Transposons are mobile genetic elements that can be cut out of and inserted into DNA. They are present in most living things and make up almost half of the human genome. Transposons help to rearrange and increase the variety of DNA sequences, which can drive evolution and regulate the expression of genes. Enzymes called transposases help to move transposons, but very few genes that encode these enzymes have been studied in humans.

PiggyBac transposase—which was first discovered in the cabbage looper moth—helps to move transposons of the piggyBac family. Humans and many other animals have genes that encode similar enzymes. In particular, the gene that encodes the human PGBD5 transposase is expressed in the developing embryo and particular areas of the brain and is highly similar to genes found in other vertebrate animals. These intriguing features prompted Henssen et al. to investigate PGBD5.

The experiments reveal that PGBD5 is able to move piggyBac-like transposons in human cells and insert them into sites that contain similar DNA sequences that are preferred by other PiggyBac transposases. Henssen et al. compared human PGBD5 to the piggyBac transposases from other organisms, including insects, bats, and frogs. They found that PGBD5 is deeply conserved among vertebrate organisms, and is distinct from other piggyBac transposases.

These findings suggest that PGBD5 is indeed a fully working piggyBac transposase. Further work is needed to understand what portions of the human genome may be rearranged by PGBD5, and how this may contribute to human brain development or disease.

Transposons are genetic elements that are found in nearly all living organisms (Feschotte and Pritham, 2007). They can contribute to the developmental and adaptive regulation of gene expression and are a major source of genetic variation that drives genome evolution (Cordaux and Batzer, 2009). In humans and other mammals, they comprise about half of the nuclear genome (Smit, 1999). The majority of primate-specific sequences that regulate gene expression are derived from transposons (Jacques et al., 2013), and transposons are a major source of structural genetic variation in human populations (Stewart et al., 2011).

While the majority of genes that encode transposase enzymes tend to become catalytically inactive and their transposon substrates tend to become immobile in the course of organismal evolution, some can maintain their transposition activities (Liu et al., 2007; Munoz-Lopez and Garcia-Perez, 2010). In humans, at least one hundred L1 long interspersed repeated sequences (LINEs) actively transpose in human genomes and induce structural variation (Kazazian, 2004), including somatic rearrangements in neurons that may contribute to neuronal plasticity (Erwin et al., 2014). The human Transib-like transposase RAG1 catalyzes somatic recombination of the V(D)J receptor genes in lymphocytes and is essential for adaptive immunity (Hiom et al., 1998). The Mariner-derived transposase SETMAR functions in single-stranded DNA resection during DNA repair and replication in human cells and can catalyze DNA transposition in vitro (Liu et al., 2007; Shaheen et al., 2010).

Among transposase enzymes that can catalyze excision and insertion of transposon sequences, DNA transposases are distinct in their dependence only on the availability of competent genomic substrates and cellular repair enzymes that ligate and repair excision sites, as compared to retrotransposons, which require transcription of the mobilized sequences (Berg and Howe, 1989). Most DNA transposases utilize an RNase H-like domain with three aspartate or glutamate residues (so-called DDD or DDE motif) that catalyze magnesium-dependent hydrolysis of phosphodiester bonds and strand exchange (Keith et al., 2008; Mitra et al., 2008; Dyda et al., 2012). The IS4 transposase family, which includes piggyBac transposases, is additionally distinguished by precise excisions without modifications of the transposon flanking sequences (De Palmenaer et al., 2008). The piggyBac transposase and its transposon were originally identified as an insertion in lepidopteran Trichoplusia ni cells (Fraser et al., 1985). The piggyBac transposon consists of 13-bp inverted terminal repeats (ITRs) and 19-bp subterminal inverted repeats located 3 and 31 base pairs from the 5′ and 3′ ITRs, respectively (Elick et al., 1997). PiggyBac transposase can mobilize a variety of ITR-flanked sequences and has a preference for integration at TTAA target sites in the host genome (Fraser et al., 1983; Beames and Summers, 1990; Wang and Fraser, 1993; Fraser et al., 1995; Elick et al., 1997; Handler et al., 1998; Mitra et al., 2008).

