Leucine zipper-bearing kinase (LZK) is overexpressed in 20% of head and neck squamous cell carcinoma (HNSCC) cases and has emerged as a promising therapeutic target in this cancer subtype. LZK promotes HNSCC survival and proliferation by stabilizing c-MYC and GOF-p53 in kinase-dependent and -independent manners, respectively. Herein, we developed a new series of LZK degraders utilizing proteolysis-targeting chimera (PROTAC) technology by modulating the linker region or LZK warhead of LZK-targeting PROTAC-21A, previously developed by our laboratory. Among the 27 PROTACs synthesized and tested, PROTAC 17 was found to be the most potent, degrading LZK at 250 nM and suppressing HNSCC viability at 500 nM. In summary, our lead PROTAC effectively targeted LZK for proteasomal degradation and inhibited oncogenic activity in HNSCC cell lines with amplified LZK.

The worldwide annual frequency and lethality of head and neck squamous cell carcinoma (HNSCC) is not improving, and thus, new therapeutic approaches are needed. Approximately 70% of HNSCC cases have either amplification or overexpression of MAP3K13, which encodes the kinase LZK. Here, we found that LZK is a therapeutic target in HNSCC and that small-molecule inhibition of its catalytic function decreased the viability of HNSCC cells with amplified MAP3K13. Inhibition of LZK suppressed tumor growth in MAP3K13-amplified xenografts derived from HNSCC patients. LZK stabilized the transcription factor c-MYC through its kinase activity and gain-of-function mutants of p53 in a kinase-independent manner. We designed a proteolysis-targeting chimera (PROTAC) that induced LZK degradation, leading to decreased abundance of both c-MYC and gain-of-function p53, and reduced the viability of HNSCC cells. Our findings demonstrate that LZK-targeted therapeutics, particularly PROTACs, may be effective in treating HNSCCs with MAP3K13 amplification.

Mammalian cells entering the cell cycle favour glycolysis to rapidly generate ATP and produce the biosynthetic intermediates that are required for rapid biomass accumulation1. Simultaneously, the ubiquitin-ligase anaphase-promoting complex/cyclosome and its coactivator CDH1 (APC/CCDH1) remains active, allowing origin licensing and blocking premature DNA replication. Paradoxically, glycolysis is reduced by APC/CCDH1 through the degradation of key glycolytic enzymes2, raising the question of how cells coordinate these mutually exclusive events to ensure proper cell division. Here we show that cells resolve this paradox by transiently inactivating the APC/C during cell cycle entry, which allows a transient metabolic shift favouring glycolysis. After mitogen stimulation, rapid mTOR-mediated phosphorylation of the APC/C adapter protein CDH1 at the amino terminus causes it to partially dissociate from the APC/C. This partial inactivation of the APC/C leads to the accumulation of PFKFB3, a rate-limiting enzyme for glycolysis, promoting a metabolic shift towards glycolysis. Delayed accumulation of phosphatase activity later removes CDH1 phosphorylation, restoring full APC/C activity, and shifting cells back to favouring oxidative phosphorylation. Thus, cells coordinate the simultaneous demands of cell cycle progression and metabolism through an incoherent feedforward loop, which transiently inhibits APC/C activity to generate a pulse of glycolysis that is required for mammalian cell cycle entry.

Cyclin-dependent kinases (CDKs) regulate proliferation dynamics and cell fate in response to extracellular inputs. It remains largely unknown how CDK activity fluctuates and influences cell commitment during early mammalian development. Here, we generated a mouse model expressing a CDK translocation reporter that enabled quantification of CDK activity in live single cells. By examining pre- and post-implantation mouse embryos at different stages, we observed a progressive decrease in CDK activity in cells from the trophectoderm (TE) prior to implantation. This drop seems to correlate with the available levels of ICM-derived FGF4 as CDK activity downregulation is rescued by exogenous FGF4. Furthermore, we showed that cell fate decisions in the pre-implantation embryo are not determined by the establishment of oscillatory CDK activity or overall changes in CDK activity. Finally, we uncovered the existence of conserved regulatory mechanisms in mammals by revealing lineage-specific regulation of CDK activity in TE-like human cells.

