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PURPOSE For patients with hormone receptor–positive (HR+), human epidermal growth factor receptor 2–negative (HER2–) metastatic breast cancer (MBC), first-line treatment is endocrine therapy (ET) plus cyclin-dependent kinase 4/6 inhibition (CDK4/6i). After disease progression, which often comes with ESR1 resistance mutations (ESR1-MUT), which therapies to use next and for which patients are open questions. An active area of exploration is treatment with further CDK4/6i, particularly abemaciclib, which has distinct pharmacokinetic and pharmacodynamic properties compared with the other approved CDK4/6 inhibitors, palbociclib and ribociclib. We investigated a gene panel to prognosticate abemaciclib susceptibility in patients with ESR1-MUT MBC after palbociclib progression. METHODS We examined a multicenter retrospective cohort of patients with ESR1-MUT MBC who received abemaciclib after disease progression on ET plus palbociclib. We generated a panel of CDK4/6i resistance genes and compared abemaciclib progression-free survival (PFS) in patients without versus with mutations in this panel (CDKi-R[–] v CDKi-R[+]). We studied how ESR1-MUT and CDKi-R mutations affect abemaciclib sensitivity of immortalized breast cancer cells and patient-derived circulating tumor cell lines in culture. RESULTS In ESR1-MUT MBC with disease progression on ET plus palbociclib, the median PFS was 7.0 months for CDKi-R(–) (n = 17) versus 3.5 months for CDKi-R(+) (n = 11), with a hazard ratio of 2.8 ( P = .03). In vitro, CDKi-R alterations but not ESR1-MUT induced abemaciclib resistance in immortalized breast cancer cells and were associated with resistance in circulating tumor cells. CONCLUSION For ESR1-MUT MBC with resistance to ET and palbociclib, PFS on abemaciclib is longer for patients with CDKi-R(–) than CDKi-R(+). Although a small and retrospective data set, this is the first demonstration of a genomic panel associated with abemaciclib sensitivity in the postpalbociclib setting. Future directions include testing and improving this panel in additional data sets, to guide therapy selection for patients with HR+/HER2– MBC.

In metastatic hormone receptor-positive breast cancer, ESR1 mutations are a common cause of acquired resistance to the backbone of therapy, estrogen deprivation by aromatase inhibition. How these mutations affect tumor sensitivity to established and novel therapies are active areas of research. These therapies include estrogen receptor-targeting agents, such as selective estrogen receptor modulators, covalent antagonists, and degraders (including tamoxifen, fulvestrant, and novel agents), and combination therapies, such as endocrine therapy plus CDK4/6, PI3K, or mTORC1 inhibition. In this review, we summarize existing knowledge surrounding the mechanisms of action of ESR1 mutations and roles in resistance to aromatase inhibition. We then analyze the recent literature on how ESR1 mutations affect outcomes in estrogen receptor-targeting and combination therapies. For estrogen receptor-targeting therapies such as tamoxifen and fulvestrant, ESR1 mutations cause relative resistance in vitro but do not clearly lead to resistance in patients, making novel agents in this category promising. Regarding combination therapies, ESR1 mutations nullify any aromatase inhibitor component of the combination. Thus, combinations using endocrine alternatives to aromatase inhibition, or combinations where the non-endocrine component is efficacious as monotherapy, are still effective against ESR1 mutations. These results emphasize the importance of investigating combinatorial resistance, challenging as these efforts are. We also discuss future directions and open questions, such as studying the differences among distinct ESR1 mutations, asking how to adjust clinical decisions based on molecular surveillance testing, and developing novel therapies that are effective against ESR1 mutations.

Aging impairs tissue repair. This is pronounced in skeletal muscle, whose regeneration by muscle stem cells (MuSCs) is robust in young adult animals but inefficient in older organisms. Despite this functional decline, old MuSCs are amenable to rejuvenation through strategies that improve the systemic milieu, such as heterochronic parabiosis. One such strategy, exercise, has long been appreciated for its benefits on healthspan, but its effects on aged stem cell function in the context of tissue regeneration are incompletely understood. Here we show that exercise in the form of voluntary wheel running accelerates muscle repair in old animals and improves old MuSC function. Through transcriptional profiling and genetic studies, we discovered that the restoration of old MuSC activation ability hinges on restoration of Cyclin D1, whose expression declines with age in MuSCs. Pharmacologic studies revealed that Cyclin D1 maintains MuSC activation capacity by repressing TGFβ signaling. Taken together, these studies demonstrate that voluntary exercise is a practicable intervention for old MuSC rejuvenation. Furthermore, this work highlights the distinct role of Cyclin D1 in stem cell quiescence.

