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We reviewed response to immune checkpoint inhibitors (ICI) of 207 patients with diagnoses of lung or head and neck cancer treated with chemotherapy/ICI combination therapy and ICI monotherapy between 2015 and 2020 at one of three clinical pavilions associated with the Dan L. Duncan Comprehensive Cancer Center at Baylor College of Medicine (Houston, TX). Two of these pavilions (Harris Health System and the Michael E. DeBakey Veterans Affairs Medical Center) serve large minority populations and provide equal access to care regardless of means. 174 patients had a diagnosis of lung cancer (non–small cell or small cell) and 33 had a diagnosis of head and neck squamous cell carcinoma (HNSCC). 38% self-identified as Black, 45% as non-Hispanic White, and 18% as Hispanic. The objective response rate (ORR) was similar for patients with lung cancer (35.057%) and HNSCC (30.3%; P = 0.894). The ORR for Hispanic and Black patients was lower compared with non-Hispanic White patients (H 27.0%, B 32.5%, W 38.7%; H vs. W P = 0.209; B vs. W P = 0.398). When considering only patients treated with ICI monotherapy, the ORR for Hispanic patients dropped further to 20.7% while the ORR of Black and non-Hispanic White patients remained about the same (B 29.3% and W 35.9%, H vs. W P = 0.133; B vs. W P = 0.419). Immune-related adverse events were the lowest in the Hispanic population occurring in only 30% of patients compared with 40% of patients in the Black cohort and 50% of the non-Hispanic White cohorts. Significance: To our knowledge, this report is the first to compare ICI effectiveness within a diverse patient population with a substantial Black and Hispanic NSCLC and HNSCC patient population treated in the context of equal access to care. The data presented in this article suggests reduced effectiveness of ICI monotherapy in Hispanic patients and thereby underscores the need for improved access and representation of racial/ethnic minority patients in ICI clinical trials.

Combined supplementary data file.

We reviewed response to immune checkpoint inhibitors (ICI) of 207 patients with diagnoses of lung or head and neck cancer treated with chemotherapy/ICI combination therapy and ICI monotherapy between 2015 and 2020 at one of three clinical pavilions associated with the Dan L. Duncan Comprehensive Cancer Center at Baylor College of Medicine (Houston, TX). Two of these pavilions (Harris Health System and the Michael E. DeBakey Veterans Affairs Medical Center) serve large minority populations and provide equal access to care regardless of means. 174 patients had a diagnosis of lung cancer (non–small cell or small cell) and 33 had a diagnosis of head and neck squamous cell carcinoma (HNSCC). 38% self-identified as Black, 45% as non-Hispanic White, and 18% as Hispanic. The objective response rate (ORR) was similar for patients with lung cancer (35.057%) and HNSCC (30.3%; P = 0.894). The ORR for Hispanic and Black patients was lower compared with non-Hispanic White patients (H 27.0%, B 32.5%, W 38.7%; H vs. W P = 0.209; B vs. W P = 0.398). When considering only patients treated with ICI monotherapy, the ORR for Hispanic patients dropped further to 20.7% while the ORR of Black and non-Hispanic White patients remained about the same (B 29.3% and W 35.9%, H vs. W P = 0.133; B vs. W P = 0.419). Immune-related adverse events were the lowest in the Hispanic population occurring in only 30% of patients compared with 40% of patients in the Black cohort and 50% of the non-Hispanic White cohorts.Significance:To our knowledge, this report is the first to compare ICI effectiveness within a diverse patient population with a substantial Black and Hispanic NSCLC and HNSCC patient population treated in the context of equal access to care. The data presented in this article suggests reduced effectiveness of ICI monotherapy in Hispanic patients and thereby underscores the need for improved access and representation of racial/ethnic minority patients in ICI clinical trials.