The worldwide frequency of head and neck squamous cell carcinoma (HNSCC) is approximately 800,000 new cases, with 430,000 deaths annually. We determined that LZK (encoded by MAP3K13) is a therapeutic target in HNSCC and showed that inhibition with small molecule inhibitors decreases the viability of HNSCC cells with amplified MAP3K13. A drug-resistant mutant of LZK blocks decreases in cell viability due to LZK inhibition, indicating on-target activity by two separate small molecules. Inhibition of LZK catalytic activity suppressed tumor growth in HNSCC PDX models with amplified MAP3K13. We found that the kinase activity of LZK stabilized c-MYC and that LZK stabilized gain-of-function (GOF) p53 through a kinase-independent mechanism. Therefore, we designed proteolysis-targeting chimeras (PROTACs) and demonstrate that our lead PROTAC promotes LZK degradation and suppresses expression of GOF p53 and c-MYC leading to impaired viability of HNSCC cell lines. This research provides a strong basis for development of therapeutics targeting LZK in HNSCCs with amplification of the gene.

Abstract The cell cycle is tightly regulated by checkpoints, playing a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anti-cancer treatments, allowing for DNA repair prior to cell division. Therefore, manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies such as alkylating agents and radiation. In this study, we conducted a forward genome scale CRISPR/Cas9 chemogenomic screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. Our findings revealed that canonical DNA repair pathways, including ATM/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of the membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase, known as Myt1 (encoded by PKMYT1), in ensuring cell survival. Analysis on patient cohort demonstrated that PKMYT1 is not only highly presented in malignancy, but also predisposes an unfavorable disease outcome. Genetic depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between TMZ and other clinically used alkylating agents, such as carmustine, busulfan, and dacarbazine, when combined with a novel Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage, as indicated by a substantial increase in γH2AX-positive cells in mitosis. Moreover, RP-6306 dramatically altered cell cycle timing, reducing the threshold time for mitotic entry. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities, including unaligned and partially condensed chromosomes, lagging chromosomes, chromosome bridges, and multipolar spindles. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Importantly, preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes. Citation Format: Fengchao Lang, James A. Cornwell, Karambir Kaur, Mirit I. Aladjem, Michael Aregger, Steven D. Cappell, Chunzhang Yang. Reckless mitotic entry as a chemosensitization approach for alkylating agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4565.

Background The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies.Methods In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress.Results Our findings revealed that canonical DNA repair pathways, including the Ataxia-telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice.Conclusions Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as an effective strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes. Graphical Abstract

Abstract The cell cycle is tightly regulated by checkpoints, playing a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anti-cancer treatments, allowing for DNA repair prior to cell division. Therefore, manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies such as alkylating agents and radiation. In this study, we conducted a forward genome wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. Our findings revealed that canonical DNA repair pathways, including ATM/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of the membrane-associated tyrosine- and threonine-specific cdc2-inhibitory kinase, known as Myt1 (encoded by PKMYT1), in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between TMZ and other clinically used alkylating agents, such as carmustine, busulfan, and dacarbazine, when combined with a novel Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage, as indicated by a substantial increase in γH2AX-positive cells in mitosis. Moreover, RP-6306 dramatically altered cell cycle timing, reducing the threshold time for mitotic entry. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities, including unaligned and partially condensed chromosomes, lagging chromosomes, chromosome bridges, and multipolar spindles. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Importantly, preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice. Collectively, our findings highlight the potential of targeting cell cycle timing through Myt1 inhibition as a novel strategy to enhance the efficacy of current standard cancer therapies, potentially leading to improved disease outcomes. Citation Format: Fengchao Lang, James Cornwell, Karambir Kaur, Omar Elmogazy, Wei Zhang, Meili Zhang, Hua Song, Zhonghe Sun, Xiaolin Wu, Mirit I. Aladjem, Michael Aregger, Steven Cappell, Chunzhang Yang. Reckless mitotic entry as a novel chemosensitization approach for alkylating agents [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr A006.

Abstract BACKGROUND The cell cycle is tightly regulated by checkpoints, which play a vital role in controlling its progression and timing. Cancer cells exploit the G2/M checkpoint, which serves as a resistance mechanism against genotoxic anticancer treatments, allowing for DNA repair prior to cell division. Manipulating cell cycle timing has emerged as a potential strategy to augment the effectiveness of DNA damage-based therapies. METHODS In this study, we conducted a forward genome-wide CRISPR/Cas9 screening with repeated exposure to the alkylating agent temozolomide (TMZ) to investigate the mechanisms underlying tumor cell survival under genotoxic stress. The screening hits were validated through in patient derived glioma cells and xenografts. RESULTS Our findings revealed that canonical DNA repair pathways, including the Ataxia–telangiectasia mutated (ATM)/Fanconi and mismatch repair, determine cell fate under genotoxic stress. Notably, we identified the critical role of PKMYT1, in ensuring cell survival. Depletion of PKMYT1 led to overwhelming TMZ-induced cytotoxicity in cancer cells. Isobologram analysis demonstrated potent drug synergy between alkylating agents and a Myt1 kinase inhibitor, RP-6306. Mechanistically, inhibiting Myt1 forced G2/M-arrested cells into an unscheduled transition to the mitotic phase without complete resolution of DNA damage. This forced entry into mitosis, along with persistent DNA damage, resulted in severe mitotic abnormalities. Ultimately, these aberrations led to mitotic exit with substantial apoptosis. Preclinical animal studies demonstrated that the combination regimen involving TMZ and RP-6306 prolonged the overall survival of glioma-bearing mice.

