Systematic autoantigen analysis identifies a distinct subtype of scleroderma with coincident cancer

Xu, George; Shah, Ami A.; Li, Mamie Z.; Xu, Qikai; Rosen, Antony; Casciola‐Rosen, Livia; Elledge, Stephen J.

doi:10.1073/pnas.1615990113

Cited by 85 publications

(78 citation statements)

References 23 publications

(25 reference statements)

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“…To assess the specificity of antibodies for L1Hs, we employed phage-display immunoprecipitation sequencing (PhIP-seq) [45][46][47]. We obtained all annotated repeats in the human genome from RepeatMasker and constructed a representational phage-display library which was used in combination with a previously constructed pan human proteome library [48]. The RepeatMasker peptidome was tiled from N-to C-terminus using 56 amino acid peptides with 28 amino acid overlaps.…”

Section: Orf2p Antibodies Are Specific For Human-specific Line-1 (L1hs)mentioning

confidence: 99%

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Ardeljan

Wang

Oghbaie

et al. 2019

Preprint

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Background: Long interspersed element-1 (LINE-1, L1) is the major driver of mobile DNA activity in modern humans. When expressed, LINE-1 loci produce bicistronic transcripts encoding two proteins essential for retrotransposition, ORF1p and ORF2p. Many types of human cancers are characterized by L1 promoter hypomethylation, L1 transcription, L1 ORF1p protein expression, and somatic L1 retrotransposition. ORF2p encodes the endonuclease and reverse transcriptase activities required for L1 retrotransposition. Its expression is poorly characterized in human tissues and cell lines. Results:We report mass spectrometry based tumor proteome profiling studies wherein ORF2p eludes detection. To test whether ORF2p could be detected with specific reagents, we developed and validated five rabbit monoclonal antibodies with immunoreactivity for specific epitopes on the protein. These reagents readily detect ectopic ORF2p expressed from bicistronic L1 constructs. However, endogenous ORF2p is not detected in human tumor samples or cell lines by western blot, immunoprecipitation, or immunohistochemistry despite high levels of ORF1p expression. Moreover, we report endogenous ORF1p-associated interactomes, affinity isolated from colorectal cancers, wherein we similarly fail to detect ORF2p. These samples include primary tumors harboring hundreds of somatically-acquired L1 insertions. The new data are available via ProteomeXchange with identifier PXD013743. Conclusions:Although somatic retrotransposition provides unequivocal genetic evidence for the expression of ORF2p in human cancers, we are unable to directly measure its presence using several standard methods. Experimental systems have previously indicated an unequal stoichiometry between ORF1p and ORF2p, but in vivo, the expression of these two proteins may be more strikingly uncoupled. These findings are consistent with observations that ORF2p is not tolerable for cell growth. References

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Section: Orf2p Antibodies Are Specific For Human-specific Line-1 (L1hs)mentioning

confidence: 99%

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Ardeljan

Wang

Oghbaie

et al. 2019

Preprint

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“…These individuals likely represent a heterogenous population of scleroderma patients targeting different autoantigens, both known and novel. We recently utilized Phage-Immunoprecipitation Sequencing (PhIP-Seq) and PLATO (Parallel Analysis of in vitro Translated ORFs) (9, 10) to identify unique autoantibodies in CTP-negative scleroderma patients with a clustering of cancer diagnosis and scleroderma onset (11). Specifically, 16 CTP-negative patients with scleroderma, cancer, and a short cancer-scleroderma interval (≤ 5 years) were studied.…”

Section: Introductionmentioning

confidence: 99%

Brief Report: Anti–RNPC‐3 Antibodies As a Marker of Cancer‐Associated Scleroderma