Intensity modulated radiation therapy (RT) for spine metastases using a simultaneous integrated boost (SSIB) was shown as an alternative to the treatment of select osseous metastases that are not amenable to spine stereotactic radiosurgery. We sought to update our clinical experience using SSIB in patients for whom dose escalation was warranted but spine stereotactic radiosurgery was not feasible.A total of 58 patients with 63 spinal metastatic sites treated with SSIB between 2012 and 2021 were retrospectively reviewed. The gross tumor volume and clinical target volume were prescribed 40 and 30 Gy in 10 fractions, respectively.The median follow-up time was 31 months. Of 79% of patients who reported pain before RT with SSIB, 82% reported an improvement following treatment. Patient-reported pain scores on a 10-point scale revealed a significant decrease in pain at 1, 3, 6, and 12 months after SSIB (P.0001). Additionally, there were limited toxicities; only 1 patient suffered grade 3 toxicity (pain) following RT. There were no reports of radiation-induced myelopathy at last follow-up, and 8 patients (13%) experienced a vertebral column fracture post-treatment. Local control was 88% (95% confidence interval [CI], 80%-98%) and 74% (95% CI, 59%-91%) at 1 and 2 years, respectively. Overall survival was 64% (95% CI, 53%-78%) and 45% (95% CI, 34%-61%) at 1 and 2 years, respectively. The median overall survival was 18 months (95% CI, 13-27 months). Multivariable analysis using patient, tumor, and dosimetric characteristics revealed that a higher Karnofsky performance status before RT (hazard ratio, 0.44, 0.22-0.89; P = .02) was associated with longer survival.These data demonstrate excellent pain relief and local control with limited acute toxicities following treatment with RT using SSIB to 40 Gy. Collectively, our data suggest that dose escalation to spine metastases using SSIB can be safe and efficacious for patients, especially those with radioresistant disease. Further investigation is warranted to validate these findings.

ImportancePrimary end point (PEP) changes to an active clinical trial raise questions regarding trial quality and the risk of outcome reporting bias. It is unknown how the frequency and transparency of the reported changes depend on reporting method and whether the PEP changes are associated with trial positivity (ie, the trial met the prespecified statistical threshold for PEP positivity).ObjectivesTo assess the frequency of reported PEP changes in oncology randomized clinical trials (RCTs) and whether these changes are associated with trial positivity.Design, Setting, and ParticipantsThis cross-sectional study used publicly available data for complete oncology phase 3 RCTs registered in ClinicalTrials.gov from inception through February 2020.Main Outcomes and MeasuresThe main outcome was change between the initial PEP and the final reported PEP, assessed using 3 methods: (1) history of tracked changes on ClinicalTrials.gov, (2) self-reported changes noted in the article, and (3) changes reported within the protocol, including all available protocol documents. Logistic regression analyses were performed to evaluate whether PEP changes were associated with US Food and Drug Administration approval or trial positivity.ResultsOf 755 included trials, 145 (19.2%) had PEP changes found by at least 1 of the 3 detection methods. Of the 145 trials with PEP changes, 102 (70.3%) did not have PEP changes disclosed within the manuscript. There was significant variability in rates of PEP detection by each method (χ2 = 72.1; P χ2 = 18.7; P P = .003).Conclusions and RelevanceThis cross-sectional study revealed substantial rates of PEP changes among active RCTs; PEP changes were markedly underreported in published articles and mostly occurred after reported study completion dates. Significant discrepancies in the rate of detected PEP changes call into question the role of increased protocol transparency and completeness in identifying key changes occurring in active trials.