Background Fusobacterium bacteria are fastidious commensals in the human oropharyngeal microbiome. As pathogens, Fusobacterium are associated with diverse infectious and inflammatory conditions, including Lemierre's syndrome, colorectal cancer, and inflammatory bowel disease. Portal and mesenteric thromboses associated with Fusobacterium are case-reportable; due to the rarity of these reports, the full spectrum of presentations and outcomes remains unknown. Case presentation A 71-year-old man presented with two weeks of abdominal pain and night sweats. He was found to have extensive portal and mesenteric venous thromboses and pancolitis. Initial workup for hypercoagulable risk factors was negative; after discharge, blood cultures became positive for Fusobacterium nucleatum. The patient was treated with antibiotics and anticoagulation with complete resolution of symptoms, thromboses, and colitis. Conclusions This report demonstrates the protean nature of Fusobacterium-associated disease, which in this patient was portal and mesenteric venous thromboses without oropharyngeal disease. We discuss the modes of transmission, mechanisms of infection, and what remains unknown about the causes and consequences of Fusobacterium-associated disease. This report also demonstrates that cases with atypical abdominal thromboses, despite a negative comprehensive workup, deserve a high suspicion for other causal factors that are not part of standard diagnostic evaluation.

Summary Adult skeletal muscle stem cells (MuSCs) are important for muscle regeneration and constitute a potential source of cell therapy. However, upon isolation, MuSCs rapidly exit quiescence and lose transplantation potency. Maintenance of the quiescent state in vitro preserves MuSC transplantation efficiency and provides an opportunity to study the biology of quiescence. Here we show that Tubastatin A (TubA), an Hdac6 inhibitor, prevents primary cilium resorption, maintains quiescence, and enhances MuSC survival ex vivo. Phenotypic characterization and transcriptomic analysis of TubA-treated cells revealed that TubA maintains most of the biological features and molecular signatures of quiescence. Furthermore, TubA-treated MuSCs showed improved engraftment ability upon transplantation. TubA also induced a return to quiescence and improved engraftment of cycling MuSCs, revealing a potentially expanded application for MuSC therapeutics. Altogether, these studies demonstrate the ability of TubA to maintain MuSC quiescence ex vivo and to enhance the therapeutic potential of MuSCs and their progeny., Highlights • TubA prevents primary cilium resorption and maintains MuSC quiescence ex vivo • TubA treatment maintains the transcriptional characteristics of quiescent MuSCs • Treatment with TubA sustains MuSC engraftment potential for transplantation studies • TubA induces a return to quiescence of cycling MuSC progeny, In this article, Arjona et al. show that TubA, an HDAC6 inhibitor, can inhibit primary cilium resorption and preserve MuSC quiescence and stem cell potency ex vivo. Indeed, treatment with TubA maintains MuSC engraftment potential and induces a return to quiescence in cycling MuSCs, revealing a potentially valuable approach to enhancing the therapeutic potential of MuSCs.

