Merna Sitto

// Paramita Ray 1 , Yee Sun Tan 1 , Vishal Somnay 1 , Ranjit Mehta 1 , Merna Sitto 1 , Aarif Ahsan 1, 4 , Shyam Nyati 1 , John P. Naughton 1, 5 , Alexander Bridges 2 , Lili Zhao 3 , Alnawaz Rehemtulla 1 , Theodore S. Lawrence 1 , Dipankar Ray 1 , Mukesh K. Nyati 1 1 Department of Radiation Oncology, University of Michigan, Ann Arbor, MI 48109, USA 2 School of Pharmacy, University of Michigan, Ann Arbor, MI 48109, USA 3 Department of Biostatistics, University of Michigan, Ann Arbor, MI 48109, USA 4 Current address: Oncology Research Unit East, Pfizer, Pearl River, NY 10965, USA 5 Current address: Department of Otorhinolaryngology-Head and Neck Surgery, Albert Einstein College of Medicine, Montefiore Medical Center, Bronx, NY 10467, USA Correspondence to: Mukesh K. Nyati, email: nyati@umich.edu Dipankar Ray, email: dipray@umich.edu Theodore S. Lawrence, email: tsl@umich.edu Keywords: EGFR, erlotinib, protein stability, ubiquitination, TKI sensitivity Received: April 29, 2016     Accepted: August 25, 2016     Published: September 06, 2016 ABSTRACT Non-small cell lung cancer (NSCLC) patients carrying specific EGFR kinase activating mutations (L858R, delE746-A750) respond well to tyrosine kinase inhibitors (TKIs). However, drug resistance develops within a year. In about 50% of such patients, acquired drug resistance is attributed to the enrichment of a constitutively active point mutation within the EGFR kinase domain (T790M). To date, differential drug-binding and altered ATP affinities by EGFR mutants have been shown to be responsible for differential TKI response. As it has been reported that EGFR stability plays a role in the survival of EGFR driven cancers, we hypothesized that differential TKI-induced receptor degradation between the sensitive L858R and delE746-A750 and the resistant T790M may also play a role in drug responsiveness. To explore this, we have utilized an EGFR- null CHO overexpression system as well as NSCLC cell lines expressing various EGFR mutants and determined the effects of erlotinib treatment. We found that erlotinib inhibits EGFR phosphorylation in both TKI sensitive and resistant cells, but the protein half-lives of L858R and delE746-A750 were significantly shorter than L858R/T790M. Third generation EGFR kinase inhibitor (AZD9291) inhibits the growth of L858R/T790M-EGFR driven cells and also induces EGFR degradation. Erlotinib treatment induced polyubiquitination and proteasomal degradation, primarily in a c-CBL-independent manner, in TKI sensitive L858R and delE746-A750 mutants when compared to the L858R/T790M mutant, which correlated with drug sensitivity. These data suggest an additional mechanism of TKI resistance, and we postulate that agents that degrade L858R/T790M-EGFR protein may overcome TKI resistance.

PURPOSE: Treatment approaches using Hsp90 inhibitors at their maximum tolerated doses (MTDs) have not produced selective tumor toxicity. Inhibition of Hsp90 activity causes degradation of client proteins including those involved in recognizing and repairing DNA lesions. We hypothesized that if DNA repair proteins were degraded by concentrations of an Hsp90 inhibitor below those required to cause nonspecific cytotoxicity, significant tumor-selective radiosensitization might be achieved. EXPERIMENTAL DESIGN: radiosensitization was assessed using a clonogenic assay. Pharmacokinetics profiling was performed in mice bearing xenografts. Finally, the effect of low-dose AT13387 on the radiosensitization of three tumor models was assessed. RESULTS: A subcytotoxic concentration of AT13387 reduced levels of DNA repair proteins, without affecting the majority of Hsp90 clients. The pharmacokinetics study using one-third of the MTD showed 40-fold higher levels of AT13387 in tumors compared with plasma. This low dose enhanced Hsp70 expression in peripheral blood mononuclear cells (PBMCs), which is a biomarker of Hsp90 inhibition. Low dose monotherapy was ineffective, but when combined with radiotherapy, produced significant tumor growth inhibition. CONCLUSIONS: This study shows that a significant therapeutic ratio can be achieved by a low dose of Hsp90 inhibitor in combination with radiotherapy. Hsp90 inhibition, even at a low dose, can be monitored by measuring Hsp70 expression in PBMCs in human studies.