For children, craniospinal irradiation (CSI) with photons is associated with significant toxic effects. The use of electrons for spinal fields is hypothesized to spare anterior structures but the long-term effects remain uncertain. We studied late effects of CSI using electrons for spinal radiation therapy (RT).Records of 84 consecutive patients treated with CSI using electrons for the spine at a single institution between 1983 and 2014 were reviewed. Median age at RT was 5 (range, 1-14) years. The most common histologies were medulloblastoma/primitive neuroectodermal tumor (59%) and ependymoma (8%). The median prescribed dose to the entire spine was 30 Gy (range, 6-45). A subset of 48 (57%) patients aged 2 to 14 at RT with clinical follow-up for ≥5 years was analyzed for late effects. Height z scores adjusted for age before and after CSI were assessed using stature-for-age charts and compared with a t test.At median follow-up of 19 years (range, 0-38 years), the median survival was 22 years (95% confidence interval, 12-28 years) after RT, with 47 patients (56%) alive at last follow-up. On subset analysis for late effects, 19 (40%) patients developed hypothyroidism and 5 (10%) developed secondary malignancies. Other complications reported were esophageal stricture and periaortic hemorrhage in 1 and restrictive pulmonary disease in 1 patient. Median height z score before treatment was -0.4 (36th percentile; interquartile range, -1.0 to 0.0) and at last follow-up was -2.2 (first percentile; interquartile range, -3.1 to -1.6; P.001). Of 44 patients with spinal curvature assessments, 15 (34%) had scoliosis with median Cobb angle 15° (range, 10°-35°) and 1 (2%) required surgery.Frequent musculoskeletal toxic effects and predominantly decreased height were seen with long-term follow-up. Scoliosis and hypothyroidism were each seen in at least one-third of long-term survivors. However, clinically evident esophageal, pulmonary, and cardiac toxic effects were infrequent.

Immune checkpoint inhibitors (ICIs) have revolutionized the treatment of both lung cancer and head and neck squamous cell carcinoma demonstrating clear benefit over traditional chemotherapy alone in the metastatic setting. While the overwhelming majority of ICI trial participants have been White patients, results of these trials have been broadly applied to patients of all ethnic/racial backgrounds. It has, therefore, not been well defined if response to ICIs differs between ethnic/racial populations or socio-economic groups.We reviewed response to ICI of 208 patients with diagnoses of lung or head and neck cancers treated with ICI between 2015 and 2020 at one of three clinical pavilions associated with the Dan L. Duncan Comprehensive Cancer Center at Baylor College of Medicine in Houston, TX. Two of these pavilions (Harris Health System and the Michael E. DeBakey Veterans Affairs Medical Center) serve large minority patient populations and provide equal access of care to patients regardless of means. Of the 208 patients, 175 had a diagnosis of lung cancer [non-small cell lung carcinoma (NSCLC) or small cell lung cancer (SCLC)] and 33 had a diagnosis of head and neck squamous cell carcinoma (HNSCC); 38% self-identified as Black, 45% as non-Hispanic White, and 18% as Hispanic. The objective response rate (ORR) was similar for lung cancer (31.4%) and HNSCC patients (27.3%) (p=0.894). Statistically, the ORR for Hispanic and Black patients did not differ compared to non-Hispanic White patients (H 23.7%, B 28.6%, W 35.5%; H vs. W p=0.189; B vs. W p=0.338). When considering patients treated with ICI monotherapy, the ORR for Hispanic patients dropped to 13.3% and was significantly lower than the ORR of the non-Hispanic White patients while the ORR of Black and non-Hispanic White patients remained about the same (B 29.3% and W 34.6%, H vs. W p=0.0285; B vs. W p=0.5131). Immune related adverse events (irAEs) were the lowest in the Hispanic population occurring in only 30% of patients compared to 50% of patients exhibiting irAEs in the Black and non-Hispanic white cohorts.ICIs demonstrate comparable anti-tumor effects in lung cancer (NSCLC + SCLC) and HNSCC during routine clinical practice regardless of race or ethnicity. The significantly lower ORR observed in our cohort for Hispanic patients, particularly when used as monotherapy, is an unexpected finding and will require additional study to identify potential biological and non-biological confounders which could contribute to reduced ICI effectiveness in this patient population.

