AUTHOR CONTRIBUTIONS E.A. conceived and designed the experiments, collected the data, performed and interpreted the analyses, and wrote the manuscript. D.M.L and A.P.M planned experiments, and collected and analyzed data. I.K. conceived of and designed the hmMHC algorithm and performed analyses using it, and wrote the methodological description found in this manuscript. M.D. generated the KP9025 sarcoma cell line. A.M.L provided technical assistance and helped plan experiments using MHC class II tetramers. W.M. and C.F.L. planned, performed and analyzed mass spectrometry experiments. E.E. assisted with bioinformatics analyses. A.N.V. assisted with the generation of the CD4 + T cell hybridomas, and helped design and perform experiments using them. D.R. designed, collected, and analyzed data for experiments involving multi-color flow cytometry. J.P.W. provided technical support for MHC class I tetramer staining. M.M.G assisted in experiment planning. R.F.V.M. collected and analyzed data for experiments involving multi-color flow cytometry. C.D.A., K.C.F.S. and J.M.W. provided technical assistance throughout the study. A.C. collected data. K.W.W. provided mITGB1-MHC class II monomers and provided assistance in experimental design. T.J. provided support in experimental design and data analysis regarding the KP9025 sarcoma line. M.N.A. conceived and designed the hmMHC algorithm and provided bioinformatics support. E.R.U. provided assistance in experimental design. R.D.S. conceived experiments, interpreted data, and wrote the manuscript. All authors contributed to manuscript revision. R.D.S. is a cofounder, scientific advisory board member, stockholder, and royalty recipient of Jounce Therapeutics and Neon Therapeutics and is a scientific advisory board member for A2 Biotherapeutics, BioLegend, Codiak Biosciences, Constellation Pharmaceuticals, NGM Biopharmaceuticals and Sensei Biotherapeutics. K.W.W. serves on the scientific advisory board of Tscan Therapeutics and Nextechinvest and receives sponsored research funding from Bristol-Myers Squibb and Novartis; these activities are not related to the findings described in this publication. T.J. is a member of the Board of Directors of Amgen and Thermo Fisher Scientific. He is also a co-Founder of Dragonfly Therapeutics and T2 Biosystems. T.J. serves on the Scientific Advisory Board of Dragonfly Therapeutics, SQZ Biotech, and Skyhawk Therapeutics. None of these affiliations represent a conflict of interest with respect to the design or execution of this study or interpretation of data presented in this manuscript. Dr. Jacks's laboratory currently also receives funding from the Johnson & Johnson Lung Cancer Initiative and Calico, but this funding did not support the research described in this manuscript. DATA AVAILABILITY Nucleotide variant calls generated from cDNA capture sequencing of the T3 and KP9025 sarcoma lines and used in the prediction of antigens shown in Figure 1a, Extended Data Figure 3a-b, and Extended Data Figure 6b are available within the article as Supplem...

Myoblast cell cycle exit and differentiation are mediated in part by down-regulation of cyclin D1 and associated cyclin-dependent kinase (Cdk) activity. Because rhabdomyosarcoma may represent a malignant tumor composed of myoblast-like cells failing to exit the cell cycle and differentiate, we considered whether excess Cdk activity might contribute to this biology. Cyclin D -dependent Cdk4 and Cdk6 were expressed in most of a panel of six human rhabdomyosarcoma-derived cell lines. Cdk4 was expressed in 73% of alveolar and embryonal rhabdomyosarcoma tumors evaluated using a human tissue microarray. When challenged to differentiate by mitogen deprivation in vitro, mouse C2C12 myoblasts arrested in G 1 phase of the cell cycle, whereas four in the panel of rhabdomyosarcoma cell lines failed to do so. C2C12 myoblasts maintained in mitogen-rich media and exposed to a Cdk4/Cdk6 inhibitor PD 0332991 accumulated in G 1 cell cycle phase. Similar treatment of rhabdomyosarcoma cell lines caused G 1 arrest and prevented cell accumulation in vitro, and it delayed growth of rhabdomyosarcoma xenografts in vivo. Consistent with a role for Cdk4/Cdk6 activity as a regulator of myogenic differentiation, we observed that PD 0332991 exposure promoted morphologic changes and enhanced the expression of musclespecific proteins in cultured myoblasts and in the Rh30 cell line. Our findings support the concept that pharmacologic inhibition of Cdk4/Cdk6 may represent a useful therapeutic strategy to control cell proliferation and possibly promote myogenic differentiation in rhabdomyosarcoma.

