Decreased but persistent epigenetic age acceleration is associated with changes in T-cell subsets after initiation of highly active antiretroviral therapy in persons living with HIV.

Introduction: Persons living with HIV (PLWH) experience the early onset of age-related illnesses, even in the setting of successful human immunodeficiency virus (HIV) suppression with highly active antiretroviral therapy (HAART). HIV infection is associated with accelerated epigenetic aging as measured using DNA methylation (DNAm)-based estimates of biological age and of telomere length (TL). Methods: DNAm levels (Infinium MethylationEPIC BeadChip) from peripheral blood mononuclear cells from 200 PLWH and 199 HIV-seronegative (SN) participants matched on chronologic age, hepatitis C virus, and time intervals were used to calculate epigenetic age acceleration, expressed as age-adjusted acceleration residuals from 4 epigenetic clocks [Horvath's pan-tissue age acceleration residual (AAR), extrinsic epigenetic age acceleration (EEAA), phenotypic epigenetic age acceleration (PEAA), and grim epigenetic age acceleration (GEAA)] plus age-adjusted DNAm-based TL (aaDNAmTL). Epigenetic age acceleration was compared for PLWH and SN participants at two visits: up to 1.5 years prior and 2-3 years after HAART (or equivalent visits). Flow cytometry was performed in PLWH and SN participants at both visits to evaluate T-cell subsets. Results: Epigenetic age acceleration in PLWH decreased after the initiation of HAART but remained greater post-HAART than that in age-matched SN participants, with differences in medians of 6.6, 9.1, and 7.7 years for AAR, EEAA, and PEAA, respectively, and 0.39 units of aaDNAmTL shortening (all p < 0.001). Cumulative HIV viral load after HAART initiation was associated with some epigenetic acceleration (EEAA, PEAA, and aaDNAmTL), but even PLWH with undetectable HIV post-HAART showed persistent epigenetic age acceleration compared to SN participants (p < 0.001). AAR, EEAA, and aaDNAmTL showed significant associations with total, naïve, and senescent CD8 T-cell counts; the total CD4 T-cell counts were associated with AAR, EEAA, and PEAA (p = 0.04 to <0.001). In an epigenome-wide analysis using weighted gene co-methylation network analyses, 11 modules demonstrated significant DNAm differences pre- to post-HAART initiation. Of these, nine were previously identified as significantly different from pre- to post-HIV infection but in the opposite direction. Discussion: In this large longitudinal study, we demonstrated that, although the magnitude of the difference decreases with HAART is associated with the cumulative viral load, PLWH are persistently epigenetically older than age-matched SN participants even after the successful initiation of HAART, and these changes are associated with changes in T-cell subsets.

Effects of highly active antiretroviral therapy initiation on epigenomic DNA methylation in persons living with HIV.

Introduction: Highly active antiretroviral therapy (HAART) helps improve some measures of accelerated epigenetic aging in persons living with HIV (PLWH), but its overall impact on the epigenome is not fully understood. Methods: In this study, we analyzed the DNA methylation profiles of PLWH (n = 187) shortly before and approximately 2-3 years after they started HAART, as well as matched seronegative (SN) controls (n = 187), taken at two time intervals. Our aim was to identify specific CpGs and biologic pathways associated with HIV infection and initiation of HAART. Additionally, we attempted to identify epigenetic changes associated with HAART initiation that were independent of HIV-associated changes, using matched HIV seronegative (SN) controls (matched on age, hepatitis C status, and interval between visits) to identify CpGs that did not differ between PLWH and SN pre-HAART but were significantly associated with HAART initiation while being unrelated to HIV viral load. Epigenome-wide association studies (EWAS) on >850,000 CpG sites were performed using pre- and post-HAART samples from PLWH. The results were then annotated using the Genomic Regions Enrichment of Annotations Tool (GREAT). Results: When only pre- and post-HAART visits in PLWH were compared, gene ontologies related to immune function and diseases related to immune function were significant, though with less significance for PLWH with detectable HIV viral loads (>50 copies/mL) at the post-HAART visit. To specifically elucidate the effects of HAART separately from HIV-induced methylation changes, we performed EWAS of HAART while also controlling for HIV viral load, and found gene ontologies associated with transplant rejection, transplant-related diseases, and other immunologic signatures. Additionally, we performed a more focused analysis that examined CpGs reaching genome-wide significance (p < 1 × 10-7) from the viral load-controlled EWAS that did not differ between all PLWH and matched SN controls pre-HAART. These CpGs were found to be near genes that play a role in retroviral drug metabolism, diffuse large B cell lymphoma proliferation, and gastric cancer metastasis. Discussion: Overall, this study provides insight into potential biological functions associated with DNA methylation changes induced by HAART initiation in persons living with HIV.