In this work, we develop a drug-mimicking nanofibrous peptide hydrogel that shows long-term bioactivity comparable to a small-molecule inhibitor of inducible nitric oxide synthase (iNOS). The iNOS inhibitor, N(6)-(1-iminoethyl)-l-lysine (l-NIL), is a positively charged amino acid whose structure could be readily integrated into the framework of a positively charged multidomain peptide (MDP) through the modification of lysine side chains. This new l-NIL-MDP maintains the self-assembling properties of the base peptide, forming β-sheet nanofibers, which entangle into a thixotropic hydrogel. The l-NIL-MDP hydrogel supports cell growth in vitro and allows syringe-directed delivery that persists in a targeted location in vivo for several weeks. Multiple characterization assays demonstrate the bioactivity of the l-NIL-MDP hydrogel to be comparable to the l-NIL small molecule. This includes iNOS inhibition of macrophages in vitro, reduced nitrotyrosine immunostaining in murine subcutaneous histology, and reduced serum levels of vascular endothelial growth factor in vivo. This study expands the toolbox of available peptide hydrogel scaffold designs that can modify biological activity without the need for any additional small-molecule drugs, proteins, or cells.

When innate immune cells and hematopoietic stem and progenitor cells (HSPCs) are exposed to pathogenic agents, they develop heightened responses to subsequent infection through a process called trained immunity. After exposure to a pathogen, bone marrow derived macrophages (BMDMs) generated from trained HSPCs are capable of enhanced pathogen clearance and cytokine production, while exhibiting persistent metabolic rewiring. Because some models of HSPC trained immunity are dependent on interferon gamma (IFNγ) signaling, and our lab has described extensive changes in HSC self-renewal and differentiation upon IFNγ exposure, we hypothesized that persistent IFNγ signaling induced by chronic infection results in reprogramming of HSPCs, causing improved non-specific immunity. To test our hypothesis, we generated a chimeric mouse model of trained immunity by transplanting control or M. avium-exposed HSPCs into naïve recipient mice. Mice that received M. avium trained HSPCs had decreased bacterial load, less splenomegaly, and fewer granulomas upon subsequent M. avium infection, indicating improved immunity. Furthermore, BMDMs generated from mice trained with a single dose of recombinant IFNγ (rIFNγ) exhibited increased pathogen clearance and metabolic profiles ex vivo. To test if rIFNγ training was sufficient to induce HSPC trained immunity in vivo, we isolated HSPCs from rIFNγ-exposed mice and challenged the recipients 4 months later. These in vivo experiments demonstrated that rIFNγ training was insufficient to induce substantial protection against subsequent M. avium challenge, but still induced BMDM metabolic rewiring four months post HSPC training. Collectively, our studies indicate that there are degrees of training that occur upon IFNγ exposure, likely related to the concentration and duration of the primary stimulus. To assess the specificity of cross protection of HSPC trained immunity, we utilized our chimeric mouse model and tested two different training and infection pathogens: M. avium and influenza. When we challenged M. avium-trained HSPC recipients with influenza, we found that although there was mildly decreased lung histopathology and increased production of IFNγ and TNFα, mice succumbed to infection like untrained controls. When we swapped the order of pathogens, we observed that mice receiving influenza-trained HSPCs produced BMDMs with increased killing capability and systemically higher IL-6 and RANTES levels, but these features were insufficient to significantly reduce bacterial CFU counts upon M. avium challenge. These transplant experiments indicate that trained immunity encoded in HSPCs is pathogen specific. To dissect the mechanism of M. avium-induced trained immunity in HSPCs, we performed RNAseq analysis on M. avium-trained HSPCs post-transplant and cross referenced it with RNAseq and WGBS data on primary M. avium-exposed HSPCs. These studies showed consistent differences in cellular signaling, metabolism, immunity, and antigen processing and presentation in the trained HSPCs, indicating that epigenetic and transcriptional reprogramming induced by M. avium exposure is durable following transplant and secondary challenge. To ascertain whether transcriptional changes are homogeneous throughout the HSC compartment, we completed scRNA-seq on naïve and M. avium-exposed hematopoietic cells. We found that genes upregulated upon M. avium exposure in HSCs, including Batf2 and Cxcl9, were induced in a subset of HSPCs, indicating that there is a heterogeneous response to training within the HSPC pool. Strikingly, the trained immunity signature was maintained in neutrophils and macrophages but lost in mature B cells, indicating specific propagation of genetic signatures induced by training in certain lineages. Finally, we found an emerging population of HSCs with B cell gene signatures upon M. avium exposure. Emergence of this HSPC subpopulation may suggest the development of a cell that acts as a direct intermediate between HSC and B cells following training. Our work shows that trained immunity induced by M. avium and persistent IFNγ signaling is pathogen-specific and heterogeneous among primitive HSPCs. Emergence of specific responder cell populations within the HSPC pool may be responsible for enhanced protection against specific infection stimuli, whereas the presence of non-responders may insure long term health of the HSPC pool. Disclosures No relevant conflicts of interest to declare.

