Biology subjects

Spencer, S. L.

Publications and source records attributed to Spencer, S. L..

14 recordsLinked to original sources

bioRxiv2026

Targeted therapy-induced chromosomal instability dictates mitotic dependency on Aurora Kinase A

Targeted therapies eliminate cancer cells by inhibiting oncogenic signaling; however, tumor cells often evade cytotoxicity through proteomic and epigenetic reprogramming that enables survival. These adaptive responses may create collateral cellular stresses, such as DNA damage, that can be therapeutically exploited. When unresolved, DNA damage leads to chromosomal instability (CIN), a potential source of vulnerability. Whether KRAS inhibition induces DNA damage or CIN in KRAS-mutant non-small cell lung cancer (NSCLC) has not been established. Here, we show that the KRASG12C inhibitor LY3499446 induces CIN in KRAS-mutant NSCLC cell lines. A targeted compound screen revealed that the extent of CIN induction by KRASG12C inhibition strongly correlates with therapeutic synergy with the selective Aurora kinase A inhibitor LSN3321213. Mechanistically, KRASG12C inhibition stabilizes cyclin B1 during mitosis through activation of mitotic ATR/ATM signaling. In the presence of Aurora Kinase A inhibition, cyclin B1 stabilization delays mitotic exit and diverts cell fate from mitotic slippage or division toward mitotic catastrophe. Together, our findings identify CIN as a predictive marker of response to combined KRASG12C and Aurora Kinase A inhibition, providing mechanistic rationale to enhance the therapeutic window of AURKA inhibitors when used with targeted therapies.

cancer biology↗

bioRxiv2025

MAPK and mTORC1 signaling converge on Cyclin D to enable cell-cycle re-entry in melanoma persister cells

Resistance to targeted therapy in BRAF-mutant melanomas can arise from drug-tolerant persister cells, which can non-genetically escape drug-induced quiescence to resume proliferation. To investigate how melanoma cells escape drug to re-enter the cell cycle within 3-4 days of BRAF and MEK inhibition, we computationally reconstructed single-cell lineages from time-lapse imaging data, linking key signaling pathways to cell-cycle fate outcomes. We found that ERK reactivation, while necessary, is insufficient for cell-cycle re-entry under MAPK inhibition. Instead, mTORC1 emerged as an additional critical mediator, enhancing translation and cell growth in cells destined for drug escape. We further found that ERK and mTORC1 signaling converge to produce Cyclin D1 protein levels, a key bottleneck for cell-cycle re-entry. Using CRISPR to tag endogenous Cyclin D1, we found that future escapees markedly upregulate Cyclin D1 at least 15 hours prior to drug escape, compared to non-escapees. Importantly, this early upregulation enables accurate prediction of future escapees from non-escapees, underscoring how differences in Cyclin D1 accumulation precede and govern the timing and likelihood of cell-cycle re-entry in persister cells. Our findings suggest that variability in ERK and mTORC1 activity underlies the heterogeneous Cyclin D1 levels observed, influencing the proliferative potential of persister cells and ultimately shaping the diverse cell-cycle behaviors observed under drug treatment. Cyclin D1 protein therefore emerges as both a critical biomarker and a therapeutic target for preventing cell-cycle re-entry in BRAF/MEK-treated melanoma. One Sentence SummaryCyclin D1 accumulation, driven by the integration of heterogeneous MAPK and mTORC1 signaling, is a critical bottleneck for cell-cycle re-entry in drug-treated melanoma cells.

systems biology↗

bioRxiv2025

Single-cell RNA sequencing reveals distinct senotypes and a quiescence-senescence continuum at the transcriptome level following chemotherapy

Quiescence (reversible cell-cycle arrest) and senescence (irreversible arrest) are challenging to distinguish due to a lack of specific biomarkers, yet both arise simultaneously after chemotherapy. While senescence suppresses tumors by limiting proliferation and recruiting the immune system, quiescent cancer cells evade future therapies and may resume proliferation. Here, we pair time-lapse imaging of cell-cycle dynamics with single-cell RNA-sequencing after etoposide treatment to differentiate these states, linking heterogeneous cell-cycle phenotypes to the transcriptomic landscape. We identify diverse senescent types (senotypes) and link them to two arrest pathways - a gradual path arising after a standard mitosis-to-G0 transition, and a rarer but direct path driven by a mitotic slip. Using pseudotime trajectory analysis, we find that senescent phenotypes begin to manifest early and gradually along the first trajectory, even in shallow quiescent cells. These data support a model wherein, following chemotherapy, quiescence and senescence exist on a continuum of cell-cycle withdrawal at a transcriptome-wide level.