Inhibition of Ephrin B2 Reverse Signaling Abolishes Multiple Myeloma Pathogenesis.

Bone marrow vascular endothelial cells (BM EC) regulate multiple myeloma pathogenesis. Identification of the mechanisms underlying this interaction could lead to the development of improved strategies for treating multiple myeloma. Here, we performed a transcriptomic analysis of human ECs with high capacity to promote multiple myeloma growth, revealing overexpression of the receptor tyrosine kinases, EPHB1 and EPHB4, in multiple myeloma-supportive ECs. Expression of ephrin B2 (EFNB2), the binding partner for EPHB1 and EPHB4, was significantly increased in multiple myeloma cells. Silencing EPHB1 or EPHB4 in ECs suppressed multiple myeloma growth in coculture. Similarly, loss of EFNB2 in multiple myeloma cells blocked multiple myeloma proliferation and survival in vitro, abrogated multiple myeloma engraftment in immune-deficient mice, and increased multiple myeloma sensitivity to chemotherapy. Administration of an EFNB2-targeted single-chain variable fragment also suppressed multiple myeloma growth in vivo. In contrast, overexpression of EFNB2 in multiple myeloma cells increased STAT5 activation, increased multiple myeloma cell survival and proliferation, and decreased multiple myeloma sensitivity to chemotherapy. Conversely, expression of mutant EFNB2 lacking reverse signaling capacity in multiple myeloma cells increased multiple myeloma cell death and sensitivity to chemotherapy and abolished multiple myeloma growth in vivo. Complementary analysis of multiple myeloma patient data revealed that increased EFNB2 expression is associated with adverse-risk disease and decreased survival. This study suggests that EFNB2 reverse signaling controls multiple myeloma pathogenesis and can be therapeutically targeted to improve multiple myeloma outcomes. SIGNIFICANCE: Ephrin B2 reverse signaling mediated by endothelial cells directly regulates multiple myeloma progression and treatment resistance, which can be overcome through targeted inhibition of ephrin B2 to abolish myeloma.

Erratum: Accelerated aging with HIV begins at the time of initial HIV infection.

[This corrects the article DOI: 10.1016/j.isci.2022.104488.].

DNA Methylation, Aging, and Cancer Risk: A Mini-Review.

Accumulation of somatic mutations and genomic instability are hallmarks of both aging and cancer. Epigenetic alterations occur across cell types and tissues with advancing age. DNA methylation-based estimates of biologic age can predict important age-related outcomes, including risk of frailty and mortality, and most recently have been shown to be associated with risk of developing cancer. In this mini-review, we examine pathways known to exhibit altered methylation in aging tissues, pre-malignant lesions, and tumors and review methodologies of epigenetic clocks that reliably predict cancer risk, including those derived from methylation studies of peripheral blood, as well as those methylation levels from within the tissues at high risk of cancer.

Accelerated aging with HIV begins at the time of initial HIV infection.

Living with HIV infection is associated with early onset of aging-related chronic conditions, sometimes described as accelerated aging. Epigenetic DNA methylation patterns can evaluate acceleration of biological age relative to chronological age. The impact of initial HIV infection on five epigenetic measures of aging was examined before and approximately 3 years after HIV infection in the same individuals (n=102). Significant epigenetic age acceleration (median 1.9-4.8 years) and estimated telomere length shortening (all p≤ 0.001) were observed from pre-to post-HIV infection, and remained significant in three epigenetic measures after controlling for T cell changes. No acceleration was seen in age- and time interval-matched HIV-uninfected controls. Changes in genome-wide co-methylation clusters were also significantly associated with initial HIV infection (p≤ 2.0 × 10-4). These longitudinal observations clearly demonstrate an early and substantial impact of HIV infection on the epigenetic aging process, and suggest a role for HIV itself in the earlier onset of clinical aging.

The impact of reproductive factors on DNA methylation-based telomere length in healthy breast tissue.

Estrogen promotes breast tissue proliferation and telomerase activation. We investigated the effects of reproductive history on cell cycling and telomere length using a DNA methylation-based estimate of telomere length (DNAmTL) in breast and blood from healthy women donors. We demonstrate that DNAmTL is shorter in breast than in blood, and that nulliparous women have longer age-adjusted DNAmTL in both breast and blood, potentially explaining their higher risk of breast cancer.

