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Therapy-related myeloid neoplasm (tMN) is considered a direct consequence of DNA damage in hematopoietic stem cells. Despite increasing recognition that altered stroma can also drive leukemogenesis, the functional biology of the tMN microenvironment remains unknown. We performed multiomic (transcriptome, DNA damage response, cytokine secretome and functional profiling) characterization of bone marrow stromal cells from tMN patients. Critically, we also compared (i) patients with myeloid neoplasm and another cancer but without cytotoxic exposure, (ii) typical primary myeloid neoplasm, and (iii) age-matched controls to decipher the microenvironmental changes induced by cytotoxics vs. neoplasia. Strikingly, tMN exhibited a profoundly senescent phenotype with induction of CDKN1A and β-Galactosidase, defective phenotype, and proliferation. Moreover, tMN stroma showed delayed DNA repair and defective adipogenesis. Despite their dormant state, tMN stromal cells were metabolically highly active with a switch toward glycolysis and secreted multiple pro-inflammatory cytokines indicative of a senescent-secretory phenotype that inhibited adipogenesis. Critically, senolytics not only eliminated dormant cells, but also restored adipogenesis. Finally, sequential patient sampling showed senescence phenotypes are induced within months of cytotoxic exposure, well prior to the onset of secondary cancer. Our data underscores a role of senescence in the pathogenesis of tMN and provide a valuable resource for future therapeutics.

AbstractSystems of Influence in Global Health:Examining the Impact of Familial, Community, and Societal Structures on Health OutcomesbyBrigid Ellen CakourosDoctor of Public HealthUniversity of California, BerkeleyProfessor Lia Fernald, ChairThis dissertation is a collection of three essays exploring topics in global health. Each essay aims to examine the associations between an individual, the systems in which they live, and potential ways to intervene on the systems in order to achieve better health and cognitive outcomes worldwide.The first study uses qualitative data from Zimbabwe to examine the experiences of women with partners of unknown human immunodeficiency virus (HIV) status accessing both HIV testing and preexposure prophylaxis (PrEP) in Zimbabwe. This analysis aimed to identify themes of structural vulnerability and social drivers within this population in order to understand the best ways in which to conduct outreach for this community. Results showed that the majority of women wanted to be tested for HIV and use PrEP as a result of their partner’s non-monogamous lifestyle and their lack of ability to negotiate condom use. In order to increase access to PrEP, these women pointed to the need to carry out sensitization efforts within the community. Specifically, many women spoke about the need for PrEP to be discussed more frequently by clinicians at any point of contact in a healthcare setting. In addition, community health workers were considered a reliable support system within many communities. Findings suggest that community health workers could function as a key group for delivering information for accessing PrEP while also further desensitizing the community to HIV prevention and care.The second study included in this dissertation was a descriptive analysis of the Young Lives Study, using longitudinal datasets from four countries (Ethiopia, India, Peru and Vietnam) in order to explore patterns of family structure trajectories for children between ages of approximately 1 and 15 years. We categorized the children into seven household structures and documented the proportion of children who experienced household stability, those who had transitions, and the frequency of transitions. In all four countries, fewer than half of the children lived in nuclear households throughout childhood. In most countries, co-residence with a grandparent was typical in early childhood, with transition into family types without grandparents as time passed. Ethiopia had larger proportion of children who have lived in single-mother households and in households without their biological mother. These results suggest that research on childhood in lower- and middle-income countries ought to consider the dynamics of household structure as a variable that could have an association with child outcomes.The third study, building on the second study, is a secondary analysis of the Young Lives Study longitudinal dataset from Ethiopia in order to explore associations between family structure trajectories and cognitive outcomes (vocabulary, math, and reading scores) at age 15 years. We categorized children as either having both parents present or not always having both parents present in all five rounds of data collection. We also explored the occasional presence or absence of grandparents. Our results show that urban children in family structures with an absent parent had statistically significant lower average test scores at age 15 when compared to results from children who always had both parents present. However, the addition of wealth index measures explained this association, reinforcing the link between socioeconomic status and cognitive measures. When a parent was absent, children with grandparents’ present had higher cognitive outcomes than children who did not have grandparent’s present. These findings suggest that educational attainment in Ethiopia could have associations with a child’s family structure beyond just the parental structure.

