The co-option of vertebrate orthologous ATP1B4 genes in placental mammals has radically altered the properties of the encoded BetaM proteins, which are genuine β-subunits of Na,K-ATPases in lower vertebrates. Eutherian BetaM acquired an extended Glu-rich N-terminal domain resulting in complete loss of its ancestral function and became skeletal and cardiac muscle-specific component of the inner nuclear membrane. BetaM is expressed at the highest level during perinatal development and is implicated in gene regulation (Pestov et al., Proc Natl Acad Sci U S A. 2007). Here we report the long-term consequences of the Atp1b4 ablation on metabolic parameters in adult mice. BetaM deficient (Atp1b4-/Y) mice have significantly lower body weight and remarkably low adiposity. They exhibit lower fasting blood glucose, enhanced insulin sensitivity, and improved glucose tolerance as compared to their wild type littermates. Knockout mice display higher heat production, increased food intake, elevated oxygen consumption especially in darkness, and higher locomotor activity. The lower respiratory exchange ratio of knockout mice indicates that fat from the diet is metabolized rather than deposited as storage. These robust changes in mouse metabolic parameters induced by Atp1b4 disruption clearly demonstrate that eutherian BetaM plays an important role in the regulation of adult mouse metabolism. Ablation of Atp1b4, leading to the loss of evolutionarily acquired BetaM functions, serves as a model for a potential alternative pathway in mammalian evolution. Essentially, Atp1b4 ablation simulates a scenario where a specific stage in mammalian evolution is bypassed. Our results suggest that bypassing the co-option of Atp1b4 potentially reduces susceptibility to obesity. ### Competing Interest Statement The authors have declared no competing interest.

Immuno-oncology has transformed the treatment of cancer, with several immunotherapies becoming the standard treatment across histologies. Despite these advancements, the majority of patients do not experience durable clinical benefits, highlighting the imperative for ongoing advancement in immuno-oncology. Computational immuno-oncology emerges as a forefront discipline that draws on biomedical data science and intersects with oncology, immunology, and clinical research, with the overarching goal to accelerate the development of effective and safe immuno-oncology treatments from the laboratory to the clinic. In this review, we outline 10 critical challenges and opportunities in computational immuno-oncology, emphasizing the importance of robust computational strategies and interdisciplinary collaborations amid the constantly evolving interplay between clinical needs and technological innovation.

The Immuno-Oncology Translational Network (IOTN) was established in 2018 as part of the Cancer Moonshot. In 2022, President Joe Biden set new goals to reduce the cancer death rate by half within 25 years and improve the lives of people with cancer and cancer survivors. The IOTN is focused on accelerating translation of cancer immunology research, from bench to bedside, and improving immunotherapy outcomes across a wide array of cancers in the adult population. The unique structure and team science approach of the IOTN is designed to accelerate discovery and evaluation of novel immune-based therapeutic and prevention strategies. In this article, we describe IOTN progress to date, including new initiatives and the development of a robust set of resources to advance cancer immunology research. We summarize new insights by IOTN researchers, some of which are ripe for translation for several types of cancers. Looking to the future, we identify barriers to the translation of immuno-oncology concepts into clinical trials and key areas for action and improvements that are suitable for high-yield investments. Based on these experiences, we recommend novel National Institutes of Health funding mechanisms and development of new resources to address these barriers.

PDF file, 267KB, Supplementary figure 1. a) Heat maps of RKIP, EZH2, Snail and E-cad expression profiles obtained by interrogating publicly available DNA microarray expression datasets (33). The abbreviations are as follows: NAP: normal adjacent prostate tissue, BPH: benign prostate hyperplasia, PCA: prostate cancer adenocarcinoma, and MPC: metastatic prostate carcinoma. Rows correspond to individual genes and columns represent individual patients. Color density is arranged in order from greatest decrease in expression at left (red) to greatest increase at right (light yellow). EZH2 and Snail mRNA are strongly expressed in MPC, in contrast to RKIP and E-cad. b) DU145 or MDA-MB231 cells were infected with indicated siRNA expressing retroviruses. The siRNA expressing cells were assayed for their ability to proliferate in growth medium containing 0.3% agar and the formation of multi-cellular colonies. Colonies were stained with MTT and quantified after 21 days from plates in triplicate (lower panel). Data shown are representative of three independent experiments and are expressed as mean colony number per plate +/- s.d. c) The effect of the indicated siRNA on DU145 cells proliferation was assayed by direct cell counting. Quiescent cells were counted by coulter counter at the indicated times after stimulation with 20% FBS. Data shown are representative of three independent experiments.