Chronic infections are prevalent worldwide and contribute to bone marrow failure syndromes. Long-term homeostatic production of all blood cells is dependent on the ability of hematopoietic stem cells (HSCs) to remain mostly quiescent and, upon activation, must carefully balance between their ability to self-renew and differentiate. However, infections trigger inflammatory cytokines, such as interferon-gamma (IFNy), that facilitate HSC proliferative exhaustion by disrupting both quiescence and self-renewal, thereby contributing to cytopenias and bone marrow failure syndromes. Our previously published results indicate that the IFN-induced surface protein, Bone Marrow Stromal Antigen 2 (BST2), facilitates HSC activation, and the loss of BST2 protected against the depletion of HSCs during chronic infection. However, the mechanism by which BST2 impacts HSCs activation is unknown. BST2 is known to promote activation of intracellular signaling in both dendritic cells and cancer cell types. In particular BST2 is a known activator of the NFkB signaling pathway. We hypothesized that BST2 could promote activation of HSCs through the induction of intracellular signaling pathways. Intracellular signaling pathways have been shown to be important for the activation of not only HSCs but also downstream immune cells. Indeed, the loss of BST2 has been shown to impair the function of T cells, NK cells, and antigen presenting cells in the presence of viral infections via impaired induction of NFkB signaling. However, there have been no studies on the role of BST2 during bacterial infections. To evaluate the role of BST2 in hematopoiesis during a bacterial infection, we obtained BST2KO mice and characterized hematopoietic populations in the presence or absence of Mycobacterium avium infection, a model bacterial infection that generates high levels of IFNy. We observed no changes in activation or production of cytokines from macrophage, NK, CD8, or neutrophil populations. Surprisingly we observed enhanced dendritic cell activation at day 15, 30, and 42 as measured by co-stimulatory molecules and MHC II expression. Additionally, we observed increased tetramer-specific T cell numbers early on during the infection. Interestingly, these results pair with decreased bacterial load at day 30 and day 42 despite having the similar spleen sizes. These results suggest that BST2 regulates the activation of dendritic cells, which may trigger more robust earlier T cell responses early in the course of infection. Our previous work showed that the loss of BST2 protects against HSC activation by displacement of HSCs from CXCL12-abundant reticular (CAR) cells in the niche. To determine whether HSC activation is related to distancing from the vascular niche or increased proximity to an activated osteoblastic niche, we performed intravital imaging using osteocalcin-GFP reporter mice. We observed no change in proximity to the osteoblastic niche after administration of recombinant IFNy using either WT or BST2KO HSCs. These results suggest that the effects of BST2 may be more related to distancing from CAR cells than migration towards other niche locations. To evaluate the mechanisms underlying BST2-dependent HSC activation, we performed RNA sequencing on WT versus BST2KO HSCs in the presence of IFNy. GSEA-pathway analysis of differentially expressed genes revealed that BST2KO HSCs have a decreased response to IFNy and diminished activation of the P13K-AKT-MTOR signaling pathways. Western blot analysis on hematopoietic stem cell progenitor cells (HSPCs) indicate a decrease in phosphorylation of the ERK signaling pathway in BST2KO HSPCs. These results were analogous to work performed in 32D cells in vitro, in which ERK-phosphorylation has been shown to be important for both HSC maintenance and proliferation. Future studies are aimed at assessing the impact of ERK inhibition on HSC activation during various inflammatory stresses. Collectively our results show that the loss of BST2 in downstream immune cells surprisingly enhances dendritic cell and T cell activation in response to bacterial infection. BST2 enhances HSC activation through activation of downstream ERK signaling. Future targeting of BST2 and/or ERK signaling could have important consequences for patients that suffer from cytopenias, bone marrow failure, and engraftment failure due to high levels of inflammatory stimuli. Disclosures No relevant conflicts of interest to declare.