Purpose of ReviewAlthough many signalling pathways have been discovered to be essential in mesenchymal stem/stromal (MSC) differentiation, it has become increasingly clear in recent years that epigenetic regulation of gene transcription is a vital component of lineage determination, encompassing diet, lifestyle and parental influences on bone, fat and cartilage development.Recent FindingsThis review discusses how specific enzymes that modify histone methylation and acetylation or DNA methylation orchestrate the differentiation programs in lineage determination of MSC and the epigenetic changes that facilitate development of bone related diseases such as osteoporosis. The review also describes how environmental factors such as mechanical loading influence the epigenetic signatures of MSC, and how the use of chemical agents or small peptides can regulate epigenetic drift in MSC populations during ageing and disease.SummaryEpigenetic regulation of MSC lineage commitment is controlled through changes in enzyme activity, which modifies DNA and histone residues leading to alterations in chromatin structure. The co-ordinated epigenetic regulation of transcriptional activation and repression act to mediate skeletal tissue homeostasis, where deregulation of this process can lead to bone loss during ageing or osteoporosis.

The purpose of this study was to identify causal pathways between HIV infection and severe mental illness. Data were collected through open-ended, semi-structured interviews. An integrated approach to the analysis, using inductive and deductive coding, was used to identify patterns among respondents. Standardized instruments were used to characterize the sample in terms of risk behaviors, physical and mental functioning and depression. Twenty-six men and women with a diagnosis of HIV and unipolar depression (n = 11) or schizophrenia/schizoaffective versus bipolar disorder (n = 15) participated. For persons with unipolar depression, the HIV diagnosis often preceded depressive symptoms. For persons with schizophrenia/schizoaffective versus bipolar disorder, mania and psychosis symptoms often preceded HIV. Substance use, incarceration and adverse childhood experiences were common across diagnoses. Attention to the directionality of effects between mental illness and HIV has important implications for anticipatory guidance for infectious disease specialists, primary care providers and public health practitioners as well as policymakers.

BackgroundDuring development, excessive osteogenic differentiation of mesenchymal progenitor cells (MPC) within the cranial sutures can lead to premature suture fusion or craniosynostosis, leading to craniofacial and cognitive issues. Saethre-Chotzen syndrome (SCS) is a common form of craniosynostosis, caused byTWIST-1gene mutations. Currently, the only treatment option for craniosynostosis involves multiple invasive cranial surgeries, which can lead to serious complications.MethodsThe present study utilizedTwist-1haploinsufficient (Twist-1del/+) mice as SCS mouse model to investigate the inhibition of Kdm6a and Kdm6b activity using the pharmacological inhibitor, GSK-J4, on calvarial cell osteogenic potential.ResultsThis study showed that the histone methyltransferaseEZH2, an osteogenesis inhibitor, is downregulated in calvarial cells derived fromTwist-1del/+mice, whereas the counter histone demethylases,Kdm6aandKdm6b, known promoters of osteogenesis, were upregulated. In vitro studies confirmed that siRNA-mediated inhibition ofKdm6aandKdm6bexpression suppressed osteogenic differentiation ofTwist-1del/+calvarial cells. Moreover, pharmacological targeting of Kdm6a and Kdm6b activity, with the inhibitor, GSK-J4, caused a dose-dependent suppression of osteogenic differentiation byTwist-1del/+calvarial cells in vitro and reduced mineralized bone formation inTwist-1del/+calvarial explant cultures. Chromatin immunoprecipitation and Western blot analyses found that GSK-J4 treatment elevated the levels of the Kdm6a and Kdm6b epigenetic target, the repressive mark of tri-methylated lysine 27 on histone 3, on osteogenic genes leading to repression ofRunx2andAlkaline Phosphataseexpression. Pre-clinical in vivo studies showed that local administration of GSK-J4 to the calvaria ofTwist-1del/+mice prevented premature suture fusion and kept the sutures open up to postnatal day 20.ConclusionThe inhibition of Kdm6a and Kdm6b activity by GSK-J4 could be used as a potential non-invasive therapeutic strategy for preventing craniosynostosis in children with SCS.Graphical abstractPharmacological targeting of Kdm6a/b activity can alleviate craniosynostosis in Saethre-Chotzen syndrome. Aberrant osteogenesis by Twist-1 mutant cranial suture mesenchymal progenitor cells occurs via deregulation of epigenetic modifiers Ezh2 and Kdm6a/Kdm6b. Suppression of Kdm6a- and Kdm6b-mediated osteogenesis with GSK-J4 inhibitor can prevent prefusion of cranial sutures.