Members of the piggyBac superfamily of transposons have colonized a wide range of organisms (Sarkar et al., 2003), including a recent and likely ongoing invasion of the bat Myotis lucifugus (Mitra et al., 2013). The human genome contains five paralogous genes derived from piggyBac transposases, PGBD1-5 (Smit and Riggs, 1996; Sarkar et al., 2003). PGBD1 and PGBD2 invaded the common mammalian ancestor, and PGBD3 and PGBD4 are restricted to primates, but are all contained as single coding exons, fused in frame with endogenous host genes, such as the Cockayne syndrome B gene (CSB)-PGBD3 fusion (Sarkar et al., 2003; Newman et al., 2008). Thus far, only the function of PGBD3 has been investigated. CSB-PGBD3 is capable of binding DNA, including endogenous piggyBac-like transposons in the human genome, but has no known catalytic activity, though biochemical and genetic evidence indicates that it may participate in DNA damage response (Bailey et al., 2012; Gray et al., 2012).

PGBD5 is distinct from other human piggyBac-derived genes by having been domesticated much earlier in vertebrate evolution approximately 500 million years (My) ago, in the common ancestor of cephalochordates and vertebrates (Sarkar et al., 2003; Pavelitz et al., 2013). PGBD5 is transcribed as a multi-intronic but non-chimeric transcript predicted to encode a full-length transposase (Pavelitz et al., 2013). Furthermore, PGBD5 expression in both human and mouse appears largely restricted to the early embryo and certain areas of the embryonic and adult brain (Sarkar et al., 2003; Pavelitz et al., 2013). These intriguing features prompted us to investigate whether human PGBD5 has retained the enzymatic capability of mobilizing DNA.

Human PGBD5 contains a C-terminal RNase H-like domain that has approximately 20% sequence identity and 45% similarity to the active lepidopteran piggyBac, ciliate piggyMac, and mammalian piggyBat transposases (Figure 1) (Sarkar et al., 2003; Baudry et al., 2009; Mitra et al., 2013). In addition, the human genome contains 2358 sequence elements with similarity to the piggyBac transposable elements (Table 1 and Figure 2A). Specifically, MER75 (MER75, MER75A, MER75B) and MER85 elements show considerably similar ITR sequences as compared to lepidopteran piggyBac transposons (Table 2 and Figure 2B). A total of 328 piggyBac-like elements in the human genome have intact ITRs and exhibit duplications of their presumed TTAA target sites (Table 1 and Figure 2C). We reasoned that even though the ancestral transposon substrates of PGBD5 cannot be predicted due to its very ancient evolutionary origin (∼500 My), preservation of its transposase activity should confer residual ability to mobilize distantly related piggyBac-like transposons. To test this hypothesis, we used a synthetic transposon reporter PB-EF1-NEO comprised of a neomycin resistance gene flanked by T. ni piggyBac ITRs (Figure 3B) (Cary et al., 1989; Fraser et al., 1995). We transiently transfected human embryonic kidney (HEK) 293 cells, which lack endogenous PGBD5 expression with the PB-EF1-NEO transposon reporter plasmid in the presence of a plasmid expressing PGBD5, and assessed genomic integration of the reporter using clonogenic assays in the presence of G418 to select cells with genomic integration conferring neomycin resistance (Figure 3C, Figure 3—figure supplement 1). Given the absence of suitable antibodies to monitor PGBD5 expression, we expressed PGBD5 as an N-terminal fusion with the green fluorescent protein (GFP). We observed significant rates of neomycin resistance of cells conferred by the transposon reporter with GFP-PGBD5, but not in cells expressing control GFP or mutant GFP-PGBD5 lacking the transposase domain (Figure 3C), despite equal expression of all transgenes (Figure 3—figure supplement 2). The efficiency of neomycin resistance conferred by the transposon reporter with GFP-PGBD5 was approximately 4.5-fold less than that of the T. ni piggyBac-derived transposase (Figure 3D), consistent with their evolutionary divergence. These results suggest that human PGBD5 can promote genomic integration of a piggyBac-like transposon.