An examination of school reopening strategies during the SARS-CoV-2 pandemic.

The SARS-CoV-2 pandemic led to closure of nearly all K-12 schools in the United States of America in March 2020. Although reopening K-12 schools for in-person schooling is desirable for many reasons, officials understand that risk reduction strategies and detection of cases are imperative in creating a safe return to school. Furthermore, consequences of reclosing recently opened schools are substantial and impact teachers, parents, and ultimately educational experiences in children. To address competing interests in meeting educational needs with public safety, we compare the impact of physical separation through school cohorts on SARS-CoV-2 infections against policies acting at the level of individual contacts within classrooms. Using an age-stratified Susceptible-Exposed-Infected-Removed model, we explore influences of reduced class density, transmission mitigation, and viral detection on cumulative prevalence. We consider several scenarios over a 6-month period including (1) multiple rotating cohorts in which students cycle through in-person instruction on a weekly basis, (2) parallel cohorts with in-person and remote learning tracks, (3) the impact of a hypothetical testing program with ideal and imperfect detection, and (4) varying levels of aggregate transmission reduction. Our mathematical model predicts that reducing the number of contacts through cohorts produces a larger effect than diminishing transmission rates per contact. Specifically, the latter approach requires dramatic reduction in transmission rates in order to achieve a comparable effect in minimizing infections over time. Further, our model indicates that surveillance programs using less sensitive tests may be adequate in monitoring infections within a school community by both keeping infections low and allowing for a longer period of instruction. Lastly, we underscore the importance of factoring infection prevalence in deciding when a local outbreak of infection is serious enough to require reverting to remote learning.

The Effects of Lifetime Estrogen Exposure on Breast Epigenetic Age.

BACKGROUND: Estrogens are thought to contribute to breast cancer risk through cell cycling and accelerated breast aging. We hypothesize that lifetime estrogen exposure drives early epigenetic breast aging observed in healthy women. In this study, we examined associations between hormonal factors and epigenetic aging measures in healthy breast tissues. METHODS: We extracted DNA from breast tissue specimens from 192 healthy female donors to the Susan G. Komen Tissue Bank at the Indiana University Simon Cancer Center. Methylation experiments were performed using the Illumina EPIC 850K array platform. Age-adjusted regression models were used to examine for associations between factors related to estrogen exposure and five DNA methylation-based estimates: Grim age, pan-tissue age, Hannum age, phenotypic age, and skin and blood clock age. RESULTS: Women were aged 19-90 years, with 95 premenopausal, and 97 nulliparous women. The age difference (Grim age - chronologic age) was higher at earlier ages close to menarche. We found significant associations between earlier age at menarche and age-adjusted accelerations according to the Grim clock, the skin and blood clock, and between higher body mass index (BMI) and age-adjusted accelerations in the Grim clock, Hannum clock, phenotypic clock, and skin and blood clock. CONCLUSIONS: Earlier age at menarche and higher BMI are associated with elevations in DNA methylation-based age estimates in healthy breast tissues, suggesting that cumulative estrogen exposure drives breast epigenetic aging. IMPACT: Epigenetic clock measures may help advance inquiry into the relationship between accelerated breast tissue aging and an elevated incidence of breast cancer in younger women.

Increased Rate of Epigenetic Aging in Men Living With HIV Prior to Treatment.

Background: Epigenetic aging is accelerated in tissues of persons living with HIV (PLWH) and may underlie the early onset of age-related illnesses. This study examines the rate-of-change in epigenetic age in PLWH following HIV infection but before HAART, using archived longitudinal samples from the Multicenter AIDS Cohort Study. Methods: DNA was isolated from cryopreserved peripheral blood mononuclear cells from 101 men living with HIV, with baseline visit <2.5 years after HIV seroconversion (Visit 1) and follow-up visit <1.5 years before the initiation of HAART (Visit 2), and 100 HIV-uninfected men matched on age and visits with comparable time intervals. DNA methylation (DNAm) age was estimated for five clocks (Pan-tissue, Extrinsic, Phenotypic, Grim, and Skin & Blood age), and a DNAm-based estimate of telomere length (DNAmTL). Multivariate linear regression models were used to examine baseline factors associated with rate-of-aging, defined as (DNAm age visit 2-DNAm age visit 1)/(age visit 2-age visit 1). Results: Epigenetic age increased approximately twice as fast in PLWH as uninfected controls (Pan-tissue, Extrinsic, and Phenotypic clocks). Shortening of DNAmTL was nearly 3-fold faster in PLWH than controls. Faster rate-of-aging was associated with HIV status (Pan-Tissue, Extrinsic, Phenotypic, and DNAmTL), white race (Extrinsic, DNAmTL), higher cumulative HIV viral load (Grim), and lower baseline DNAm age (Phenotypic, Skin & Blood). Conclusion: Epigenetic rates-of-aging were significantly faster for untreated PLWH. Our findings expand on the important impact of HIV infection on biologic aging, both in elevating epigenetic age and increasing the rate-of-aging in the years following infection.