cell biology↗

bioRxiv2025

p16 expression confers sensitivity to CDK2 inhibitors

Blocking the cell cycle is a promising avenue for cancer therapy, with Cyclin-Dependent Kinase 2 (CDK2) emerging as a key target. However, in multiple cell types, CDK4/6 activity compensates for CDK2 inhibition and sustains the proliferative program, enabling CDK2 reactivation. Thus, we hypothesized that sensitivity to CDK2 inhibition is linked to the absence of this CDK4/6-mediated compensatory mechanism. Here we show that Cyclin E1-driven ovarian cancers often co-express the tumor suppressor p16, which inhibits CDK4/6. We show that ovarian cancer cells expressing p16 exhibit heightened sensitivity to CDK2 inhibitors and that depletion of p16 renders them significantly more resistant. Multiplexed immunofluorescence of 225 ovarian patient tumors reveals that at least 18% of tumors express high Cyclin E1 and high p16, a group that we expect to be particularly sensitive to CDK2 inhibition. Thus, p16 may be a useful biomarker for identifying the patients most likely to benefit from CDK2 inhibitors.

cancer biology↗

bioRxiv2025

Multi-omics and biochemical reconstitution reveal CDK7-dependent mechanisms controlling RNA polymerase II function at gene 5'- and 3'-ends

CDK7 regulates RNA polymerase II (RNAPII) initiation, elongation, and termination through incompletely understood mechanisms. Because contaminating kinases precluded CDK7 analysis with nuclear extracts, we completed biochemical assays with purified factors. Reconstitution of RNAPII transcription initiation showed CDK7 inhibition slowed and/or paused RNAPII promoter-proximal transcription, which reduced re-initiation. These CDK7-regulatory functions were Mediator- and TFIID-dependent. Similarly in human cells, CDK7 inhibition reduced transcription by suppressing RNAPII activity at promoters, consistent with reduced initiation and/or re-initiation. Moreover, widespread 3-end readthrough transcription was observed in CDK7-inhibited cells; mechanistically, this occurred through rapid nuclear depletion of RNAPII elongation and termination factors, including high-confidence CDK7 targets. Collectively, these results define how CDK7 governs RNAPII function at gene 5-ends and 3-ends, and reveal that nuclear abundance of elongation and termination factors is kinase-dependent. Because 3-readthrough transcription is commonly induced during stress, our results further suggest regulated suppression of CDK7 activity may enable this RNAPII transcriptional response.

biochemistry↗

bioRxiv2024

BRAF V600 and ErbB inhibitors directly activate GCN2 in an off-target manner to limit cancer cell proliferation

Targeted kinase inhibitors are well known for their promiscuity and off-target effects. Herein, we define an off-target effect in which several clinical BRAFV600 inhibitors, including the widely used dabrafenib and encorafenib, interact directly with GCN2 to activate the Integrated Stress Response and ATF4. Blocking this off-target effect by co-drugging with a GCN2 inhibitor in A375 melanoma cells causes enhancement rather than suppression of cancer cell outgrowth, suggesting that the off-target activation of GCN2 is detrimental to these cells. This result is mirrored in PC9 lung cancer cells treated with erlotinib, an EGFR inhibitor, that shares the same off-target activation of GCN2. Using an in silico kinase inhibitor screen, we identified dozens of FDA-approved drugs that appear to share this off-target activation of GCN2 and ATF4. Thus, GCN2 activation may modulate the therapeutic efficacy of some kinase inhibitors, depending on the cancer context.