The Unfolded Protein Response (UPR) plays a key role in the adaptive response to loss of protein homeostasis within the endoplasmic reticulum (ER). The UPR has an adaptive function in protein homeostasis, however, sustained activation of the UPR due to hypoxia, nutrient deprivation, and increased demand for protein synthesis, alters the UPR program such that additional perturbation of ER homeostasis activates a pro-apoptotic program. Since ubiquitination followed by proteasomal degradation of misfolded proteins within the ER is a central mechanism for restoration of ER homeostasis, inhibitors of this pathway have proven to be valuable anti-cancer therapeutics. Ubiquitin activating enzyme 1(UAE1), activates ubiquitin for transfer to target proteins for proteasomal degradation in conjunction with E2 and E3 enzymes. Inhibition of UAE1 activity in response to TAK-243, leads to an accumulation of misfolded proteins within the ER, thereby aggravating ER stress, leading to DNA damage and arrest of cells in the G2/M phase of the cell cycle. Persistent drug treatment mediates a robust induction of apoptosis following a transient cell cycle arrest. These biological effects of TAK-243 were recapitulated in mouse models of PDAC demonstrating antitumor activity at a dose and schedule that did not exhibit obvious normal tissue toxicity. In vitro as well as studies in mouse models failed to show enhanced efficacy when TAK-243 was combined with ionizing radiation or gemcitabine, providing an impetus for future studies to identify agents that synergize with this class of agents for improved tumor control in PDAC. SIGNIFICANCE: The UAE1 inhibitor TAK-243, mediates activation of the unfolded protein response, accumulation of DNA breaks and apoptosis, providing a rationale for the use as a safe and efficacious anti-cancer therapeutic for PDAC.

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. 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. Cell division is a tightly regulated process to ensure the production of two genetically identical cells and is often dysregulated in cancer, wherein oncogenic mutations perturb the cell cycle leading to uncontrolled cell division. The somatic mammalian cell cycle involves a tightly regulated, precise, and sequential phase transition through G1, S, G2, and M. Entry into the cell cycle requires the activation of G1 cyclin-dependent kinases (CDK4/6), which inactivates the retinoblastoma protein (Rb) by its phosphorylation (1Narasimha A.M. Kaulich M. Shapiro G.S. Choi Y.J. Sicinski P. Dowdy S.F. Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.Elife. 2014; 3e02872Google Scholar, 2Yao G. Lee T.J. Mori S. Nevins J.R. You L. A bistable Rb-E2F switch underlies the restriction point.Nat. Cell Biol. 2008; 10: 476-482Google Scholar). Inactive Rb releases the E2F transcription factor from inhibition leading to the transcription of E2F targets including cyclin E and early mitotic inhibitor 1 (Emi1) (3Cappell S.D. Chung M. Jaimovich A. Spencer S.L. Meyer T. Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry.Cell. 2016; 166: 167-180Google Scholar, 4Robbins J.A. Cross F.R. Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1.Mol. Biol. Cell. 2010; 21: 914-925Google Scholar). Ubiquitination of S-phase cyclins (e.g., cyclin A2 and cyclin E) and Emi1 (3Cappell S.D. Chung M. Jaimovich A. Spencer S.L. Meyer T. Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry.Cell. 2016; 166: 167-180Google Scholar, 5Malumbres M. Barbacid M. Cell cycle, CDKs and cancer: a changing paradigm.Nat. Rev. Cancer. 2009; 9: 153-166Google Scholar, 6Nakayama K.I. Nakayama K. Ubiquitin ligases: cell-cycle control and cancer.Nat. Rev. Cancer. 