Jeganathan, Karthik B.

Abstract Cellular senescence is a dynamic, multistage biological program that exerts context-dependent effects on tumor initiation, progression, and overall carcinogenesis. Our laboratory has previously demonstrated that the early accumulation of p16Ink4a-expressing (representing strong cyclin-dependent kinase inhibition) senescent alveolar macrophages during tumorigenesis actively facilitates tumor formation and progression by inhibiting the infiltration of cytotoxic T lymphocytes. However, considering the dynamic nature of cellular senescence, distinct senescent cell populations could express high amounts of the alternative cell cycle inhibitor p21Waf1/Cip1 independently of p16Ink4a to potentially catalyze distinct functions. To test this, we have employed transgenic p21-ATTAC (p21-driven apoptosis through targeted activation of caspase 8) mice to investigate the role of p21-expressing cells during Kras-driven adenoma formation. In agreement with our previous observations following clearance of p16-expressing senescent cells, we found that removal of p21+ cells attenuates early tumor development. Western blot and immunofluorescence analyses corroborates a reduction in p21, p16, and multiple members of a putative senescence-associated secretory phenotype (SASP). Although senescent alveolar macrophages remain a critical therapeutic target, here we found that senescent endothelial cells also exhibited elevated expression of p21, p16, and SASP with a high senolytic response. Future studies will include identifying the predominant p21-expressing senescent endothelial subpopulations and elucidating the mechanisms by which endothelial senescence contributes to lung tumorigenesis. Collectively, our findings have unveiled additional senescent cell populations with the potential to drive lung tumorigenesis. Citation Format: Yuanning Du, Luis I. Prieto, Karthikbabu Jeganathan, Cheng Zhang, Hu Li, Darren Baker. Elucidating the role of p21 + senescent cells in early-stage lung tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2268.

ABSTRACT His domain protein tyrosine phosphatase (HD-PTP; also known as PTPN23) facilitates function of the endosomal sorting complexes required for transport (ESCRTs) during multivesicular body (MVB) formation. To uncover its role in physiological homeostasis, embryonic lethality caused by a complete lack of HD-PTP was bypassed through generation of hypomorphic mice expressing reduced protein, resulting in animals that are viable into adulthood. These mice exhibited marked lipodystrophy and decreased receptor-mediated signaling within white adipose tissue (WAT), involving multiple prominent pathways including RAS/MAPK, phosphoinositide 3-kinase (PI3K)/AKT and receptor tyrosine kinases (RTKs), such as EGFR. EGFR signaling was dissected in vitro to assess the nature of defective signaling, revealing decreased trans-autophosphorylation and downstream effector activation, despite normal EGF binding. This corresponds to decreased plasma membrane cholesterol and increased lysosomal cholesterol, likely resulting from defective endosomal maturation necessary for cholesterol trafficking and homeostasis. The ESCRT components Vps4 and Hrs have previously been implicated in cholesterol homeostasis; thus, these findings expand knowledge on which ESCRT subunits are involved in cholesterol homeostasis and highlight a non-canonical role for HD-PTP in signal regulation and adipose tissue homeostasis.

The clock is ticking for senescent cells Senescent cells promote their own recognition and removal through the immune system by generating a bioactive secretome called the senescence-associated secretory phenotype (SASP). Sturmlechner et al . report that the cell cycle regulator p21 directs an early form of the SASP, which they call the p21-activated secretory phenotype (PASP) (see the Perspective by Reen and Gil). As part of the PASP, the chemokine CXCL14 attracts macrophages, which monitor stressed cells expressing elevated p21. If stressed cells recuperate and p21 levels return to normal within 4 days, then macrophages disengage from their targets. Otherwise, macrophages recruit cytotoxic T cells that facilitate target cell removal. Other cell cycle regulators such as p16 can induce many factors overlapping with the PASP, but p21 uniquely drives this CXCL14-mediated “timer” mechanism of senescent cell immunosurveillance. —STS

RanBP2/Nup358 is a component of the nucleocytoplasmic transport machinery that has been implicated in mitotic progression. To determine the physiological importance of RanBP2 and its role in mitosis we have generated a series of mice with graded reduction of RanBP2 levels. Mice with very low amounts of RanBP2 are viable and overtly normal, but develop severe aneuploidy. Their major mitotic abnormality is the formation of anaphase bridges, a defect linked to improper Topoisomerase II/alpha‐mediated decatenation of sister chromatids. We find that RanBP2 forms a complex with Topoisomerase II/alpha in mitosis and regulates its decatenation activity and localization to chromosomes. Furthermore, we find that RanBP2 deficient mice are prone to spontaneous and carcinogen‐induced tumors. Together, these data identify RanBP2 as a key regulator of Topoisomerase II/alpha‐mediated DNA decatenation and a suppressor of tumorigenesis.

Cyclin A2 activates the cyclin-dependent kinases Cdk1 and Cdk2 and is expressed at elevated levels from S phase until early mitosis. We found that mutant mice that cannot elevate cyclin A2 are chromosomally unstable and tumor-prone. Underlying the chromosomal instability is a failure to up-regulate the meiotic recombination 11 (Mre11) nuclease in S phase, which leads to impaired resolution of stalled replication forks, insufficient repair of double-stranded DNA breaks, and improper segregation of sister chromosomes. Unexpectedly, cyclin A2 controlled Mre11 abundance through a C-terminal RNA binding domain that selectively and directly binds Mre11 transcripts to mediate polysome loading and translation. These data reveal cyclin A2 as a mechanistically diverse regulator of DNA replication combining multifaceted kinase-dependent functions with a kinase-independent, RNA binding–dependent role that ensures adequate repair of common replication errors.