cancer biology↗

bioRxiv2024

Acute multi-level response to defective de novo chromatin assembly in S-phase

Long-term perturbation of de novo chromatin assembly during DNA replication has profound effects on epigenome maintenance and cell fate. The early mechanistic origin of these defects is unknown. Here, we combine acute degradation of Chromatin Assembly Factor 1 (CAF-1), a key player in de novo chromatin assembly, with single-cell genomics, quantitative proteomics, and live-microscopy to uncover these initiating mechanisms in human cells. CAF-1 loss immediately slows down DNA replication speed and renders nascent DNA hyper-accessible. A rapid cellular response, distinct from canonical DNA damage signaling, is triggered and lowers histone mRNAs. As a result, histone variants usage and their modifications are altered, limiting transcriptional fidelity and delaying chromatin maturation within a single S-phase. This multi-level response induces a cell-cycle arrest after mitosis. Our work reveals the immediate consequences of defective de novo chromatin assembly during DNA replication, explaining how at later times the epigenome and cell fate can be altered. HighlightsO_LIThe histone chaperone CAF-1 sustains DNA replication speed in single cells. C_LIO_LICAF-1 loss alters histone repertoire and delays chromatin maturation. C_LIO_LIH3K9me3 and H3K27me3 regions respond differently to acute CAF-1 depletion. C_LIO_LIImpaired S-phase chromatin assembly triggers an immediate response and a G0 arrest. C_LI

cell biology↗

bioRxiv2023

Multiple cancer types rapidly escape from multiple MAPK inhibitors to generate mutagenesis-prone subpopulations

Many cancers harbor pro-proliferative mutations of the mitogen-activated protein kinase (MAPK) pathway and many targeted inhibitors now exist for clinical use, but drug resistance remains a major issue. We recently showed that BRAF-driven melanoma cells treated with BRAF inhibitors can non-genetically adapt to drug within 3-4 days to escape quiescence and resume slow proliferation. Here we show that this phenomenon is not unique to melanomas treated with BRAF inhibitors but rather is widespread across many clinical MAPK inhibitors and cancer types driven by EGFR, KRAS, and BRAF mutations. In all treatment contexts examined, a subset of cells can escape drug-induced quiescence within four days to resume proliferation. These escapee cells broadly experience aberrant DNA replication, accumulate DNA lesions, spend longer in G2-M cell cycle phases, and mount an ATR-dependent stress response. We further identify the Fanconi anemia (FA) DNA repair pathway as critical for successful mitotic completion in escapees. Long-term cultures, patient samples, and clinical data demonstrate a broad dependency on ATR- and FA-mediated stress tolerance. Together, these results highlight the pervasiveness with which MAPK-mutant cancers are able to rapidly escape drug and the importance of suppressing early stress tolerance pathways to potentially achieve more durable clinical responses to targeted MAPK pathway inhibitors.

cancer biology↗

bioRxiv2023

The intensities of canonical senescence biomarkers integrate the duration of cell-cycle withdrawal

Senescence, a state of permanent cell-cycle withdrawal, is difficult to distinguish from quiescence, a transient state of cell-cycle withdrawal. This difficulty arises because quiescent and senescent cells are defined by overlapping biomarkers, raising the question of whether quiescence and senescence are truly distinct states. To address this, we used single-cell time-lapse imaging to distinguish slow-cycling quiescent cells from bona fide senescent cells after chemotherapy treatment, followed immediately by staining for various senescence biomarkers. We found that the staining intensity of multiple senescence biomarkers is graded rather than binary and primarily reflects the duration of cell-cycle withdrawal, rather than senescence per se. Together, our data suggest that quiescence and senescence are not distinct cellular states but rather fall on a continuum of cell-cycle withdrawal, where the intensities of canonical senescence biomarkers reflect the likelihood of cell-cycle re-entry.

systems biology↗

bioRxiv2023

Cyclin E/CDK2 and feedback from soluble histone protein regulate the S phase burst of histone biosynthesis

Faithful DNA replication requires that cells fine-tune their histone pool in coordination with cell-cycle progression. Replication-dependent histone biosynthesis is initiated at a low level upon cell-cycle commitment, followed by a burst at the G1/S transition, but it remains unclear how exactly the cell regulates this change in histone biosynthesis as DNA replication begins. Here, we use single-cell timelapse imaging to elucidate the mechanisms by which cells modulate histone production during different phases of the cell cycle. We find that CDK2-mediated phosphorylation of NPAT at the Restriction Point triggers histone transcription, which results in a burst of histone mRNA precisely at the G1/S phase boundary. Excess soluble histone protein further modulates histone abundance by promoting the degradation of histone mRNA for the duration of S phase. Thus, cells regulate their histone production in strict coordination with cell-cycle progression by two distinct mechanisms acting in concert.