Vertebrate ATP1B4 genes represent a rare instance of orthologous gene co-option, resulting in radically different functions of the encoded BetaM proteins. In lower vertebrates, BetaM is a Na, K-ATPase β-subunit that is a component of ion pumps in the plasma membrane. In placental mammals, BetaM lost its ancestral role and, through structural alterations of the N-terminal domain, became a skeletal and cardiac muscle-specific protein of the inner nuclear membrane, highly expressed during late fetal and early postnatal development. We previously determined that BetaM directly interacts with the transcriptional co-regulator SKI-interacting protein (SKIP) and is implicated in the regulation of gene expression. This prompted us to investigate a potential role for BetaM in the regulation of muscle-specific gene expression in neonatal skeletal muscle and cultured C2C12 myoblasts. We found that BetaM can stimulate expression of the muscle regulatory factor (MRF), MyoD, independently of SKIP. BetaM binds to the distal regulatory region (DRR) of MyoD, promotes epigenetic changes associated with activation of transcription, and recruits the SWI/SNF chromatin remodeling subunit, BRG1. These results indicate that eutherian BetaM regulates muscle gene expression by promoting changes in chromatin structure. These evolutionarily acquired new functions of BetaM might be very essential and provide evolutionary advantages to placental mammals.

Decades of research into the molecular mechanisms of cancer and the development of novel therapeutics have yielded a number of remarkable successes. However, our ability to broadly assign effective, rationally targeted therapies in a personalized manner remains elusive for many patients, and drug resistance persists as a major problem. This is in part due to the well-documented heterogeneity of cancer, including the diversity of tumor cell lineages and cell states, the spectrum of somatic mutations, the complexity of microenvironments, and immune-suppressive features and immune repertoires, which collectively require numerous different therapeutic approaches. Here, we describe a framework to understand the types and biological causes of resistance, providing translational opportunities to tackle drug resistance by rational therapeutic strategies.

Background Background: Despite regulatory approval and advancements of immune-based interventions over the last decade, significant obstacles remain to be overcome before the full potential of immunotherapy can be realized to improve outcomes for cancer patients [1,3]. The NCI Cancer Moonshot Immuno-Oncology Translational Network (IOTN) was established in 2018 with the overarching goal of improving the efficacy, durability, and safety of immunotherapy across the spectrum of human cancers, as well as developing immunoprevention approaches that will prevent cancers before they occur.1,2 Methods The IOTN consortium, consisting of 29 research project awards spanning 33 institutions, conducts translational research focused on four thematic areas: Immunotherapy, Immuno-Engineering to improve immuno-therapy (i3), Mitigating immune related adverse events (irAE) and Immuno-Prevention.2, 3 At each stage of the IOTN9s translational studies, the Data Management Resource Sharing Center (DMRC) serves as an administrative and analytic hub, focusing on lowering barriers for access to analytic expertise, improving network productivity, maintaining a high standard for data collection and management, and fostering a collaborative and supportive research community. Results The DMRC has developed centralized infrastructures to enhance the IOTN's research output, including a collection of publicly accessible and scalable resource sharing catalogs for Data, Model, Software and Clinical Trials.4 In addition to supporting intra-network collaboration, the DMRC engaged cross-moonshot communication by hosting and coordinating a number of meetings and workshops, including the inaugural Cancer Moonshot Collaborative Meeting and monthly NCI Cancer Moonshot Seminar Series.5, 6 The DMRC in collaboration with SITC has organized the highly popular SITC-NCI webinar series on "Computational Science in Immuno-Oncology", fostering collaboration, communication and outreach to the broader communities.7 Conclusions The IOTN9s consortium structure, strengthened by the DMRC9s centralized management, analytical, and data integration supports, has facilitated effective intra- and inter-moonshot communication and enabled collaborative translational studies. Acknowledgements This work was supported by IOTN Moonshot grant U24CA232979-01 References Jacks T, Jaffee E and Singer D. Blue Ribbon Panel Co-Chairs (2016) Cancer Moonshot Blue Ribbon Panel Report. National Cancer Institute, Bethesda, MD. Adult Immunotherapy Network Fact Sheet [https://www.cancer.gov/research/key-initiatives/moonshot-cancer-initiative/implementation/adult-immunotherapy-network/fact-sheet.pdf] Annapragada A, Sikora A, Bollard C, et al. Cancer Moonshot Immuno-Oncology Translational Network (IOTN): accelerating the clinical translation of basic discoveries for improving immunotherapy and immunoprevention of cancer. J Immunother Cancer 2020;8:e00079. Immuno-Oncology Translational Network. IOTN Resource Sharing Catalogs. 2022. Available from: https://www.iotnmoonshot.org/en/resources/all-resources/. National Cancer Institute. Cancer Moonshot Workshop Highlights Advances – National Cancer Institute 2020. Available from: https://www.cancer.gov/news-events/cancer-currents-blog/2020/cancer-moonshot-nci-workshop-advances-collaboration. National Cancer Institute. Cancer MoonshotSM Seminar Series [online workshop series], September 24, 2020-May 26, 2022: National Cancer Institute; 2021. Available from: https://www.cancer.gov/research/key-initiatives/moonshot-cancer-initiative/seminar-series. 18. Society for Immunotherapy of Cancer. SITC-NCI Computational Immuno-oncology Webinar Series [online workshop series], May 11, 2021-December 17, 2022: Society for Immunotherapy of Cancer (SITC) in partnership with the National Cancer Institute (NCI); 2021 Available from: https://www.sitcancer.org/education/computational.