Bone marrow failure is a significant complication of many chronic infections, which affect a third of the world's population. The inflammatory cytokine interferon-gamma (IFNy) contributes to bone marrow failure syndromes by activating hematopoietic stem cells (HSCs) and impairing their self-renewal. IFNy upregulation during many chronic infections such as tuberculosis, HIV and hepatitis B directly depletes hematopoietic stem cells (HSCs). The mechanisms by which IFNy drives the loss of quiescence and ultimate exhaustion of HSCs remain poorly understood, but may be related to changes in the interaction between HSCs and the bone marrow (BM) microenvironment, or BM niche. Current evidence suggests that quiescent HSCs reside predominantly in the vascular niche, where the production of stem cell factor (SCF) from endothelial cells and CXCL12 from perivascular CXCL12 abundant reticular (CAR) stromal cells are critical for maintaining their quiescence. The goal of our work was to determine whether IFNy signaling alters HSC interactions within the niche. We performed transcriptomic analysis of IFNy-stimulated HSCs and focused on changes in cell-surface expressed genes that may influence HSC-niche interactions. Our analysis revealed Bone Marrow Stromal Antigen 2 (BST2) as the only surface protein upregulated on HSCs upon 24-hour IFNy stimulation. To study the effects of BST2 on HSC-niche interactions, we performed intravital imaging using two reporter mouse models: CXCL12-GFP reporter mice in which CAR cells are labeled with GFP, and CXCL12-GFP Krt18-Cre LSL-tdTomato dual reporter mice in which CAR cells are labelled with GFP and HSCs are labeled with tdTomato. After exogenously labeling and transferring HSCs into CXCL12-GFP mice or endogenously labeling HSCs in the CXCL12-GFP Krt18-Cre LSL-tdTomato mice, we observed that HSCs stimulated with IFNy were significantly distanced from CAR cells compared to pre-treated controls. These findings are consistent with other reports that chemotherapeutic and inflammatory stress disrupts HSC interactions with the niche and promotes HSC migration. Conversely, we observed no change in HSC distancing from CAR cells after IFNy stimulation of IFNy-receptor deficient HSCs, suggesting that the observed HSC displacement was due to a cell autonomous mechanism. These changes were not due to a loss of CXCL12 receptor (CXCR4) expression or disrupted capacity of HSCs to migrate towards CXCL12. Interestingly, Intravital imaging using BST2-deficient HSCs revealed that BST2 KO HSCs do not re-localize from CAR cells during IFNy stimulation. Increased BST2 expression has been linked to the migration, adhesion and metastasis of various cancer cells and we explored whether it could serve a similar role in protein binding in HSCs. Using in vitro plate binding assays, we found that IFNy-treatment promoted increased HSC binding to E-selectin via BST2, as well as increased HSC homing to the bone marrow, a property that is dependent on E-selectin binding. Finally, to determine whether BST2 affects IFNy-dependent HSC activation we performed cell cycle analysis of WT and BST2 KO HSCs. We discovered that the loss of BST2 protects against HSC activation during Mycobacterium avium infection. Furthermore, HSC depletion during chronic infection was mitigated in BST2 KO mice. Our data identifies BST2 as a key protein that influences niche relocalization and activation in response to inflammatory stimulation. This study expands our understanding of factors that contribute to HSC activation and loss of quiescence. These findings could shed light on novel therapeutic interventions for patients who develop pancytopenia or bone marrow failure due to chronic inflammation. Disclosures No relevant conflicts of interest to declare.