Figure 1

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If neomycin resistance conferred by the PGBD5 and the transposon reporter is due to genomic integration and DNA transposition, then this should require specific activity on the transposon ITRs. To test this hypothesis, we generated transposon reporters with mutant ITRs and assayed them for genomic integration (Figure 3B, Figure 3—figure supplement 3). DNA transposition by the piggyBac family transposases involves hairpin intermediates with a conserved 5′-GGGTTAACCC-3′ sequence that is required for target site phosphodiester hydrolysis (Mitra et al., 2008). Thus, we generated reporter plasmids lacking ITRs entirely or containing complete ITRs with 5′-ATATTAACCC-3′ mutations predicted to disrupt the formation of productive hairpin intermediates (Mitra et al., 2008). To enable precise quantitation of mobilization activity, we developed a quantitative genomic PCR assay using primers specific for the transposon reporter and the endogenous human TK1 gene for normalization (Figure 3—figure supplements 4, 5). In agreement with the results of the clonogenic neomycin resistance assays, we observed efficient genomic integration of the donor transposons in cells transfected by GFP-PGBD5 as compared to the minimal signal observed in cells expressing GFP control (Figure 3E). Deletion of transposon ITRs from the reporter reduced genomic integration to background levels (Figure 3E). Consistent with the specific function of piggyBac family ITRs in genomic transposition, mutation of the terminal GGG sequence in the ITR significantly reduced the integration efficiency (Figure 3E). These results indicate that specific transposon ITR sequences are required for PGBD5-mediated DNA transposition.

DNA transposition by piggyBac superfamily transposases is distinguished from most other DNA transposon superfamilies by the precise excision of the transposon from the donor site and preference for insertion in TTAA sites (Cary et al., 1989; Fraser et al., 1995). To determine the structure of the donor sites of transposon reporters mobilized by PGBD5, we isolated plasmid DNA from cells 2 days after transfection, amplified the transposon reporter using PCR, and determined its sequence using capillary Sanger sequencing (Figure 4—figure supplement 1). Similar to the hyperactive T. ni piggyBac, cells expressing GFP-PGBD5, but not those expressing GFP control vector, exhibited robust excision of ITR-flanked transposon with apparently precise repair of the donor plasmid (Figure 4A,B and Figure 4—figure supplement 1). These results suggest that PGBD5 is an active cut-and-paste DNA transposase.

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To validate chromosomal integration and determine the location and precise structure of the insertion of the reporter transposons in the human genome, we isolated genomic DNA from G418-resistant HEK293 cells following transfection with PGBD5 and PB-EF1-NEO and amplified the genomic sites of transposon insertions using flanking sequence exponential anchored (FLEA) PCR, a technique originally developed for high-efficiency analysis of retroviral integrations (Pule et al., 2008). We adapted FLEA-PCR for the analysis of genomic DNA transposition by using unique reporter sequence to prime polymerase extension upstream of the transposon ITR into the flanking human genome, followed by reverse linear extension using degenerate primers, and exponential amplification using specific nested primers to generate chimeric amplicons suitable for massively parallel single-molecule Illumina DNA sequencing (Figure 5) (Henssen et al., 2015a). This method enabled us to isolate specific portions of the human genome flanking transposon insertions, as evidenced by the reduced yield of amplicons isolated from control cells lacking transposase vectors or expressing GFP (Figure 6—figure supplement 1). To identify the sequences of the transposon genomic insertions at single-base pair resolution, we aligned reads obtained from FLEA-PCR Illumina sequencing to the human hg19 reference genome and synthetic transposon reporter, and identified split reads that specifically span both (Figure 5). These data have been deposited to the Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra/, accession number SRP061649, Henssen et al., 2015c), with the processed and annotated data available from the Dryad Digital Repository (Henssen et al., 2015b).