Background: For patients with hormone receptor-positive (HR+) metastatic breast cancer (MBC) and progression on combination endocrine therapy plus palbociclib/ribociclib, abemaciclib can be effective and well-tolerated, conferring durable clinical benefit in a subset of patients (Wander SA et al. JNCCN 2021). We previously reported that, even in patients with ESR1-mutant (ESR1-MUT) HR+ MBC, some - but not all - still achieve long-term disease control with abemaciclib (Wander SA et al. SABCS 2020). How to identify which patients are likely to benefit from abemaciclib after progression on combination endocrine therapy and CDK4/6 inhibition is not known and, more generally, predicting tumor response to CDK4/6 inhibition has been an area of ongoing research. Because resistance to CDK4/6 inhibition can occur through multiple mechanisms, we hypothesized that a comprehensive resistance panel, rather than single genetic markers or immunohistochemical readouts, would provide an effective predictive tool. Methods: To determine which patients with ESR1-MUT MBC and progression on endocrine therapy plus palbociclib/ribociclib benefit from abemaciclib, we examined a multicenter cohort of such patients who had genomic profiling by standard commercially available assays, the majority of which were via plasma-based cell-free DNA (cfDNA) genotyping assays. We generated a curated list based upon prior literature of CDK4/6i resistance drivers that had been validated in tumor biopsy specimens and in laboratory models: these genes were involved in cell cycle regulation (CCNE1/2, RB1, AURKA) and growth factor signaling pathways (ERBB2, FGFR1/2, AKT1, PTEN, KRAS, FAT1). Progression-free survival (PFS) was defined as time from abemaciclib initiation to time of discontinuation due to disease progression or death; patients who discontinued due to toxicity were right-censored. To examine the cellular effects of different mutations, we also studied the impact of ESR1-MUT, RB1 loss, and KRAS activation on the growth and survival (using an ATP abundance-based assay) of patient-derived circulating tumor cell (CTC) lines treated with palbociclib and abemaciclib in vitro. Results: Among patients with ESR1-MUT MBC with disease progression on endocrine therapy plus palbociclib/ribociclib (n=28), absence of co-existing genomic alterations in our curated panel was associated with greater clinical benefit with subsequent abemaciclib. Patients lacking a mutation in this resistance panel (n=17) had a median PFS of 7.0 months (95% CI: 4.1-13.2); patients with at least one mutation in this panel (n=11) had a median PFS of 3.5 months (95% CI: 2.1-5.4). The difference in PFS was statistically significant (p=0.02, log-rank test). On univariable Cox regression the hazard ratio for patients with a mutation in the resistance panel was 2.8 (95% CI: 1.1-7.1, p=0.03). In vitro, two out of three patient-derived cell lines with ESR1-MUT remained sensitive to abemaciclib, while those with mutation in RB1 or KRAS were less sensitive to abemaciclib. Conclusions: In our study, absence of co-existing genomic alterations in a curated panel was associated with greater clinical benefit with subsequent abemaciclib among patients with ESR1-MUT MBC with prior disease progression on endocrine therapy plus palbociclib/ribociclib. While a small dataset, this is the first demonstration of a genomic panel associated with continued CDK4/6 inhibitor sensitivity. Future directions include testing this panel outside of ESR1-MUT MBC and refining the panel in additional datasets with increased sample size, to guide therapy selection for patients with HR+ MBC. Citation Format: Jamie O Brett, Taronish D Dubash, Andrzej Niemierko, Veronica Mariotti, Leslie SL Kim, Jing Xi, Apurva Pandey, Siobhan Dunne, Azadeh Nasrazadani, Maxwell R Lloyd, Laura M Spring, Douglas Micalizzi, Maristela Onozato, Dante Che, Adam Brufsky, Kevin M Kalinsky, Cynthia X Ma, Joyce O’Shaughnessy, Hyo S Han, A. John Iafrate, Shyamala Maheswaran, Daniel A Haber, Aditya Bardia, Seth A Wander. Association between co-existing genomic alterations and abemaciclib benefit in patients with metastatic hormone receptor-positive breast cancer with ESR1 mutations following disease progression on prior endocrine therapy plus palbociclib or ribociclib [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr PD2-03.