Since its discovery close to twenty years ago, the ARF tumor suppressor has played a pivotal role in the field of cancer biology. Elucidating ARF's basal physiological function in the cell has been the focal interest of numerous laboratories throughout the world for many years. Our current understanding of ARF is constantly evolving to include novel frameworks for conceptualizing the regulation of this critical tumor suppressor. As a result of this complexity, there is great need to broaden our understanding of the intricacies governing the biology of the ARF tumor suppressor. The ARF tumor suppressor is a key sensor of signals that instruct a cell to grow and proliferate and is appropriately localized in nucleoli to limit these processes. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.

In this report, we employed a lentiviral RNA interference screen to discover nucleolar DEAD/DEAH-box helicases involved in RNA polymerase I (Pol I)-mediated transcriptional activity. Our screen identified DHX33 as an important modulator of 47S rRNA transcription. We show that DHX33 is a cell cycle-regulated nucleolar protein that associates with ribosomal DNA (rDNA) loci, where it interacts with the RNA Pol I transcription factor upstream binding factor (UBF). DHX33 knockdown decreased the association of Pol I with rDNA and caused a dramatic decrease in levels of rRNA synthesis. Wild-type DHX33 overexpression, but not a DNA binding-defective mutant, enhanced 47S rRNA synthesis by promoting the association of RNA polymerase I with rDNA loci. In addition, an NTPase-defective DHX33 mutant (K94R) acted as a dominant negative mutant, inhibiting endogenous rRNA synthesis. Moreover, DHX33 deficiency in primary human fibroblasts triggered a nucleolar p53 stress response, resulting in an attenuation of proliferation. Thus, we show the mechanistic importance of DHX33 in rRNA transcription and proliferation.RNA is a highly structured macromolecule whose secondary and tertiary conformations facilitate an array of specific interactions with proteins. The DEAD/DEAH-box family of RNA helicases (here referred to as DDX/DHX) (3) is one such classification of RNA binding proteins that are capable of modifying the higher-ordered structures of RNA through the hydrolysis of ATP/nucleoside triphosphate (NTP) (41). DDX/ DHX proteins often form large multiprotein complexes that participate in fundamental biological activities such as RNA transcription, RNA editing, pre-mRNA splicing, ribosome biogenesis, and RNA decay (3).DDX/DHX helicases are named and characterized by the conserved DEAD/DEAH motif common among all family members. Through site-directed mutagenesis analysis, DEAD/ DEAH along with seven conserved peptide motifs have been found to participate in ATP/NTP binding, hydrolysis, and substrate binding (28). Despite the conservation of these peptide motifs, the remaining sequences within each RNA helicase family member vary widely. Specifically, differences exist between the two categories of DDX and DHX proteins. DDX proteins contain a unique Q motif at their N termini that distinguishes them from DHX proteins. It was proposed previously that the Q motif might sense the state of ATP in vivo (40), given that DHX-box proteins are promiscuous in their ability to utilize NTP (16).Ribosome biogenesis is a complex multistep process, the majority of which occurs in the nucleolus of the cell (24, 43). The transcription of ribosomal DNA (rDNA) is the initial and rate-limiting step in ribosome biogenesis, and as such, it is influenced by multiple levels of regulation (25). One of the key regulators of rDNA transcription is the upstream binding factor (UBF), a transcriptional transactivator that binds to the upstream core element of rDNA and subsequently bends rDNA (37). This change in the rDNA structure favors the binding of SL.1 as wel...