An examination of school reopening strategies during the SARS-CoV-2 pandemic.

The SARS-CoV-2 pandemic led to closure of nearly all K-12 schools in the United States of America in March 2020. Although reopening K-12 schools for in-person schooling is desirable for many reasons, officials understand that risk reduction strategies and detection of cases are imperative in creating a safe return to school. Furthermore, consequences of reclosing recently opened schools are substantial and impact teachers, parents, and ultimately educational experiences in children. To address competing interests in meeting educational needs with public safety, we compare the impact of physical separation through school cohorts on SARS-CoV-2 infections against policies acting at the level of individual contacts within classrooms. Using an age-stratified Susceptible-Exposed-Infected-Removed model, we explore influences of reduced class density, transmission mitigation, and viral detection on cumulative prevalence. We consider several scenarios over a 6-month period including (1) multiple rotating cohorts in which students cycle through in-person instruction on a weekly basis, (2) parallel cohorts with in-person and remote learning tracks, (3) the impact of a hypothetical testing program with ideal and imperfect detection, and (4) varying levels of aggregate transmission reduction. Our mathematical model predicts that reducing the number of contacts through cohorts produces a larger effect than diminishing transmission rates per contact. Specifically, the latter approach requires dramatic reduction in transmission rates in order to achieve a comparable effect in minimizing infections over time. Further, our model indicates that surveillance programs using less sensitive tests may be adequate in monitoring infections within a school community by both keeping infections low and allowing for a longer period of instruction. Lastly, we underscore the importance of factoring infection prevalence in deciding when a local outbreak of infection is serious enough to require reverting to remote learning.

The acute effects of adjuvant radiation and chemotherapy on peripheral blood epigenetic age in early stage breast cancer patients.

Survival has increased in early stage breast cancer (BC), and the late effects of treatment persist for decades. Molecular mechanisms underlying the acceleration of age-related diseases after chemotherapy and radiotherapy are poorly understood. We examined epigenetic changes in peripheral whole blood cells in early stage BC patients undergoing surgery followed by adjuvant radiotherapy, or surgery followed by adjuvant chemotherapy and radiotherapy. DNA methylation experiments were performed on whole blood samples collected before and after adjuvant therapy. Methylation profiles were used to estimate four measures of epigenetic age acceleration-intrinsic, extrinsic, phenotypic, and Grim-and cell counts. We found significant increases in extrinsic, phenotypic, and Grim epigenetic age acceleration and in estimated proportions of senescent T lymphocytes from pre- to post-treatment. When examining differential effects by treatment category, most of these increases were significant only in women undergoing radiation alone. Further studies are needed to examine whether these effects are related to the risk of cognitive and functional decline in BC survivors.

The Effects of Anti-retroviral Therapy on Epigenetic Age Acceleration Observed in HIV-1-infected Adults.