During chronic infection, the inflammatory cytokine interferon gamma (IFNγ) damages hematopoietic stem cells (HSCs) by disrupting quiescence and promoting excessive terminal differentiation. However, the mechanism by which IFNγ hinders HSC quiescence remains undefined. Using intravital 3-dimensional microscopy, we find that IFNγ disrupts the normally close interaction between HSCs and CXCL12-abundant reticular (CAR) cells in the HSC niche. IFNγ stimulation increases expression of the cell surface protein BST2, which we find is required for IFNγ-dependent HSC relocalization and activation. IFNγ stimulation of HSCs increases their E-selectin binding by BST2 and homing to the bone marrow, which depends on E-selectin binding. Upon chronic infection, HSCs from mice lacking BST2 are more quiescent and more resistant to depletion than HSCs from wild-type mice. Overall, this study defines a critical mechanism by which IFNγ promotes niche relocalization and activation in response to inflammatory stimulation and identifies BST2 as a key regulator of HSC quiescence. VIDEO ABSTRACT.

Stimulation of cells with TNFα can promote distinct cell death pathways, including RIPK1-independent apoptosis, necroptosis, and RIPK1-dependent apoptosis (RDA)-the latter of which we still know little about. Here we show that RDA involves the rapid formation of a distinct detergent-insoluble, highly ubiquitinated, and activated RIPK1 pool, termed "iuRIPK1." iuRIPK1 forms after RIPK1 activation in TNF-receptor-associated complex I, and before cytosolic complex II formation and caspase activation. To identify regulators of iuRIPK1 formation and RIPK1 activation in RDA, we conducted a targeted siRNA screen of 1,288 genes. We found that NEK1, whose loss-of-function mutations have been identified in 3% of ALS patients, binds to activated RIPK1 and restricts RDA by negatively regulating formation of iuRIPK1, while LRRK2, a kinase implicated in Parkinson's disease, promotes RIPK1 activation and association with complex I in RDA. Further, the E3 ligases APC11 and c-Cbl promote RDA, and c-Cbl is recruited to complex I in RDA, where it promotes prodeath K63-ubiquitination of RIPK1 to lead to iuRIPK1 formation. Finally, we show that two different modes of necroptosis induction by TNFα exist which are differentially regulated by iuRIPK1 formation. Overall, this work reveals a distinct mechanism of RIPK1 activation that mediates the signaling mechanism of RDA as well as a type of necroptosis.