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To infer the mechanism of genomic integration of transposon reporters, we analyzed the sequences of the insertion loci to determine integration preferences at base-pair resolution and identify potential sequence preferences. We found that transposon amplicons isolated from cells expressing GFP-PGBD5, but not those isolated from GFP control cells, were significantly enriched for TTAA sequences, as determined by sequence entropy analysis (Crooks et al., 2004) (Figure 6A). To discriminate between potential DNA transposition at TTAA target sites and alternative mechanisms of chromosomal integration, we classified genomic insertions based on target sites containing TTAA and those containing other sequence motifs (Table 3). Consistent with the DNA transposition activity of PGBD5, we observed significant induction of TTAA-containing insertions in cells expressing GFP-PGBD5 and transposons with intact ITRs, as compared to control cells expressing GFP, or to cells transfected with GFP-PGBD5 and mutant ITR transposons (Table 3). Sequence analysis of split reads containing transposon-human junction at TTAA sites revealed that, in almost every case examined (n = 65 out of 66), joining between TTAA host and transposon DNA occurred precisely at the GGG/CCC terminal motif of the donor transposon ITR (Figure 6C), in agreement with its requirement for efficient DNA transposition (Figure 3E). Consistent with the genome-wide transposition induced by PGBD5, we identified transposition events in all human chromosomes, including both genic and intergenic loci (Figure 6B). Thus, PGBD5 can mediate canonical cut-and-paste DNA transposition of piggyBac transposons in human cells.

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Requirement for transposon substrates with specific ITRs, their precise excision, and preferential insertion into TTAA-containing genomic locations is all consistent with the preservation of PGBD5's DNA transposase activity in human cells. Like other cut-and-paste transposases, piggyBac superfamily transposases are thought to utilize a triad of aspartate or glutamate residues to catalyze phosphodiester bond hydrolysis, but the catalytic triad of aspartates previously proposed for T. ni piggyBac is apparently not conserved in the primary sequence of PGBD5 (Figure 1) (Sarkar et al., 2003; Keith et al., 2008; Mitra et al., 2008; Nesmelova and Hackett, 2010). Thus, we hypothesized that distinct aspartic or glutamic acid residues may be required for DNA transposition mediated by PGBD5. To test this hypothesis, we used alanine-scanning mutagenesis and assessed transposition activity of GFP-PGBD5 mutants using quantitative genomic PCR (Figure 7 and Figure 7—figure supplements 1–3). This analysis indicated that simultaneous alanine mutations of D168, D194, and D386 reduced apparent transposition activity to background levels, similar to that of GFP control (Figure 7A). We confirmed that the mutant GFP-PGBD5 proteins have equivalent stability and expression as the wild-type protein in cells by immunoblotting against GFP (Figure 7B). Phylogenetic analysis of vertebrate piggyBac homologs from Danio rerio, Python bivittatus, Xenopus tropicalis, Gallus gallus, Mus musculus, and Homo sapiens showed that PGBD5 proteins are divergent from other piggyBac-like proteins (Figure 8) and include conservation of functionally important D168, D194, and D386 residues that distinguish them from lepidopteran piggyBac and human PGBD1-4 (Figure 8—figure supplement 1). These results suggest that PGBD5 represents a distinct member of the evolutionarily ancient piggyBac-like family of DNA transposases.

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Our current findings indicate that human PGBD5 is an active piggyBac transposase that can catalyze DNA transposition in human cells. DNA transposition by PGBD5 requires its C-terminal transposase domain and depends on specific ITRs derived from the lepidopteran piggyBac transposons (Figure 3). DNA transposition involves transesterification reactions mediated by DNA hairpin intermediates (Mitra et al., 2008). Consistent with the requirement of intact termini of the piggyBac, Tn10, and Mu transposons (Elick et al., 1997), elimination or mutation of the terminal GGG nucleotides from the transposon substrates also abolishes the transposition activity of PGBD5 (Figure 3). PGBD5-induced DNA transposition is precise with preference for insertions at TTAA genomic sites (Figure 4). Since our analysis was limited to ectopically expressed PGBD5 fused to GFP and episomal substrates derived from lepidopteran piggyBac transposons, it is possible that endogenous PGBD5 may exhibit different activities on chromatinized substrates in the human genome.