Cyanide is a fast-acting toxicant that inhibits aerobic cellular respiration in mitochondria. Historically, amyl nitrite has been used to treat cyanide poisoning. The objective of this study was to demonstrate the effectiveness of intranasal (IN) administration of stabilized isoamyl nitrite (SIAN) against lethal potassium cyanide (KCN) intravenous (IV) challenge in a conscious nonhuman primate (NHP) model. Animals received an IV infusion of a 1 mg/mL KCN solution at a dose of 3.25 mg/kg for 15 min. The IN SIAN treatment was administered at 10 or 12 min post-start of KCN challenge and monkeys were monitored for survival, clinical signs, respiratory and cardiovascular changes via continuous telemetry recording. In addition, blood methemoglobin (metHb) levels and blood gases were closely monitored after KCN administration. Mid to mid-high doses (13.5 or 27.0 μL/kg) of SIAN treatments at 10 min post KCN challenge significantly increased animal survival. Improvements were also observed in respiratory and cardiovascular functions. The benefits of SIAN treatment decreased when treatment was delayed to 12 min post cyanide exposure. IN administration of SIAN 10 min following initiation of a lethal dose KCN infusion produced 100% survival (at doses ≥ 13.5 μL/kg) in NHP model of lethal cyanide intoxication.

There is mounting evidence in the literature that mesenchymal stromal/stem cell (MSC) like populations derived from different tissues, undergo epigenetic changes during aging, leading to compromised connective tissue integrity and function. This body of work has linked the biological aging of MSC to changes in their epigenetic signatures affecting growth, lifespan, self-renewal and multi-potential, due to deregulation of processes such as cellular senescence, oxidative stress, DNA damage, telomere shortening and DNA damage. This review addresses recent findings examining DNA methylation, histone modifications and miRNA changes in aging MSC populations. Moreover, we explore how epigenetic factors alter cellular pathways and associated biological networks, contributing to the MSC aging phenotype. Finally we discuss the crucial areas requiring a greater understanding of these processes, in order to piece together a global picture of the changing epigenetic landscape in MSC during aging.

ObjectiveInsomnia is underrecognized and inadequately managed, with close to 60% of cancer survivors experiencing insomnia at some point in the treatment trajectory. The objective of this study was to further understand predisposing, precipitating, and perpetuating factors in the development and maintenance of insomnia in cancer survivors.MethodA heterogeneous sample of 63 patients who had completed active treatment was recruited. Participants were required to have a score >7 on the Insomnia Severity Index and meet the diagnostic criteria for insomnia disorder. Open-ended, semistructured interviews were conducted to elicit participants’ experiences with sleep problems. An a priori set of codes and a set of codes that emerged from the data were used to analyze the data.ResultThe mean age of the sample was 60.5 years, with 30% identifying as non-white and 59% reporting their sex as female. The cancer types represented were heterogeneous with the two most common being breast (30%) and prostate (21%). Participants described an inherited risk for insomnia, anxious temperament, and insufficient ability to relax as predisposing factors. Respondents were split as to whether they classified their cancer diagnosis as the precipitating factor for their insomnia. Participants reported several behaviors that are known to perpetuate problems with sleep including napping, using back-lit electronics before bed, and poor sleep hygiene. One of the most prominent themes identified was the use of sleeping medications. Participants reported that they were reluctant to take medication but felt that it was the only option to treat their insomnia and that it was encouraged by their doctors.Significance of resultsInsomnia is a prevalent, but highly treatable, disorder in cancer survivors. Patients and provider education is needed to change individual and organizational behaviors that contribute to the development and maintenance of insomnia and increase access to evidence-based nonpharmacological interventions.