Abstract Tumor cells are highly heterogeneous, exhibiting diverse responses when exposed to different environments. We have recently observed this heterogeneity in tumor cell apoptosis by which some tumor cell die for the benefits of the population’s survival. Majority of tumor cells undergo apoptosis in response to immune surveillance, various treatments, and a hostile microenvironment, both in circulation and at metastatic organ sites. However, the full range of apoptotic signals, or "last words," that dying cells communicate to the surviving cancer cell community remains incompletely understood. While apoptotic cell death is often perceived as a favorable therapeutic outcome, tumor cells can exploit it to enhance their metastatic capabilities, leading to poor prognoses in certain cancers. Here, we present a remarkable discovery of an altered apoptotic program in tumor cells characterized by nuclear expulsion and citrullinated chromatin, promoting metastatic outgrowth in the lungs in a Padi4-dependent manner. We further identified a calcium spike during apoptosis as the trigger for Padi4 activation, resulting in rapid chromatin de-condensation, nuclear content expulsion, followed by the generation of nuclear expulsion products (NEPs). This process was observed in various types of human cancer tissues. Profiling of proteins bound to the NEP complex unveiled an enrichment of receptor for advanced glycation end-products (RAGE) pathway proteins, such as S100a4 in mouse NEPs and high-mobility group-binding (HMGB) proteins in human NEPs. These chromatin-bound RAGE ligands activated RAGE receptors in neighboring surviving tumor cells, leading to Erk pathway activation. This, in turn, promotes proliferation through the RAGE-MAPK pathway. A nuclear expulsion signature was associated with worse prognoses in cancer patients, underscoring the clinical efficacy in the context of apoptosis-inducing treatments. As we continue our investigations, we are particularly interested in understanding how dying tumor cell-derived NEPs from chemotherapy may affect therapy outcomes. Taken together, our studies uncover a novel mechanism by which tumor cells come together as a community and that the apoptotic tumor cells help live tumor cells metastasize. This insight is likely a step forward to understand the mechanisms of metastatic spread, treatment resistance and relapse. Citation Format: Wooyong Park, Justin M Gray, Ronald J Holewinski, Thorkell Andresson, Jae Young So, Carmelo M Carmona-Rivera, Mariana J Kaplan, Steven D Cappell, Li Yang. Uncovering nuclear expulsion: chromatin-bound signals from dying cancer cells accelerate metastatic outgrowth through S100A4-RAGE pathway [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Breast Cancer Research; 2023 Oct 19-22; San Diego, California. Philadelphia (PA): AACR; Cancer Res 2024;84(3 Suppl_1):Abstract nr PR06.

Cells can irreversibly exit the cell cycle and become senescent to safeguard against uncontrolled proliferation. While the p53-p21 and p16-Rb pathways are thought to mediate senescence, they also mediate reversible cell cycle arrest (quiescence), raising the question of whether senescence is actually reversible or whether alternative mechanisms underly the irreversibility associated with senescence. Here, we show that senescence is irreversible and that commitment to and maintenance of senescence are mediated by irreversible MYC degradation. Senescent cells start dividing when a non-degradable MYC mutant is expressed, and quiescent cells convert to senescence when MYC is knocked down. In early oral carcinogenesis, epithelial cells exhibit MYC loss and become senescent as a safeguard against malignant transformation. Later stages of oral premalignant lesions exhibit elevated MYC levels and cellular dysplasia. Thus, irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation, but bypassing this degradation may allow tumor cells to escape during cancer initiation.

Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.