Current structure-function analysis indicates that PGBD5 requires three aspartate residues to mediate DNA transposition (Figure 7), but its transposase domain appears to be distinct from other piggyBac transposase enzymes with respect to its primary sequence (Figure 1 and Figure 8) (Keith et al., 2008). Thus, the three aspartate residues required for efficient DNA transposition by PGBD5 may form a catalytic triad that functions in phosphodiester bond hydrolysis, similar to the DDD motif in other piggyBac family transposases, or alternatively may contribute to other steps in the transposition reaction, such as synaptic complex formation, hairpin opening, or strand exchange (Elick et al., 1997; Keith et al., 2008; Mitra et al., 2008). In addition, we find that alanine mutations of the three required aspartate residues in the PGBD5 transposase domain significantly reduce but do not completely eliminate genomic integration of the transposon reporters (Figure 7). This could reflect residual catalytic activity despite these mutations, or that PGBD5 expression may affect other mechanisms of DNA integration in human cells.

The evolutionary conservation of the transposition activity of PGBD5 suggests that it may have hitherto unknown biologic functions among vertebrate organisms. DNA transposition is a major source of genetic variation that drives genome evolution, with some DNA transposases becoming extinct and others domesticated to evolve exapted functions. The evolution of transposons' activities can be highly variable, with some organisms such as Zea mays undergoing continuous genome remodeling and recent twofold expansion through endogenous retrotransposition, Drosophila and Saccharomyces owing over half of their known spontaneous mutations to transposons, and primate species including humans exhibiting relative extinction of transposons (Feschotte and Pritham, 2007).

Indeed, transposase-derived genes domesticated in humans have evolved to have endogenous functions other than genomic transposition per se. For example, human RAG1 is a domesticated Transib transposase that has retained its active transposase domain, and can transpose ITR-containing transposons in vitro, but catalyzes somatic recombination of immunoglobulin and T-cell receptor genes in lymphocytes across signal sequences that might be derived from related transposons (Landree et al., 1999; Fugmann et al., 2000). Human SETMAR is a Mariner-derived transposase with a divergent DDN transposase domain that has retained its endonuclease activity and functions in double-strand DNA repair by non-homologous end joining (Liu et al., 2007). The human genome encodes over 40 other genes derived from DNA transposases (Smit, 1999; Feschotte and Pritham, 2007), including THAP9 that was recently found to mobilize transposons in human cells with as of yet unknown function (Majumdar et al., 2013). RAG1, THAP9, and PGBD5 are, to our knowledge, the only human proteins with demonstrated transposase activity in human cells.

The distinct biochemical and structural features of PGBD5 indicated by our findings are consistent with its unique evolution and function among human piggyBac-derived transposase genes (Sarkar et al., 2003; Pavelitz et al., 2013). PGBD5 exhibits deep evolutionary conservation predating the origin of vertebrates, including a preservation of genomic synteny across lancelet, lamprey, teleosts, and amniotes (Pavelitz et al., 2013). This suggests that while PGBD5 likely derived from an autonomous mobile element, this ancestral copy was immobilized early in evolution and PGBD5 can probably no longer mobilize its own genomic locus, at least in germ line cells. The human genome contains several thousands of miniature inverted repeat transposable elements (MITEs) with similarity to piggyBac transposons (Figure 2 and Table 1) (Sarkar et al., 2003; Feschotte and Pritham, 2007). CSB-PGBD3 can bind to the piggyBac-derived MER85 elements in the human genome (Bailey et al., 2012; Gray et al., 2012). Similarly, it is possible that PGBD5 can act in trans to recognize and mobilize one or several related MITEs in the human genome. Recently, single-molecular maps of the human genome have predicted thousands of mobile element insertions, and the activity of PGBD5 or other endogenous transposases may explain some of these novel variants (Chaisson et al., 2015; Pendleton et al., 2015).