High-dose irradiation poses extreme risk of mortality from acute damage to the hematopoietic compartment and gastrointestinal tract. While bone marrow transplantation can reestablish the hematopoietic compartment, a more imminent risk of death is posed by gastrointestinal acute radiation syndrome (GI-ARS), for which there are no FDA-approved medical countermeasures. Although the mechanisms dictating the severity of GI-ARS remain incompletely understood, sialylation by ST6GAL1 has been shown to protect against radiation-induced apoptosis in vitro. Here, we used a C57BL/6 St6gal1-KO mouse model to investigate the contribution of ST6GAL1 to susceptibility to total body irradiation in vivo. Twelve gray total body ionizing gamma-irradiation (TBI) followed by bone marrow transplant is not lethal to wild-type mice, but St6gal1-KO counterparts succumbed within 7 d. Both St6gal1-KO and wild-type animals exhibited damage to the GI epithelium, diarrhea and weight loss, but these symptoms became progressively more severe in the St6gal1-KO animals while wild-type counterparts showed signs of recovery by 120 h after TBI. Increased apoptosis in the GI tracts of St6gal1-KO mice and the absence of regenerative crypts were also observed. Together, these observations highlight an important role for ST6GAL1 in protection and recovery from GI-ARS in vivo.

SWI/SNF chromatin remodeling enzymes are multisubunit complexes that contain one of two catalytic subunits, BRG1 or BRM and 9-11 additional subunits called BRG1 or BRM-associated factors (BAFs). BRG1 interacts with the microphthalmia-associated transcription factor (MITF) and is required for melanocyte development in vitro and in vivo. The subunits of SWI/SNF that mediate interactions between BRG1 and MITF have not been elucidated. Three mutually exclusive isoforms of a 60-kDa subunit (BAF60A, B, or C) often facilitate interactions with transcription factors during lineage specification. We tested the hypothesis that a BAF60 subunit promotes interactions between MITF and the BRG1-containing SWI/SNF complex. We found that MITF can physically interact with BAF60A, BAF60B, and BAF60C. The interaction between MITF and BAF60A required the basic helix-loop-helix domain of MITF. Recombinant BAF60A pulled down recombinant MITF, suggesting that the interaction can occur in the absence of other SWI/SNF subunits and other transcriptional regulators of the melanocyte lineage. Depletion of BAF60A in differentiating melanoblasts inhibited melanin synthesis and expression of MITF target genes. MITF promoted BAF60A recruitment to melanocyte-specific promoters, and BAF60A was required to promote BRG1 recruitment and chromatin remodeling. Thus, BAF60A promotes interactions between MITF and the SWI/SNF complex and is required for melanocyte differentiation.