Background For HR+ MBC, ESR1 point mutations (ESR1-MUT) are a common mechanism of acquired resistance to aromatase inhibition (AI); ESR1 fusions (ESR1-FUS) are rare and promote intrinsic resistance to ER-targeting drugs. Retrospective analyses of CDK4/6i trials suggest ESR1-MUT does not cause CDK4/6i resistance, but whether CDK4/6i is effective for ESR1-MUT, or for ESR1-FUS, in the real-world setting is unknown. Methods Real-world evidence was sourced from the GuardantInform database of commercial payer claims and ctDNA tests from 170,000+ individuals. Patients with MBC who started CDK4/6i within 30 days of ctDNA testing were categorized as ESR1-MUT vs. ESR1-WT and analyzed for time-to-next-treatment (TTNT). Separately, cases with ESR1-FUS detected by tissue RNA-Seq were extracted from a clinicopathologic database at an academic cancer center. Results There was no significant difference in TTNT on CDK4/6i for ESR1-MUT vs. ESR1-WT. As expected, ESR1-MUT had shorter overall survival (OS), even after adjustment for age, CDK4/6i drug, and prior treatment (HR 0.58 (0.42-0.82), p=0.002, multivariable Cox). Endocrine partner analysis was limited by lack of clinical annotation to 27% of cases: AI was given to 55% of ESR1-WT and 25% of ESR1-MUT; fulvestrant was given to 39% of ESR1-WT and 68% of ESR1-MUT. Additional stratified analyses will be presented. In the clinicopathologic database, we identified 4 ESR1-FUS cases, and all received CDK4/6i. Progression-free survival durations on CDK4/6i were 4, 10, 11, and 33+ months. Conclusions Using real-world evidence, we demonstrate that CDK4/6i is effective in both ESR1-MUT and ESR1-WT HR+ MBC, supporting the use of CDK4/6i in this setting. CDK4/6i may be additionally beneficial for patients with ESR1-FUS. Future directions include expanding the ESR1-FUS cohort and deciphering the heterogeneity of CDK4/6i responses in this patient population. ESR1-WT ESR1-MUT p-value n=612 n=145 TTNT, median days (95% CI) 99 (85-121) 102 (85-152) 0.84 (log-rank) OS, median years (95% CI) 5.1 (4.5-NA) 2.2 (2.0-NA) Citation Format: Jamie O. Brett, Caroline M. Weipert, Lauren L. Ritterhouse, Nicole Zhang, Junhua Yu, Lianne Y. Ryan, Laura M. Spring, Miguel N. Rivera, Jochen K. Lennerz, Dora Dias-Santagata, Leif W. Ellisen, Aditya Bardia, Seth A. Wander. CDK4/6 inhibition (CDK4/6i) is effective in the real-world setting for hormone receptor-positive metastatic breast cancer (HR+ MBC) with ESR1 mutations and fusions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5248.

A common concept in aging research is that chronological age is the most important risk factor for the development of diverse diseases, including degenerative diseases and cancers. The mechanistic link between the aging process and disease pathogenesis, however, is still enigmatic. Nevertheless, measurement of lifespan, as a surrogate for biological aging, remains among the most frequently used assays in aging research. In this review, we examine the connection between “normal aging” and age-related disease from the point of view that they form a continuum of aging phenotypes. This notion of common mechanisms gives rise to the converse postulate that diseases may be risk factors for accelerated aging. We explore the advantages and caveats associated with using lifespan as a metric to understand cell and tissue aging, focusing on the elucidation of molecular mechanisms and potential therapies for age-related diseases.

Oligodendrocytes—the myelin-forming cells of the central nervous system—can be regenerated during adulthood. In adults, new oligodendrocytes originate from oligodendrocyte progenitor cells (OPCs), but also from neural stem cells (NSCs). Although several factors supporting oligodendrocyte production have been characterized, the mechanisms underlying the generation of adult oligodendrocytes are largely unknown. Here we show that genetic inactivation of SIRT1, a protein deacetylase implicated in energy metabolism, increases the production of new OPCs in the adult mouse brain, in part by acting in NSCs. New OPCs produced following SIRT1 inactivation differentiate normally, generating fully myelinating oligodendrocytes. Remarkably, SIRT1 inactivation ameliorates remyelination and delays paralysis in mouse models of demyelinating injuries. SIRT1 inactivation leads to the upregulation of genes involved in cell metabolism and growth factor signalling, in particular PDGF receptor α (PDGFRα). Oligodendrocyte expansion following SIRT1 inactivation is mediated at least in part by AKT and p38 MAPK—signalling molecules downstream of PDGFRα. The identification of drug-targetable enzymes that regulate oligodendrocyte regeneration in adults could facilitate the development of therapies for demyelinating injuries and diseases, such as multiple sclerosis.