2006; 6: 369-381Google Scholar, 7Peters J.M. The anaphase promoting complex/cyclosome: a machine designed to destroy.Nat. Rev. Mol. Cell Biol. 2006; 7: 644-656Google Scholar), by the E3 ligase, Anaphase Promoting Complex with its co-activator Cdh1 (APC/C-Cdh1) and subsequent degradation by the proteasomal machinery provides a strict unidirectional behavior of the cell cycle, necessitating inactivation of the APC/C-Cdh1 prior to entry into the cell cycle (8Pines J. Cubism and the cell cycle: the many faces of the APC/C.Nat. Rev. Mol. Cell Biol. 2011; 12: 427-438Google Scholar, 9Watson E.R. Brown N.G. Peters J.M. Stark H. Schulman B.A. Posing the APC/C E3 ubiquitin ligase to orchestrate cell division.Trends Cell Biol. 2019; 29: 117-134Google Scholar). Accumulation of cyclin E leads to activation of the cyclin-dependent kinase CDK2, which in collaboration with Emi1 provides a feed-forward loop for APC/C-Cdh1 inactivation, a critical decision point, required for a rapid and robust accumulation of S-phase cyclins triggering the transition of cells into S phase and an irreversible commitment to proliferate (3Cappell S.D. Chung M. Jaimovich A. Spencer S.L. Meyer T. Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry.Cell. 2016; 166: 167-180Google Scholar, 8Pines J. Cubism and the cell cycle: the many faces of the APC/C.Nat. Rev. Mol. Cell Biol. 2011; 12: 427-438Google Scholar, 10Bassermann F. Eichner R. Pagano M. The ubiquitin proteasome system - implications for cell cycle control and the targeted treatment of cancer.Biochim. Biophys. Acta. 2014; 1843: 150-162Google Scholar, 11Cappell S.D. M. Meyer T. from a to an inhibitor of to the cell Scholar, Brown N.G. G. E.R. A. of mechanism of E3 ligase Mol. Biol. Scholar, Cyclin E in and cell Scholar, A. and the as a 2006; Scholar, Emi1 ubiquitin by the Cell Biol. Scholar, H. a Mol. Biol. Scholar). Fas-associated protein with death domain (FADD) as an for death activation of the A.M. K. M. a novel death interacts with the death domain of and Scholar, A.M. K. S. is a of and tumor factor Biol. Scholar, J. A. A Fas-associated protein with to the protein is for Cell. Biol. Scholar). a function of FADD in cell wherein the of that of the is of and in with J. M. of FADD at by its Cell. Scholar, G. FADD cell cycle, and is with in S. A. Scholar, B.A. of FADD by the kinase promotes Scholar). of the FADD is in G. A. FADD cell and is with Scholar, L. and expression the Fas-associated death domain as a in the in Scholar, P. F. of Fas-associated death domain protein with in 2011; Scholar, K. and expression of FADD in cell 2010; Scholar). Using genetically of cancer, we a for FADD and its phosphorylation at for mutant oncogenic in B.A. of FADD by the kinase promotes Scholar). the for FADD’s function in to reveal that FADD promotes entry into the cell cycle and the G1 to S cell cycle to the of FADD with Cdh1 leads to the inhibition of APC/C-Cdh1 E3 ligase which is required for the G1 to S A consensus KEN-box domain The an from the D targeted by the of FADD to for with Cdh1 and the inactivation of APC/C-Cdh1. The of FADD in cell cycle entry by its to G1 to S transition in the absence of cyclin and Rb phosphorylation. demonstrate a role for FADD as an for APC/C-Cdh1 inactivation and entry into the cell cycle as as a of the role of a protein FADD in promoting cell with that FADD with in with G. FADD cell cycle, and is with in S. A. Scholar). an to the role of FADD in the transition through phase of the cell cycle at we live cell imaging the of cell cycle phase in cells with Scholar). of a to the as as a to the is G1 and upon entry into the S while is and from anaphase through G1 by APC/C-Cdh1 but upon entry into the S accumulation and degradation as for and of the G1 to S transition We cell and live cell of and degradation of A and for by a rapid in and a accumulation in of a transition into the S phase from G1 cell in accumulation of with is of phase of the cell A rapid in upon and cell production to the cell cycle a is often in cells a in that the of the thus uncontrolled cell as as a that is with to of of cell from cell and Scholar, cell Cell. Scholar). cells a mutant J. and of in Cancer. 