BubR1 is a key component of the spindle assembly checkpoint (SAC). Mutations that reduce BubR1 abundance cause aneuploidization and tumorigenesis in humans and mice, whereas BubR1 overexpression protects against these. However, how supranormal BubR1 expression exerts these beneficial physiological impacts is poorly understood. Here, we used Bub1b mutant transgenic mice to explore the role of the amino-terminal (BubR1N) and internal (BubR1I) Cdc20-binding domains of BubR1 in preventing aneuploidy and safeguarding against cancer. BubR1N was necessary, but not sufficient to protect against aneuploidy and cancer. In contrast, BubR1 lacking the internal Cdc20-binding domain provided protection against both, which coincided with improved microtubule-kinetochore attachment error correction and SAC activity. Maximal SAC reinforcement occurred when both the Phe- and D-box of BubR1I were disrupted. Thus, while under- or overexpression of most mitotic regulators impairs chromosome segregation fidelity, certain manipulations of BubR1 can positively impact this process and therefore be therapeutically exploited.

The mitotic checkpoint protein Bub1 is essential for embryogenesis and survival of proliferating cells, and bidirectional deviations from its normal level of expression cause chromosome missegregation, aneuploidy, and cancer predisposition in mice. To provide insight into the physiological significance of this critical mitotic regulator at a modular level, we generated Bub1 mutant mice that lack kinase activity using a knockin gene-targeting approach that preserves normal protein abundance. In this paper, we uncover that Bub1 kinase activity integrates attachment error correction and mitotic checkpoint signaling by controlling the localization and activity of Aurora B kinase through phosphorylation of histone H2A at threonine 121. Strikingly, despite substantial chromosome segregation errors and aneuploidization, mice deficient for Bub1 kinase activity do not exhibit increased susceptibility to spontaneous or carcinogen-induced tumorigenesis. These findings provide a unique example of a modular mitotic activity orchestrating two distinct networks that safeguard against whole chromosome instability and reveal the differential importance of distinct aneuploidy-causing Bub1 defects in tumor suppression.

Abstract During mitosis, sister chromatids are separated and equally divided over two daughter cells. If missegregation of whole chromosomes occurs, progeny cells with an abnormal number of chromosomes will arise. This is referred to as aneuploidy, a hallmark of most human cancers. We found that inactivation of a single Pten allele in mouse splenocytes and mouse embryonic fibroblasts (MEFs) is sufficient to cause aneuploidy, suggesting a role for Pten in the chromosome segregation process. Upon further study by live cell imaging of haploinsufficient and null Pten MEFs, we indeed found many missegregation errors (59% and 79% respectively, compared to 15% in wildtype MEFs). These abnormalities were independent from the AKT pathway since overexpression of a myristoylated, constitutively active form of AKT in wildtype MEFs resembled a wildtype instead of a Pten heterozygous phenotype. In addition, overexpression of a catalytic mutant Pten (C124S) in Pten+/- MEFs corrected the missegregation errors. We therefore propose a novel tumor-suppressive function of Pten to prevent whole chromosome instability, independent of the increased cell proliferation and survival caused by the activation of AKT. BubR1 is a regulator of kinetochore-microtubule attachment and a potent inhibitor of Cdc20, a key co-activator of the anaphase promoting complex/cyclosome (APC/C) that drives cells into anaphase by targeting the anaphase inhibitors cyclin B1 and securin for degradation by the 26S proteosome. We found that overexpression of BubR1 completely restores accurate chromosome segregation in Pten+/- MEFs, alluding to a specific role for Pten in mitosis. As a first step to test the hypothesis that Pten's mitotic role in preventing aneuploidization represents a key tumor suppressive function of this phosphatase, we bred a Flag-tagged BubR1 transgene into Pten+/- mice. We are currently in the process of studying the impact of the transgene on the development of prostatic intraepithelial neoplasia (PIN). Consistent with our hypothesis, preliminary analysis of two mice shows that BubR1 overexpression considerably decreases PIN lesion multiplicity. Data from the complete analysis of our double transgenic mouse cohort will be presented at the meeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2011-2992

RanBP2/Nup358, the major component of the cytoplasmic filaments of the nuclear pore complex (NPC), is essential for mouse embryogenesis and is implicated in both macromolecular transport and mitosis, but its specific molecular functions are unknown. Using RanBP2 conditional knockout mouse embryonic fibroblasts and a series of mutant constructs, we show that transport, rather than mitotic, functions of RanBP2 are required for cell viability. Cre-mediated RanBP2 inactivation caused cell death with defects in M9- and classical nuclear localization signal (cNLS)–mediated protein import, nuclear export signal–mediated protein export, and messenger ribonucleic acid export but no apparent mitotic failure. A short N-terminal RanBP2 fragment harboring the NPC-binding domain, three phenylalanine-glycine motifs, and one Ran-binding domain (RBD) corrected all transport defects and restored viability. Mutation of the RBD within this fragment caused lethality and perturbed binding to Ran guanosine triphosphate (GTP)–importin-β, accumulation of importin-β at nuclear pores, and cNLS-mediated protein import. These data suggest that a critical function of RanBP2 is to capture recycling RanGTP–importin-β complexes at cytoplasmic fibrils to allow for adequate cNLS-mediated cargo import.