Background: Therapy-related myeloid neoplasms (tMN) is a second haematological malignancy associated with distinct molecular profile (Singhal et al Leukemia 2019) and dismal outcome. tMN is believed to arise from cytotoxic DNA damage to haematopoietic stem cells (HSC). Although, cytotoxic therapy (CT) can damage bone marrow (BM) microenvironment, its role in tMN pathogenesis remains unknown. Aim and Methods: We performed comprehensive multiomic profiling (transcriptomic, cytokine quantification, phenotype, DNA damage) of BM stromal cells (BMSC) from (i) tMN patients previously exposed to CT. Critically, we compared it with (ii) patients with MN and a history of another cancer without CT (pMN+Ca), (iii) primary MN (pMN) and (iv) age-matched controls (Healthy). Results: To decipher microenvironmental changes induced by CT from that of MN and age-related changes, whole transcriptome analysis was performed on BMSC isolated from tMN and compared with all control cohorts. Twenty-nine genes were differentially expressed in tMN compared to Healthy (FDR < 0.1, P < 0.05). Unexpectedly 146 genes were differentially expressed in tMN BMSC compared to other MN and interestingly, ~90% of differentially expressed genes were involved in senescence. Moreover, functional enrichment and GO analysis suggest DNA damage repair, cell cycle regulation, and senescence pathways were deregulated in tMN BMSC (Fig. 1A). Genes such as CDKN1A (a critical cyclin dependent kinase inhibitor orchestrating cell cycle arrest), TNFRSF10D (senescence associated), and FGF-2 (a key player in cell proliferation) were highly expressed (P < 0.001). These findings were validated by demonstrating other features of senescence in tMN BMSC: (i) enlarged/flattened cellular morphology, (ii) decreased cell proliferation and colony-forming potential, (iii) increased β-galactosidase expression, and (iv) defective DNA damage repair (Fig. 1Bi-iv). Interestingly, within the tMN cohort there was no correlation between latency period (the interval between completion of CT until tMN diagnosis) and senescence, indicating that higher senescence is persistent even after several years of CT. Senescence associated secretory phenotype (SASP), a mixture of inflammatory cytokines and chemokines such as IL-7, IL-1β, IL-13, and IL-6, were significantly higher in conditioned media of tMN BMSC (9/14, 64%) (Fig. 1 Bv). Despite reduced proliferation and senescence, transcriptome analysis showed enrichment of metabolic and energy production pathways in tMN BMSC compared to controls. TXNRD1, regulator of glucose and lipid metabolism, and BNIP3, a negative regulator of mitochondrial potential, were highly expressed in tMN BMSC (P < 0.001). These findings were further verified by Seahorse bioenergetic analyses. The overall energetic rate (as assessed by ATP production) was higher in tMN compared to Healthy BMSC (P = 0.002), with higher proportion of ATP generated by glycolysis (77% versus 35.5%) (Fig. 1C). Adipogenic differentiation potential of senescent BMSC is not well known. Transcriptome analysis showed reduced expression of genes involved in adipogenesis in tMN BMSC. This was further validated by two independent in vitro assays showing reduced adipogenesis (Fig. 1D). Interestingly, PNPLA2, a catalyst of the first lipolysis reaction, were significantly de-regulated in tMN BMSC (P < 0.001). The key difference in tMN and other MN is prior exposure to CT. Hence, we hypothesise that prior CT leads to long-term irreversible damage to BM microenvironment and induced senescence, which in turn propagate senescence in surrounding normal cells and promote clonal abnormalities in HSC. Other possibility is that tMN clone can induce these changes in BM microenvironment. To decipher it, we assessed serial BMSC. We observed aberrant stroma proliferation and bi-differentiation capacity, following CT, well before the diagnosis of tMN. Conclusions: By multiple orthogonal indices, our results show that tMN BMSC lie on an extreme trajectory away from normal and typical MN, with massive defect in senescence and distinct metabolic phenotype. Importantly, prior CT leads to long-term irreversible damage to the BM microenvironment which potentially contributes to tMN pathogenesis. Together, these data provide a valuable resource for future strategies to delay or prevent the onset of tMN and assist in marrow regeneration in patients undergoing CT. Figure 1 Figure 1. Disclosures Hughes: BMS: Research Funding; Novartis: Honoraria, Research Funding; Takeda: Honoraria. Hiwase: Novartis: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees.

