Spencer Lab Publications
CDK2 activity crosstalk on the ERK kinase translocation reporter can be resolved computationally (Cell Systems 2025)
The mitogen-activated protein kinase (MAPK) pathway integrates growth factor signaling through extracellular signal-regulated kinase (ERK) to control cell proliferation. To study ERK dynamics, many researchers use an ERK activity kinase translocation reporter (KTR). Our study reveals that this ERK KTR also partially senses cyclin-dependent kinase 2 (CDK2) activity, making it appear as if ERK activity rises as cells progress through the cell cycle. Through single-cell time-lapse imaging, we identified a resi...
Cells use multiple mechanisms for cell-cycle arrest upon withdrawal of individual amino acids (Cell Reports 2023)
Amino acids are required for cell growth and proliferation, but it remains unclear when and how amino acid availability impinges on the proliferation-quiescence decision. Here, we used time-lapse microscopy and single-cell tracking of cyclin-dependent kinase 2 (CDK2) activity to assess the response of individual cells to withdrawal of single amino acids and found strikingly different cell-cycle effects depending on the amino acid. For example, upon leucine withdrawal, MCF10A cells complete two cell cycles a...
Multiple cancers escape from multiple MAPK pathway inhibitors and use DNA replication stress signaling to tolerate aberrant cell cycles (Science Signaling 2023)
Many cancers harbor pro-proliferative mutations of the mitogen-activated protein kinase (MAPK) pathway. In BRAF-driven melanoma cells treated with BRAF inhibitors, subpopulations of cells escape drug-induced quiescence through a nongenetic manner of adaptation and resume slow proliferation. Here, we found that this phenomenon is common to many cancer types driven by EGFR, KRAS, or BRAF mutations in response to multiple, clinically approved MAPK pathway inhibitors. In 2D cultures and 3D spheroid models of va...
The intensities of canonical senescence biomarkers integrate the duration of cell-cycle withdrawal (Nature Communications 2023)
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 frombona fidesenescent cells after chemotherapy treatment, followed immediately by...
Rapid adaptation to CDK2 inhibition exposes intrinsic cell-cycle plasticity (Cell 2023)
CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. Here, we use several CDK2 inhibitors in clinical development to interrogate CDK2 substrate phosphorylation, cell-cycle progression, and drug adaptation in preclinical models. Whereas CDK1 is known to compensate for loss of CDK2 inCdk2−/−mice, this is not true of acute inhibition of CDK2. Upon CDK2 inhib...
Melanoma subpopulations that rapidly escape MAPK pathway inhibition incur DNA damage and rely on stress signalling (Nature Communications 2021)
Despite the increasing number 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 responses of thousands of single melanoma cells to BRAF inhibitors and show that a subset of cells escapes drug via non-genetic mechanisms within the first three days of treatment. Cells...
Replication-dependent histone biosynthesis is coupled to cell-cycle commitment (PNAS 2021)
The current model of replication-dependent (RD) histone biosynthesis posits that RD histone gene expression is coupled to DNA replication, occurring only in S phase of the cell cycle once DNA synthesis has begun. However, several key factors in the RD histone biosynthesis pathway are up-regulated by E2F or phosphorylated by CDK2, suggesting these processes may instead begin much earlier, at the point of cell-cycle commitment. In this study, we use both fixed- and live-cell imaging of human cells to address ...
EllipTrack: A Global-Local Cell-Tracking Pipeline for 2D Fluorescence Time-Lapse Microscopy (Cell Reports 2020)
Time-lapse microscopy provides an unprecedented opportunity to monitor single-cell dynamics. However, tracking cells for long periods 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 t...
Temporal integration of mitogen history in mother cells controls proliferation of daughter cells (Science 2020)
Multicellular organisms use mitogens to regulate cell proliferation, but how fluctuating mitogenic signals are converted into proliferation-quiescence decisions is poorly understood. In this work, we combined live-cell imaging with temporally controlled perturbations to determine the time scale and mechanisms underlying this system in human cells. Contrary to the textbook model that cells sense mitogen availability only in the G1cell cycle phase, we find that mitogenic signaling is temporally integrated thr...
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 mech...
Senescence Evasion in Chemotherapy: A Sweet Spot for p21 (Cell 2019)
Time-lapse imaging reveals a nuanced role forp21in cancer cells challenged with chemotherapeutic drugs: cells with either high or low p21 are biased toward senescence, whereas intermediate p21 allows cells to re-enter the cell cycle after drug treatment. [Article Link]
Spontaneously slow-cycling subpopulations of human cells originate from activation of stress-response pathways (PLOS Biology 2019)
Slow-cycling subpopulations exist in bacteria, yeast, and mammalian systems. In the case of cancer, slow-cycling subpopulations have been proposed to give rise to drug resistance. However, the origin of slow-cycling human cells is poorly studied, in large part due to lack of markers to identify these rare cells. Slow-cycling cells pass through a noncycling period marked by low CDK2 activity and high p21 levels. Here, we use this knowledge to isolate these naturally slow-cycling cells from a heterogeneous po...
Ki67 is a Graded Rather than a Binary Marker of Proliferation versus Quiescence (Cell Reports 2018)
Ki67 staining is widely used as a proliferation indicator in the clinic, despite poor understanding of this protein’s function or dynamics. Here, we track Ki67 levels under endogenous control in single cells over time and find that Ki67 accumulation occurs only during S,G2, and M phases. Ki67 is degraded continuously inG1 and G0 phases, regardless of the cause of entry into G0/quiescence. Consequently, the level of Ki67 during G0 and G1 in individual cells is highly heterogeneous and depends on how long an ...
A map of protein dynamics during cell-cycle progression and cell-cycle exit (PLOS Biology 2017)
The cell-cycle field has identified the core regulators that drive the cell cycle, but we do not have a clear map of the dynamics of these regulators during cell-cycle progression versus cell-cycle exit. Here we use single-cell time-lapse microscopy of Cyclin-Dependent Kinase 2 (CDK2) activity followed by endpoint immunofluorescence and computational cell synchronization to determine the temporal dynamics of key cell-cycle proteins in asynchronously cycling human cells. We identify several unexpected patter...
Control of the Restriction Point by Rb and p21 (PNAS 2017)
The Restriction Point was originally defined as the moment that cells commit to the cell cycle and was later suggested to coincide with hyperphosphorylation of the retinoblastoma protein (Rb). Current cell cycle models posit that cells exit mitosis into a pre-Restriction Point state, where they have low cyclin-dependent kinase (CDK) activity and hypophosphorylated Rb; passage through the Restriction Point then occurs in late G1. Recent single-cell studies have challenged the current paradigm, raising questi...
Endogenous Replication Stress in Mother Cells Leads to Quiescence of Daughter Cells (Cell Reports 2017)
Mammalian cellshave two fundamentally different states, proliferative and quiescent, but our understanding of how and why cells switch between these states is limited. We previously showed that actively proliferating populations contain a subpopulation that enters quiescence (G0) in an apparently stochastic manner. Using single-cell time-lapse imaging of CDK2 activity and DNA damage, we now show that unresolved endogenous replication stress in the previous (mother) cell cycle prompts p21-dependent entry of ...