BACKGROUND: HIV-1 infection is associated with acceleration of age-related methylation patterns in peripheral blood and brain of infected individuals although the relative contributions of HIV-1 infection versus its treatment to the observed accelerations in biological aging have not yet been investigated. METHODS: In this longitudinal study of the effects of antiretroviral therapy (ART) on epigenetic aging patterns, we extracted DNA from peripheral blood mononuclear cells from 15 HIV-1-infected individuals infected at three time points: 6 months-1year pre-ART, 6-12 months post-initiation of ART, and 18-24 months after initiating ART. We compared these trajectories with those of 15 age-matched uninfected control participants at three time points with similar intervals. Methylation studies were performed using the Infinium methylation 450 arrays. We examined four epigenetic clock measurements: Age acceleration residual (AAR), Extrinsic (EEAA), Phenotypic (PEAA), and Grim (GEAA) epigenetic age acceleration. Weighted correlation network (WGCNA) analysis was used to identify clusters of highly co-methylated CpGs. RESULTS: We found that prior to the initiation of ART all four epigenetic measures were significantly higher in HIV-1-infected individuals compared with uninfected individuals (P<0.001 for AAR, P=0.008 for EEAA, P=0.012 for GEAA, P<0.001 for PEAA using Wilcoxon rank sum tests between serostatus groups). These effects persisted after the initiation of ART, although the magnitude of these differences diminished. At 18-24 months post-ART initiation (time point 3), PEAA and GEAA were no longer significantly different between HIV-1-infected and uninfected individuals (P=0.059 for PEAA, P=0.11 for GEAA), while AAR and EEAA remained significantly higher in HIV-1-infected individuals compared with uninfected individuals. We further examined for global patterns of methylation differences between HIV-1-infected and uninfected at each time point, and found 14 groups of co-methylated CpGs that were significantly different between groups at baseline, and remained different after the initiation of ART. Conclusion: We confirm that epigenetic age acceleration associated with HIV-1 infection is most dramatic before ART initiation, and this observation is consistent across four epigenetic clock measurements, as well as in additional groups of co-methylated CpGs identified using WGCNA. Following initiation of ART, there is a partial reduction in age acceleration in all measures, with loss of any significant difference in PEAA and GEAA between serostatus groups. Our findings support the need for future studies examining for a link between epigenetic age acceleration and clinical outcomes in HIV-1-infected individuals.

BioSimulator.jl: Stochastic simulation in Julia.

BACKGROUND AND OBJECTIVES: Biological systems with intertwined feedback loops pose a challenge to mathematical modeling efforts. Moreover, rare events, such as mutation and extinction, complicate system dynamics. Stochastic simulation algorithms are useful in generating time-evolution trajectories for these systems because they can adequately capture the influence of random fluctuations and quantify rare events. We present a simple and flexible package, BioSimulator.jl, for implementing the Gillespie algorithm, τ-leaping, and related stochastic simulation algorithms. The objective of this work is to provide scientists across domains with fast, user-friendly simulation tools. METHODS: We used the high-performance programming language Julia because of its emphasis on scientific computing. Our software package implements a suite of stochastic simulation algorithms based on Markov chain theory. We provide the ability to (a) diagram Petri Nets describing interactions, (b) plot average trajectories and attached standard deviations of each participating species over time, and (c) generate frequency distributions of each species at a specified time. RESULTS: BioSimulator.jl's interface allows users to build models programmatically within Julia. A model is then passed to the simulate routine to generate simulation data. The built-in tools allow one to visualize results and compute summary statistics. Our examples highlight the broad applicability of our software to systems of varying complexity from ecology, systems biology, chemistry, and genetics. CONCLUSION: The user-friendly nature of BioSimulator.jl encourages the use of stochastic simulation, minimizes tedious programming efforts, and reduces errors during model specification.

Modeling of Interactions between Cancer Stem Cells and their Microenvironment: Predicting Clinical Response.

Mathematical models of cancer stem cells are useful in translational cancer research for facilitating the understanding of tumor growth dynamics and for predicting treatment response and resistance to combined targeted therapies. In this chapter, we describe appealing aspects of different methods used in mathematical oncology and discuss compelling questions in oncology that can be addressed with these modeling techniques. We describe a simplified version of a model of the breast cancer stem cell niche, illustrate the visualization of the model, and apply stochastic simulation to generate full distributions and average trajectories of cell type populations over time. We further discuss the advent of single-cell data in studying cancer stem cell heterogeneity and how these data can be integrated with modeling to advance understanding of the dynamics of invasive and proliferative populations during cancer progression and response to therapy.

Potential Mechanisms of Age Acceleration Caused by Estrogen Deprivation: Do Endocrine Therapies Carry the Same Risks?