Given that both human RAG1 and ciliate piggyMac domesticated transposases catalyze the elimination of specific genomic DNA sequences (Hiom et al., 1998; Baudry et al., 2009), it is reasonable to hypothesize that PGBD5's biological function may similarly involve the excision of as of yet unknown ITR-flanked sequences in the human genome or another form of DNA recombination. Since DNA transposition by piggyBac family transposases requires substrate chromatin accessibility and DNA repair, we anticipate that additional cellular factors are required for and regulate PGBD5 functions in cells. Likewise, just as RAG1-mediated DNA recombination of immunoglobulin loci is restricted to B lymphocytes, and rearrangements of T-cell receptor genes to T lymphocytes, potential DNA rearrangements mediated by PGBD5 may be restricted to specific cell types and developmental periods.

PGBD5 localizes to the cell nucleus and is expressed during embryogenesis and neurogenesis, but its physiological function is not yet known (Pavelitz et al., 2013). Generation of molecular diversity through DNA recombination during nervous system development has been a long-standing hypothesis (Dreyer et al., 1967; Wu and Maniatis, 1999). The recent discovery of somatic retrotransposition in human neurons (Coufal et al., 2009; Evrony et al., 2012; Upton et al., 2015), combined with our finding of DNA transposition activity by human PGBD5, which is highly expressed in neurons, suggests that additional mechanisms of somatic genomic diversification may contribute to vertebrate nervous system development.

Because DNA transposition is inherently topological and orientation of transposons can affect the arrangements of reaction products (Claeys Bouuaert et al., 2011), potential activities of PGBD5 can depend on the arrangements of accessible genomic substrates, leading to both conservative DNA transposition involving excision and insertion of transposon elements, as well as irreversible reactions such as DNA elimination and chromosomal breakage-fusion-bridge cycles, as originally described by McClintock (1942). Finally, given the potentially mutagenic activity of active DNA transposases, we anticipate that unlicensed activity of PGBD5 and other domesticated transposases can be pathogenic in specific disease states, particularly in cases of aberrant chromatin accessibility, such as cancer.

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Author details

1. Anton G Henssen

   Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   AGH, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

2. Elizabeth Henaff

   Institute for Computational Biomedicine, Weill Cornell Medical College, New York, United States

   Contribution

   EH, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

   "This ORCID iD identifies the author of this article:" 0000-0001-7906-9681

3. Eileen Jiang

   Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   EJ, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

4. Amy R Eisenberg

   Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   ARE, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

5. Julianne R Carson

   Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   JRC, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

6. Camila M Villasante

   Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   CMV, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

7. Mondira Ray

   Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   MR, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

8. Eric Still

   Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States

   Contribution

   ES, Acquisition of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

9. Melissa Burns

   Boston Children's Hospital, Harvard Medical School, Boston, United States

   Contribution

   MB, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

   Competing interests

   The authors declare that no competing interests exist.

10. Jorge Gandara

    Institute for Computational Biomedicine, Weill Cornell Medical College, New York, United States

    Contribution

    JG, Acquisition of data, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

11. Cedric Feschotte

    Department of Human Genetics, University of Utah School of Medicine, Salt Lake City, United States

    Contribution

    CF, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

12. Christopher E Mason

    Institute for Computational Biomedicine, Weill Cornell Medical College, New York, United States

    Contribution

    CEM, Analysis and interpretation of data, Drafting or revising the article

    Competing interests

    The authors declare that no competing interests exist.

13. Alex Kentsis

    1. Molecular Pharmacology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, New York, United States
    2. Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, United States
    3. Weill Cornell Medical College, Cornell University, New York, United States

    Contribution

    AK, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article

    For correspondence

    kentsisresearchgroup@gmail.com

    Competing interests

    The authors declare that no competing interests exist.

    "This ORCID iD identifies the author of this article:" 0000-0002-8063-9191

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