Mutations in SOX10 cause neurocristopathies which display varying degrees of hypopigmentation. Using a sensitized mutagenesis screen, we identified Smarca4 as a modifier gene that exacerbates the phenotypic severity of Sox10 haplo-insufficient mice. Conditional deletion of Smarca4 in SOX10 expressing cells resulted in reduced numbers of cranial and ventral trunk melanoblasts. To define the requirement for the Smarca4 -encoded BRG1 subunit of the SWI/SNF chromatin remodeling complex, we employed in vitro models of melanocyte differentiation in which induction of melanocyte-specific gene expression is closely linked to chromatin alterations. We found that BRG1 was required for expression of Dct, Tyrp1 and Tyr, genes that are regulated by SOX10 and MITF and for chromatin remodeling at distal and proximal regulatory sites. SOX10 was found to physically interact with BRG1 in differentiating melanocytes and binding of SOX10 to the Tyrp1 distal enhancer temporally coincided with recruitment of BRG1. Our data show that SOX10 cooperates with MITF to facilitate BRG1 binding to distal enhancers of melanocyte-specific genes. Thus, BRG1 is a SOX10 co-activator, required to establish the melanocyte lineage and promote expression of genes important for melanocyte function.

Vol. 213, No. 3, March 7, 2016. Pages [433–449][1]. The authors regret that in their original paper, the gene Zfp521 appeared as Zfp251 . The online HTML and PDF versions of this article have been corrected. Figs. 1, 3, and 5 have also been edited to correct this mistake. The error only remains

Introduction: Hematopoiesis involves the hierarchical generation of the major blood lineages from a common ancestor, the Hematopoietic Stem Cell (HSC). HSC also have the intrinsic ability to repopulate an ablated hematopoietic compartment when introduced into the periphery of a recipient. This has allowed Hematopoietic Stem Cell transplantation (HSCT) to be used as a cell therapy over the last 45 years, benefiting thousands of patients. Unfortunately many patients succumb to disease while waiting for an adequate donor. Others have to undergo unrelated donor transplants, putting themselves at a risk of developing graft-versus-host disease. Improving HSC engraftment could ameliorate transplant morbidity. Thus, understanding mechanisms regulating HSC engraftment is key. Results: We used our recently published gene expression profiles of developing HSC and other public databases to prioritize 58 genes as putative regulators of adult HSC function. We confirmed by qRT-PCR that 51/58 candidates were enriched for gene expression in Lineage-Sca-1+c-Kit+ (LSK) bone marrow cells relative to downstream progeny, suggesting a role in hematopoietic stem and progenitor cells (HSPC). To functionally assay a role for each gene of interest (GOI) in HSC engraftment, we designed and validated ≥2 independent shRNAs/GOI that effected a >75% knockdown in gene expression in LSK cells. LSK cells were lentivirally transduced with control or individual gene-specific shRNAs and transplanted into lethally irradiated recipients along with mock-transduced LSK competitor cells congenic at the CD45 allele. In contrast to previous functional screens, transplant was performed within 24-hours of LSK cell isolation, avoiding extensive ex vivo culture. This minimal manipulation allowed us to detect genes critical for efficient HSC engraftment. Peripheral blood chimerism was analyzed for at least 16 weeks post-transplant. The major bone marrow hematopoietic compartments were also analyzed. 17 of 48 genes tested were identified as necessary for optimal HSPC engraftment (i.e. knockdown induced a significant loss of repopulation) and the knockdown of three genes enhanced HSPC repopulation. shRNAs targeting each “Hit” were interrogated ex vivo for non-specific effects on LSK cell viability and expansion. A 2° screen was performed to validate the results of this primary screen. Here, CD45.2 LSK cells transduced with control or individual gene-specific shRNAs were sorted 48 hours post-transduction for mCherry+ cells and then transplanted into lethally irradiated mice with mock-transduced and mock-sorted CD45.1 congenic LSK cells. 18 “Hits” were confirmed to perturb HSC repopulating potential in this 2° screen, including three whose loss enhanced HSPC repopulation. The transcription factor, Foxa3, is one hit identified here as necessary for HSC repopulation. We further found that that Foxa3-/- bone marrow displays a significant loss of repopulating potential >16 weeks post-transplant, confirming the results of our screen. As Foxa3-/- long-term HSC also display reduced colony forming potential in vitro and fail to contribute to downstream progenitor compartments in transplant recipients, we propose that Foxa3 is a novel regulator of HSC differentiation post-transplant. Foxa3 has never before been implicated in hematopoiesis or HSPC biology. Conclusions: Our novel functional screen has revealed 15 genes required for optimal HSPC engraftment and three genes whose knockdown improved HSPC engraftment. We further validated Foxa3 as a novel regulator of HSC activity by demonstrating that Foxa3-/- HSC are also deficient in repopulating activity. We are currently investigating the molecular mechanism of Foxa3’s role in HSC and, given that Foxa genes are known transcriptional pioneering factors, pursuing the hypothesis that Foxa3 functions as a novel epigenetic regulator of HSC activation and differentiation. Each gene identified in our screen represents a window into the discovery of novel mechanisms regulating HSC biology and engraftment. Disclosures No relevant conflicts of interest to declare.