cell biology↗

bioRxiv2022

Multi range ERK responses shape the proliferative trajectory of single cells following oncogene induced senescence

Oncogene-induced senescence (OIS) is a phenomenon in which aberrant oncogene expression causes non-transformed cells to enter a non-proliferative state. Cells undergoing OIS display phenotypic heterogeneity, with some cells senescing and others remaining proliferative. The causes of the heterogeneity remain poorly understood. We studied the sources of heterogeneity in the responses of human epithelial cells to oncogenic BRAFV600E expression. We found that a narrow expression range of BRAFV600E generated a wide range of activities of its downstream effector ERK. In population-level and single cell assays, ERK activity displayed a non-monotonic relationship to proliferation, with intermediate ERK activities leading to maximal proliferation. We profiled gene expression across a range of ERK activities over time and characterized four distinct ERK response classes, which we propose act in concert to generate the unique ERK-proliferation response. Altogether, our studies mapped the input-output relationships between ERK activity and proliferation providing important insights into how heterogeneity can be generated during OIS.

systems biology↗

bioRxiv2022

Stress granules promote chemoresistance by triggering cellular quiescence

Cells respond to cellular stress by forming stress granules, molecular condensates containing non-translating messenger ribonucleoproteins. Stress granules form during chemotherapy and promote cell survival and chemoresistance, although the mechanism of this effect is not understood. We provide several lines of evidence that stress granules enhance cell survival by promoting cellular quiescence. First, we see a correlation between spontaneous stress granule formation and cell-cycle exit under non-stress conditions. Second, cells deficient in proteins required for stress granule formation (G3BP1/2) are less likely to exit the cell cycle under non-stress, stress, and chemotherapeutic conditions. Third, rescuing stress granule formation in G3BP1/2 knockout cells restores the fraction of cycling cells to wild-type levels. Finally, cells with enhanced stress granule formation (ddx6 knockout cells) show an increased propensity to exit the cell cycle. These results suggest that stress granules are important regulators of cellular quiescence, which could enable the identification of new anti-chemoresistance therapies that target stress granules.

molecular biology↗

bioRxiv2020

EllipTrack: A Global-Local Cell-Tracking Pipeline for 2D Fluorescence Time-Lapse Microscopy

Time-lapse microscopy provides an unprecedented opportunity to monitor single-cell dynamics. However, tracking cells for long periods of time remains a technical challenge, especially for multi-day, large-scale movies with rapid cell migration, high cell density, and drug treatments that alter cell morphology/behavior. Here, we present EllipTrack, a global-local cell-tracking pipeline optimized for tracking such movies. EllipTrack first implements a global track-linking algorithm to construct tracks that maximize the probability of cell lineages, and then corrects tracking mistakes with a local track-correction module where tracks generated by the global algorithm are systematically examined and amended if a more probable alternative can be found. Through benchmarking, we show that EllipTrack outperforms state-of-the-art cell trackers and generates nearly error-free cell lineages for multiple large-scale movies. In addition, EllipTrack can adapt to time- and cell density-dependent changes in cell migration speeds, requires minimal training datasets, and provides a user-friendly interface. EllipTrack is available at github.com/tianchengzhe/EllipTrack.

systems biology↗

bioRxiv2020

Rapidly induced drug adaptation mediates escape from BRAF inhibition in single melanoma cells

Despite increasing numbers of effective anti-cancer therapies, successful treatment is limited by the development of drug resistance. While the contribution of genetic factors to drug resistance is undeniable, little is known about how drug-sensitive cells first evade drug action to proliferate in drug. Here we track the response of thousands of single melanoma cells to BRAF inhibitors and show that a subset escapes drug within the first 3 days of treatment. Cell-cycle re-entry occurs via a non-genetic mechanism involving activation of mTORC1 and ATF4, validated in cultures of patient biopsies. These escapees cycle periodically in drug, incur significant DNA damage, and out-proliferate non-escapees over extended treatment. Our work reveals a mutagenesis-prone, expanding subpopulation of early drug escapees that may seed development of permanent drug resistance.

cell biology↗