Interferon gamma (IFNy) is a pro-inflammatory cytokine that is upregulated during chronic infections and chronic diseases, such as aplastic anemia, and has been associated with pancytopenia and diminished hematopoiesis. Studies have shown that IFNy negatively regulates hematopoietic stem cell (HSC) homeostasis by decreasing self-renewal and promoting terminal differentiation. The tight regulation of HSC homeostasis is dependent upon the bone marrow (BM) microenvironment, or BM niche. The BM niche is composed of a network of cell types that provide elaborate cell-cell interactions, cellular metabolites, transcriptional regulators, and local and distant humoral and neural signals that allow for hematopoietic homeostasis. In particular, CXCL12-abundant reticular (CAR) cells are vital to HSC maintenance, as depletion of CXCL12, or its receptor, leads to HSC depletion. However, the mechanism which IFNy activates HSCs and influences its interaction with the BM niche is unknown. We hypothesize that IFNy promotes HSC terminal differentiation and loss of quiescence by altering HSC interactions with the BM niche. To assess changes in HSC interactions with the BM niche upon IFNy stimulation, we performed intravital imaging using CXCL12 GFP reporter mice before and after administration of recombinant IFNy. We found that HSCs stimulated with IFNy were significantly distanced from CAR cells compared to pre-treated controls. There was no change in distance with IFNy-receptor deficient HSCs, suggesting that movement away from the CAR cells was due to a cell autonomous IFNy-dependent mechanism. We performed gene expression analysis and transwell migration assays on HSCs from IFNy treated mice, and determined that there was no change in CXCL12 receptor (CXCR4) expression upon IFNy treatment, and IFNy did not alter migration towards CXCL12. These results suggest that HSC re-localization upon IFNy is independent of CXCL12 signaling. To explore the mechanism by which IFNy induces re-localization of HSCs, we first performed microarray analysis on HSCs from IFNy stimulated mice to assess what surface proteins were changed upon IFNy treatment. While there was no change in common HSC receptors thought to influence HSC homeostasis (cKit, Cdh2, Mpl, Itgb1, Itbg2, Itga4, and Itga1), we observed an increase in expression of bone marrow stromal antigen 2 (BST2). To explore the impact of BST2 on HSC homeostasis, quantification and proliferation analysis was performed on HSCs from Bst2-/- mice. Interestingly, Bst2-/- HSCs were significantly less proliferative and more abundant compared to controls. These studies suggest that BST2 may play a role in maintaining HSC homeostasis. The functional role of BST2 in cellular movement and adhesion has been studied in cancer. Increased BST2 expression has been associated with promoting the migration, adhesion and metastasis of various cancer cells. Since migration and adhesion is important for HSC homing, we assessed the effects of IFNy on HSC homing. Hematopoietic progenitors from IFNy-treated mice homed to the bone marrow with greater efficiency than PBS-treated controls, whereas progenitors from IFNy-receptor-deficientmice showed a decrease in homing. Additionally, WBM from IFNyR-/- had reduced engraftment than wildtype, consistent with a role for IFNy signaling in promoting HSC homing. The impact of BST2 on homing is currently being explored. In summary, we show that IFNy induces re-localization of HSCs away from quiescence-promoting CAR cells within the bone marrow niche via a mechanism that is independent of CXCL12 signaling. We further show that IFNy promotes HSC homing. The increased expression of BST2 on IFNy-stimulated HSCs appears to impact HSC proliferation and abundance in the bone marrow. Thus, BST2 may play a role in HSC activation and exit from quiescence. Expanding our understanding of the mechanism that drives HSC activation and terminal differentiation has important implications for patients who develop pancytopenia or bone marrow failure due to chronic inflammation. Disclosures No relevant conflicts of interest to declare.

Axonal pathology and necroptosis in ALS Necroptosis, a non–caspase-dependent form of cell death, can be reduced in disease states by inhibiting a kinase called RIPK1. Until now, no human mutations have been linked to necroptosis. Ito et al. show that loss of optineurin, which is encoded by a gene that has been implicated in the human neurodegenerative disorder ALS (amyotrophic lateral sclerosis), results in sensitivity to necroptosis and axonal degeneration. When RIPK1-kinase dependent signaling is disrupted in mice that lack optineurin, necroptosis is inhibited and axonal pathology is reversed. Science , this issue p. 603

Acute respiratory distress syndrome (ARDS) is a common and often fatal inflammatory lung condition without effective targeted therapies. Regulatory T cells (Tregs) resolve lung inflammation, but mechanisms that enhance Tregs to promote resolution of established damage remain unknown. DNA demethylation at the forkhead box protein 3 (Foxp3) locus and other key Treg loci typify the Treg lineage. To test how dynamic DNA demethylation affects lung injury resolution, we administered the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (DAC) to wild-type (WT) mice beginning 24 hours after intratracheal LPS-induced lung injury. Mice that received DAC exhibited accelerated resolution of their injury. Lung CD4(+)CD25(hi)Foxp3(+) Tregs from DAC-treated WT mice increased in number and displayed enhanced Foxp3 expression, activation state, suppressive phenotype, and proliferative capacity. Lymphocyte-deficient recombinase activating gene-1-null mice and Treg-depleted (diphtheria toxin-treated Foxp3(DTR)) mice did not resolve their injury in response to DAC. Adoptive transfer of 2 × 10(5) DAC-treated, but not vehicle-treated, exogenous Tregs rescued Treg-deficient mice from ongoing lung inflammation. In addition, in WT mice with influenza-induced lung inflammation, DAC rescue treatment facilitated recovery of their injury and promoted an increase in lung Treg number. Thus, DNA methyltransferase inhibition, at least in part, augments Treg number and function to accelerate repair of experimental lung injury. Epigenetic pathways represent novel manipulable targets for the treatment of ARDS.