Introduction: Therapy-related myeloid neoplasms (t-MN) are associated with extremely poor clinical outcomes in otherwise long-term cancer survivors. t-MN accounts for ~20% of cases of myeloid neoplasms and is expected to rise due to the increased use of chemotherapy/radiotherapy (CT/RT) and improved cancer survivorship. Historically, t-MN was considered a direct consequence of DNA damage induced in normal hematopoietic stem cells (HSC) by DNA damaging cytotoxics. However, these studies have largely ignored the bone marrow (BM) microenvironment and the effects of age and concurrent/previous cancers. Aim: We performed an exhaustive functional study of mesenchymal stromal cells (MSC) obtained from a comparatively large cohort of t-MN patients and carefully selected control populations to evaluate the long-term damage induced by cytotoxic therapy to BM microenvironment and its impact on malignant and normal haematopoiesis. Methods: Four different cohorts were used: (1) t-MN, in which myeloid malignancy occurred after CT/RT for a previous cancer (n=18); (2) patients with multiple cancer and in which a myeloid neoplasm developed following an independent cancer which was not treated with CT/RT (MC-MN; n=10); (3) primary MN (p-MN; n=7) untreated and without any prior cancer or CT/RT; (4) age-matched controls (HC; n=17). Morphology, proliferation, cellular senescence, differentiation potential and γH2AX DNA damage response was performed. Stem/progenitor supportive capacity was assessed by co-culturing haematopoietic stem cells on MSC feeder-layer in long-term culture initiating assay (LTC-IC). Cytokine measurements were performed using 38-plex magnetic bead panel (Millipore) and RNA sequencing libraries were prepared with Illumina TruSeq Total RNA protocol for 150bp paired-end sequencing on a NextSeq500 instrument. Functional enrichment analysis was performed using EnrichR software. Results: MSC cultured from t-MN patients were significantly different from HC, p-MN and MC-MN MSC according to multiple parameters. They exhibited aberrant morphology consisting of large, rounded and less adhesive cells compared to typical spindle-shaped morphology observed with controls. MSC from myeloid neoplasm also showed impaired proliferation, senescence, osteo- and adipogenic differentiation with t-MN MSC showing the greatest differences. DNA repair was dramatically impaired compared to p-MN and HC (Fig.1A). Importantly, these aberrant t-MN MSC were not able to support normal or autologous in vitro long-term haematopoiesis (Fig.1B). The biological characteristic and poor haematopoietic supportive capacity of MSC could be "cell-intrinsic" or driven by an altered paracrine inflammatory microenvironment. Interestingly, several inflammatory cytokines were higher in t-MN compared with marrow interstitial fluid obtained from p-MN patients (Fig.1Ci) and many of these including Fractalkine, IFNα2, IL-7 and G-CSF were also significantly higher in t-MN MSC conditional media (Fig.1Cii). Together, this data suggest that t-MN microenvironment is distinct from p-MN with paracrine production of pro-inflammatory milieu that may contribute to poor HSC supportive capacity. Preliminary whole transcriptome analysis revealed differential gene expression between t-MN and HC (Fig.1Di) and p-MN MSC. Importantly, the deregulated genes play critical role in cell cycle, DNA damage repair, and cellular senescence pathways explaining phenotypical characteristic of t-MN MSC (Fig.1Dii). Moreover CXCL12 expression, a key regulator of haematopoiesis, was significantly lower in t-MN compared to HC (p=0.002) and p-MN MSC (p=0.009), thus explaining poor HSC supportive capacity. The key difference between the p-MN, MC-MN and t-MN is prior exposure to CT/RT. To study this we obtained MSC from two t-MN patients for whom we had samples at the time of their primary cancer, post high-dose chemotherapy and at the time of t-MN. MSC displayed aberrant proliferation and differentiation capacity after high-dose cytotoxic therapy (2 to 4 years prior to developing t-MN) and remained aberrant at t-MN diagnosis (Fig.1E). Conclusions: BM-MSC from t-MN patients are significantly abnormal compared with age-matched controls and typical myeloid neoplasm. Importantly, prior CT/RT leads to long-term irreversible damage to the BM microenvironment which potentially contributes to t-MN pathogenesis. Disclosures Hughes: Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Hiwase:Novartis Australia: Research Funding.