Shah

Rosen

et al. 2017

Arthritis & Rheumatology

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Introduction Prior studies have demonstrated an increased risk of cancer-associated scleroderma in patients with RNA polymerase III (POL) autoantibodies and in patients negative for anti-centromere (CENP), anti-topoisomerase-1 (TOPO), and anti-POL antibodies (referred to as CENP/TOPO/POL (CTP)-Negative). In a recent study of 16 CTP-negative scleroderma patients with coincident cancer, we found that 25% had autoantibodies to RNPC3, a member of the minor spliceosome complex. In this investigation, we validated the relationship between anti-RNPC3 antibodies and cancer and examined the associated clinical phenotype in a large sample of scleroderma patients. Methods Scleroderma patients with cancer were assayed for CENP, TOPO, POL and RNPC3 autoantibodies. Disease characteristics and the cancer-scleroderma interval were compared across autoantibody groups. The relationship between autoantibody status and cancer-associated scleroderma was assessed by logistic regression. Results Of 318 patients with scleroderma and cancer, 70 (22.0%) were positive for anti-POL, 54 (17.0%) for anti-TOPO, and 96 (30.2%) for anti-CENP. Twelve patients (3.8% of overall group or 12.2% of CTP-negatives) were positive for anti-RNPC3. Patients with anti-RNPC3 had a short cancer-scleroderma interval (median 0.9 years). Relative to patients with anti-CENP, patients with anti-RNPC3 (OR 4.3; 95%CI 1.10–16.9; p=0.037) and anti-POL (OR 4.49; 95%CI 1.98–10.2; p<0.001) had a >4-fold increased risk of cancer within 2 years of scleroderma onset. Patients with anti-RNPC3 had severe restrictive lung and gastrointestinal disease, Raynaud’s, and myopathy. Conclusion Anti-RNPC3 autoantibodies associate with an increased risk of cancer at scleroderma onset, similar to POL autoantibodies. These data suggest the possibility of cancer-induced autoimmunity in this scleroderma subset.

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“…There is early anecdotal evidence that rituximab is effective in certain refractory cases of PAH, including SSc-PAH: For example, B cell depletion with rituximab is effective in a model of T helper 2 cell–mediated PAH (18), and an SSc patient with mild ILD and very substantial PAH who was refractory to standard PAH therapy was successfully treated with rituximab twice (19). The B cell basis of SSc has previously been studied (20) with respect to subset sizes (5), cytokine levels (21), response regulators (5, 22) and autoantibody specificities (23, 24) but not at the level of sequence-based B cell phylogeny. Reconstitution of the immunoglobulin heavy chain (IGH) repertoire after rituximab administration has been studied before (25), but the sample size was small (two participants) and the sequencing depth was low (on the order of 680 sequences).…”

Section: Introductionmentioning

confidence: 99%

Dynamics of the human antibody repertoire after B cell depletion in systemic sclerosis

et al. 2017

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Systemic sclerosis with pulmonary arterial hypertension (SSc-PAH) is a debilitating and frequently lethal disease of unknown cause lacking effective treatment options. Lymphocyte anomalies and autoantibodies observed in systemic sclerosis have suggested an autoimmune character. We study the clonal structure of the B cell repertoire in SSc-PAH using immunoglobulin heavy chain (IGH) sequencing before and after B cell depletion. We found SSc-PAH to be associated with anomalies in B cell development, namely, altered VDJ rearrangement frequencies (reduced IGHV2-5 segment usage) and an increased somatic mutation–fixation probability in expanded B cell lineages. SSc-PAH was also characterized by anomalies in B cell homeostasis, namely, an expanded immunoglobulin D–positive (IgD+) proportion with reduced mutation loads and an expanded proportion of highly antibody-secreting cells. Disease signatures pertaining to IGHV2-5 segment usage, IgD proportions, and mutation loads were temporarily reversed after B cell depletion. Analyzing the time course of B cell depletion, we find that the kinetics of naïve replenishment are predictable from baseline measurements alone, that release of plasma cells into the periphery can precede naïve replenishment, and that modes of B cell receptor diversity are highly elastic. Our findings reveal humoral immune signatures of SSc-PAH and uncover determinism in the effects of B cell depletion on the antibody repertoire.

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Systematic autoantigen analysis identifies a distinct subtype of scleroderma with coincident cancer

Cited by 85 publications

References 23 publications

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

LINE-1 ORF2p Expression is Nearly Imperceptible in Human Cancers

Brief Report: Anti–RNPC‐3 Antibodies As a Marker of Cancer‐Associated Scleroderma

Dynamics of the human antibody repertoire after B cell depletion in systemic sclerosis

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