A promising therapeutic strategy for diverse genetic disorders involves transplantation of autologous stem cells that have been genetically corrected ex vivo. A major challenge in such approaches is a loss of stem cell potency during culture. Here we describe an artificial niche for maintaining muscle stem cells (MuSCs) in vitro in a potent, quiescent state. Using a machine learning method, we identified a molecular signature of quiescence and used it to screen for factors that could maintain mouse MuSC quiescence, thus defining a quiescence medium (QM). We also engineered muscle fibers that mimic the native myofiber of the MuSC niche. Mouse MuSCs maintained in QM on engineered fibers showed enhanced potential for engraftment, tissue regeneration and self-renewal after transplantation in mice. An artificial niche adapted to human cells similarly extended the quiescence of human MuSCs in vitro and enhanced their potency in vivo. Our approach for maintaining quiescence may be applicable to stem cells isolated from other tissues.

In adult mammals, neural stem cells (NSCs) generate new neurons that are important for specific types of learning and memory. Controlling adult NSC number and function is fundamental for preserving the stem cell pool and ensuring proper levels of neurogenesis throughout life. Here we study the importance of the microRNA gene cluster miR-106b~25 (miR-106b, miR-93, and miR-25) in primary cultures of neural stem/progenitor cells (NSPCs) isolated from adult mice. We find that knocking down miR-25 decreases NSPC proliferation, whereas ectopically expressing miR-25 promotes NSPC proliferation. Expressing the entire miR-106b~25 cluster in NSPCs also increases their ability to generate new neurons. Interestingly, miR-25 has a number of potential target mRNAs involved in insulin/insulin-like growth factor-1 (IGF) signaling, a pathway implicated in aging. Furthermore, the regulatory region of miR-106b~25 is bound by FoxO3, a member of the FoxO family of transcription factors that maintains adult stem cells and extends lifespan downstream of insulin/IGF signaling. These results suggest that miR-106b~25 regulates NSPC function and is part of a network involving the insulin/IGF-FoxO pathway, which may have important implications for the homeostasis of the NSC pool during aging.

SummaryIn the nervous system, neural stem cells (NSCs) are necessary for the generation of new neurons and for cognitive function. Here we show that FoxO3, a member of a transcription factor family known to extend lifespan in invertebrates, regulates the NSC pool. We find that adult FoxO3−/− mice have fewer NSCs in vivo than wild-type counterparts. NSCs isolated from adult FoxO3−/− mice have decreased self-renewal and an impaired ability to generate different neural lineages. Identification of the FoxO3-dependent gene expression profile in NSCs suggests that FoxO3 regulates the NSC pool by inducing a program of genes that preserves quiescence, prevents premature differentiation, and controls oxygen metabolism. The ability of FoxO3 to prevent the premature depletion of NSCs might have important implications for counteracting brain aging in long-lived species.

A mouse study reveals that the stem cell quiescent state is composed of two distinct phases, G0 and GAlert; stem cells reversibly transition between these two phases in response to systemic environmental stimuli acting through the mTORC1 pathway. This paper demonstrates that the in vivo stem-cell quiescent state is composed of distinct phases, much like the phases of the cell cycle. Thomas Rando and colleagues studied stem-cell quiescence in muscle stem cells in vivo and identified a quiescent phase that they call GAlert, which is distinct from the familiar G0 state and is stable, reversible and regulated by environmental stimuli through mTORC1. The authors propose that the transition of quiescent stem cells into GAlert functions as an 'alerting' mechanism by which stem cells can rapidly respond to injury and stress without requiring cell cycle entry or a cell fate commitment. A unique property of many adult stem cells is their ability to exist in a non-cycling, quiescent state1. Although quiescence serves an essential role in preserving stem cell function until the stem cell is needed in tissue homeostasis or repair, defects in quiescence can lead to an impairment in tissue function2. The extent to which stem cells can regulate quiescence is unknown. Here we show that the stem cell quiescent state is composed of two distinct functional phases, G0 and an ‘alert’ phase we term GAlert. Stem cells actively and reversibly transition between these phases in response to injury-induced systemic signals. Using genetic mouse models specific to muscle stem cells (or satellite cells), we show that mTORC1 activity is necessary and sufficient for the transition of satellite cells from G0 into GAlert and that signalling through the HGF receptor cMet is also necessary. We also identify G0-to-GAlert transitions in several populations of quiescent stem cells. Quiescent stem cells that transition into GAlert possess enhanced tissue regenerative function. We propose that the transition of quiescent stem cells into GAlert functions as an ‘alerting’ mechanism, an adaptive response that positions stem cells to respond rapidly under conditions of injury and stress, priming them for cell cycle entry.