The E3 ubiquitin ligase APC/C-Cdh1 maintains the G0/G1 state, and its inactivation is required for cell cycle entry. We reveal a novel role for Fas-associated protein with death domain (FADD) in the cell cycle through its function as an inhibitor of APC/C-Cdh1. Using real-time, single-cell imaging of live cells combined with biochemical analysis, we demonstrate that APC/C-Cdh1 hyperactivity in FADD-deficient cells leads to a G1 arrest despite persistent mitogenic signaling through oncogenic EGFR/KRAS. We further show that FADDWT interacts with Cdh1, while a mutant lacking a consensus KEN-box motif (FADDKEN) fails to interact with Cdh1 and results in a G1 arrest due to its inability to inhibit APC/C-Cdh1. Additionally, enhanced expression of FADDWT but not FADDKEN, in cells arrested in G1 upon CDK4/6 inhibition, leads to APC/C-Cdh1 inactivation and entry into the cell cycle in the absence of retinoblastoma protein phosphorylation. FADD's function in the cell cycle requires its phosphorylation by CK1α at Ser-194 which promotes its nuclear translocation. Overall, FADD provides a CDK4/6-Rb-E2F-independent "bypass" mechanism for cell cycle entry and thus a therapeutic opportunity for CDK4/6 inhibitor resistance.

Most tumor cells undergo apoptosis in circulation and at the metastatic organ sites due to host immune surveillance and a hostile microenvironment. It remains to be elucidated whether dying tumor cells have a direct effect on live tumor cells during the metastatic process and what the underlying mechanisms are. Here we report that apoptotic cancer cells enhance the metastatic outgrowth of surviving cells through Padi4-mediated nuclear expulsion. Tumor cell nuclear expulsion results in an extracellular DNA-protein complex that is enriched with receptor for advanced glycation endproducts (RAGE) ligands. The chromatin-bound RAGE ligand S100a4 activates RAGE receptors in neighboring surviving tumor cells, leading to Erk activation. In addition, we identified nuclear expulsion products in human patients with breast, bladder and lung cancer and a nuclear expulsion signature correlated with poor prognosis. Collectively, our study demonstrates how apoptotic cell death can enhance the metastatic outgrowth of neighboring live tumor cells.

In mammalian cells, the decision to proliferate is thought to be irreversibly made at the restriction point of the cell cycle 1 , 2 , when mitogen signalling engages a positive feedback loop between cyclin A2/cyclin-dependent kinase 2 (CDK2) and the retinoblastoma protein 3 – 5 . Contrary to this textbook model, here we show that the decision to proliferate is actually fully reversible. Instead, we find that all cycling cells will exit the cell cycle in the absence of mitogens unless they make it to mitosis and divide first. This temporal competition between two fates, mitosis and cell cycle exit, arises because cyclin A2/CDK2 activity depends upon CDK4/6 activity throughout the cell cycle, not just in G1 phase. Without mitogens, mitosis is only observed when the half-life of cyclin A2 protein is long enough to sustain CDK2 activity throughout G2/M. Thus, cells are dependent on mitogens and CDK4/6 activity to maintain CDK2 activity and retinoblastoma protein phosphorylation throughout interphase. Consequently, even a 2-h delay in a cell’s progression towards mitosis can induce cell cycle exit if mitogen signalling is lost. Our results uncover the molecular mechanism underlying the restriction point phenomenon, reveal an unexpected role for CDK4/6 activity in S and G2 phases and explain the behaviour of all cells following loss of mitogen signalling.

The F-box protein beta-transducin repeat containing protein (β-TrCP) acts as a substrate adapter for the SCF E3 ubiquitin ligase complex, plays a crucial role in cell physiology, and is often deregulated in many types of cancers. Here, we develop a fluorescent biosensor to quantitatively measure β-TrCP activity in live, single cells in real-time. We find β-TrCP remains constitutively active throughout the cell cycle and functions to maintain discreet steady-state levels of its substrates. We find no correlation between expression levels of β-TrCP and β-TrCP activity, indicating post-transcriptional regulation. A high throughput screen of small-molecules using our reporter identifies receptor-tyrosine kinase signaling as a key axis for regulating β-TrCP activity by inhibiting binding between β-TrCP and the core SCF complex. Our study introduces a method to monitor β-TrCP activity in live cells and identifies a key signaling network that regulates β-TrCP activity throughout the cell cycle.

The G(1)-S checkpoint is thought to prevent cells with damaged DNA from entering S phase and replicating their DNA and efficiently arrests cells at the G(1)-S transition. Here, using time-lapse imaging and single-cell tracking, we instead find that DNA damage leads to highly variable and divergent fate outcomes. Contrary to the textbook model that cells arrest at the G(1)-S transition, cells triggering the DNA damage checkpoint in G(1) phase route back to quiescence, and this cellular rerouting can be initiated at any point in G(1) phase. Furthermore, we find that most of the cells receiving damage in G(1) phase actually fail to arrest and proceed through the G(1)-S transition due to persistent cyclin-dependent kinase (CDK) activity in the interval between DNA damage and induction of the CDK inhibitor p21. These observations necessitate a revised model of DNA damage response in G(1) phase and indicate that cells have a G(1) checkpoint.