Microphthalmia-associated transcription factor (MITF) is a survival factor in melanocytes and melanoma cells. MITF regulates expression of antiapoptotic genes and promotes lineage-specific survival in response to ultraviolet (UV) radiation and to chemotherapeutics. SWI/SNF chromatin-remodeling enzymes interact with MITF to regulate MITF target gene expression. We determined that the catalytic subunit, BRG1, of the SWI/SNF complex protects melanoma cells against UV-induced death. BRG1 prevents apoptosis in UV-irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (ML-IAP). Down-regulation of ML-IAP compromises BRG1-mediated survival of melanoma cells in response to UV radiation. BRG1 regulates ML-IAP expression by cooperating with MITF to promote transcriptionally permissive chromatin structure on the ML-IAP promoter. The alternative catalytic subunit, BRM, and the BRG1-associated factor, BAF180, were found to be dispensable for elevated expression of ML-IAP in melanoma cells. Thus, we illuminate a lineage-specific mechanism by which a specific SWI/SNF subunit, BRG1, modulates the cellular response to DNA damage by regulating an antiapoptotic gene and implicate this subunit of the SWI/SNF complex in mediating the prosurvival function of MITF.

SOX10 is a Sry-related high mobility (HMG)-box transcriptional regulator that promotes differentiation of neural crest precursors into Schwann cells, oligodendrocytes, and melanocytes. Myelin, formed by Schwann cells in the peripheral nervous system, is essential for propagation of nerve impulses. SWI/SNF complexes are ATP dependent chromatin remodeling enzymes that are critical for cellular differentiation. It was recently demonstrated that the BRG1 subunit of SWI/SNF complexes activates SOX10 expression and also interacts with SOX10 to activate expression of OCT6 and KROX20, two transcriptional regulators of Schwann cell differentiation. To determine the requirement for SWI/SNF enzymes in the regulation of genes that encode components of myelin, which are downstream of these transcriptional regulators, we introduced SOX10 into fibroblasts that inducibly express dominant negative versions of the SWI/SNF ATPases, BRM or BRG1. Dominant negative BRM and BRG1 have mutations in the ATP binding site and inhibit gene activation events that require SWI/SNF function. Ectopic expression of SOX10 in cells derived from NIH 3T3 fibroblasts led to the activation of the endogenous Schwann cell specific gene, myelin protein zero (MPZ) and the gene that encodes myelin basic protein (MBP). Thus, SOX10 reprogrammed these cells into myelin gene expressing cells. Ectopic expression of KROX20 was not sufficient for activation of these myelin genes. However, KROX20 together with SOX10 synergistically activated MPZ and MBP expression. Dominant negative BRM and BRG1 abrogated SOX10 mediated activation of MPZ and MBP and synergistic activation of these genes by SOX10 and KROX20. SOX10 was required to recruit BRG1 to the MPZ locus. Similarly, in immortalized Schwann cells, BRG1 recruitment to SOX10 binding sites at the MPZ locus was dependent on SOX10 and expression of dominant negative BRG1 inhibited expression of MPZ and MBP in these cells. Thus, SWI/SNF enzymes cooperate with SOX10 to directly activate genes that encode components of peripheral myelin.