Longer duration of endocrine therapy decreases breast cancer recurrence and mortality, but these benefits need to be weighed against potential risks to overall health. Notable side effects of endocrine therapy include cataracts, uterine cancer, thromboembolic events, osteoporosis and fracture risk, chronic musculoskeletal complaints, as well as vaginal dryness and discharge, and vasomotor symptoms. Estrogen deprivation in healthy women younger than 50 years undergoing bilateral oophorectomy has been shown to accelerate the development of diseases related to aging, including coronary artery disease, cardiac arrhythmias, stroke, dementia, and osteoporosis, raising concern that even less dramatic modulation of estrogen homeostasis may adversely affect health outcomes. Diminished available estrogen at the cellular and molecular level may facilitate mechanisms that underlie the aging process, often termed the hallmarks of aging. In this review, we describe estrogen's role in normal physiology across tissues, review the effects of estrogen deprivation on health outcomes in the setting of both surgical and natural menopause, and examine the hallmarks of aging with attention to the effects of estrogen and estrogen blockade on each molecular mechanism underlying the aging process.

DNA methylation age is elevated in breast tissue of healthy women.

BACKGROUND: Limited evidence suggests that female breast tissue ages faster than other parts of the body according to an epigenetic biomarker of aging known as the "epigenetic clock." However, it is unknown whether breast tissue samples from healthy women show a similar accelerated aging effect relative to other tissues, and what could drive this acceleration. The goal of this study is to validate our initial finding of advanced DNA methylation (DNAm) age in breast tissue, by directly comparing it to that of peripheral blood tissue from the same individuals, and to do a preliminary assessment of hormonal factors that could explain the difference. METHODS: We utilized n = 80 breast and 80 matching blood tissue samples collected from 40 healthy female participants of the Susan G. Komen Tissue Bank at the Indiana University Simon Cancer Center who donated these samples at two time points spaced at least a year apart. DNA methylation levels (Illumina 450K platform) were used to estimate the DNAm age. RESULTS: DNAm age was highly correlated with chronological age in both peripheral blood (r = 0.94, p < 0.0001) and breast tissues (r = 0.86, p < 0.0001). A measure of epigenetic age acceleration (age-adjusted DNAm Age) was substantially increased in breast relative to peripheral blood tissue (p = 1.6 × 10-11). The difference between DNAm age of breast and blood decreased with advancing chronologic age (r = -0.53, p = 4.4 × 10-4). CONCLUSIONS: Our data clearly demonstrate that female breast tissue has a higher epigenetic age than blood collected from the same subject. We also observe that the degree of elevation in breast diminishes with advancing age. Future larger studies will be needed to examine associations between epigenetic age acceleration and cumulative hormone exposure.

Disease drivers of aging.

It has long been known that aging, at both the cellular and organismal levels, contributes to the development and progression of the pathology of many chronic diseases. However, much less research has examined the inverse relationship-the contribution of chronic diseases and their treatments to the progression of aging-related phenotypes. Here, we discuss the impact of three chronic diseases (cancer, HIV/AIDS, and diabetes) and their treatments on aging, putative mechanisms by which these effects are mediated, and the open questions and future research directions required to understand the relationships between these diseases and aging.

Modeling of Cancer Stem Cell State Transitions Predicts Therapeutic Response.

Cancer stem cells (CSCs) possess capacity to both self-renew and generate all cells within a tumor, and are thought to drive tumor recurrence. Targeting the stem cell niche to eradicate CSCs represents an important area of therapeutic development. The complex nature of many interacting elements of the stem cell niche, including both intracellular signals and microenvironmental growth factors and cytokines, creates a challenge in choosing which elements to target, alone or in combination. Stochastic stimulation techniques allow for the careful study of complex systems in biology and medicine and are ideal for the investigation of strategies aimed at CSC eradication. We present a mathematical model of the breast cancer stem cell (BCSC) niche to predict population dynamics during carcinogenesis and in response to treatment. Using data from cell line and mouse xenograft experiments, we estimate rates of interconversion between mesenchymal and epithelial states in BCSCs and find that EMT/MET transitions occur frequently. We examine bulk tumor growth dynamics in response to alterations in the rate of symmetric self-renewal of BCSCs and find that small changes in BCSC behavior can give rise to the Gompertzian growth pattern observed in breast tumors. Finally, we examine stochastic reaction kinetic simulations in which elements of the breast cancer stem cell niche are inhibited individually and in combination. We find that slowing self-renewal and disrupting the positive feedback loop between IL-6, Stat3 activation, and NF-κB signaling by simultaneous inhibition of IL-6 and HER2 is the most effective combination to eliminate both mesenchymal and epithelial populations of BCSCs. Predictions from our model and simulations show excellent agreement with experimental data showing the efficacy of combined HER2 and Il-6 blockade in reducing BCSC populations. Our findings will be directly examined in a planned clinical trial of combined HER2 and IL-6 targeted therapy in HER2-positive breast cancer.