) were CR and 10/23 (44%) PR. The ongoing response was noted in 6/23 (27%) with continued treatment. Treatment was well-tolerated with 6/23 (26%) adverse events (AE) all of which were grade 1 or 2: 1 (4%) grade 1 pruritus, 5 (21.7%) grade 2. Of the 5 grade 2 AEs, 1 was infusion-related, 2 hypothyroidism, 1 dermatitis, 1 rheumatoid arthritis flare. No grade 3-5 AEs were noted. Conclusions: To the best of our knowledge, this is the first real-world outcomes study of advanced cSCC patients treated with cemiplimab in the United States. The overall efficacy, response rate, and safety profile were better in comparison to the results of the EMPOWER-CSCC-1 trial.[Table: see text]