21: the role of FADD in the cell cycle we of FADD two to control cells a persistent wherein accumulation of to S not in of the cells for the imaging cells FADD a G1 to S transition which to to for the that the in the G1 to S transition in FADD cells due to a prior to entry into G1, we the in S and the to control cells not show an of the in S and while the subsequent in G1 from to in cells to the control cells of the for the S and in and control cells in that the S and of G1 cells to control of cells arrested in G1 at that of cells in to in the control cells of mitotic cells a that of control cells at of of FADD cells that cells demonstrate G1 to S The in the G1 to S transition upon FADD in an cell mitogenic signaling wherein a G1 a G1 as as mitotic A in accumulation in cells a in APC/C-Cdh1 inactivation a in G1 to S we cells at a cells at to the degradation and accumulation of APC/C-Cdh1 control cyclin cyclin and at upon from APC/C-Cdh1 is and at APC/C-Cdh1 is upon mitotic by of cyclin cyclin and at in control cells is with APC/C-Cdh1 inactivation and entry into S from cells to show an accumulation of cyclin cyclin at the of cyclin and cyclin A2 in a in the accumulation of APC/C-Cdh1 in the absence of results demonstrate that FADD in and cells results in a in the G1 to S due to a to APC/C-Cdh1. is a of the signaling that mitogenic to the upon cell activation is a which from its to an upon of from in that in a state, which through the a that promotes uncontrolled cell as a of uncontrolled cell cycle entry S. kinase in Scholar). FADD in the G1 to S transition in cells mutant we cells cells upon and of APC/C-Cdh1 of FADD in a G1 to S transition which required to in control and that the in S in cells that a G1 to S transition not and that the cell cycle to a G1 to S transition a G1 arrest in of the cells cell of the to results in demonstrate that cells which a G1 arrest in the absence of FADD an S and to of mitotic that of cells the to of the control cells demonstrate that the in G1 to S transition in cells is due to APC/C-Cdh1 hyperactivity in we a as in to cells arrested cells cyclin cyclin and A in the of arrested and and entry into G1, a in of of the APC/C-Cdh1 at activation of the APC/C-Cdh1. entry into the S phase at in control cells by an accumulation of of APC/C of APC/C-Cdh1 inactivation cells FADD of cyclin cyclin A2 and A upon from at the and FADD a transition from G1 into S not as the of cyclin cyclin and to to the in control cells as A to of APC/C-Cdh1 is with a to APC/C-Cdh1 at the in of cells that FADD in and at the of and to prior to entry into the S phase from G1 that FADD regulated in a cell cycle A but of FADD in a cell cycle in cells expression of a of the not cells of Emi1 identical the in the G1 to S transition in cells to cells a of we the of FADD in which in to in the that FADD results in a transition of cells from G1 into S a cell we from mutant an FADD which is by a and B.A. of FADD by the kinase promotes Scholar). that mutant for rapid upon which the expression of oncogenic that for the FADD and a of and B.A. of FADD by the kinase promotes Scholar), that FADD is required for entry into the cell cycle in in of mutant as a mitogenic The of APC/C-Cdh1 inactivation the G1 to S transition in cells S.D. M. Meyer T. from a to an inhibitor of to the cell Scholar, A. and the as a 2006; Scholar, Emi1 ubiquitin by the Cell Biol. Scholar, J. Emi1 is to with but not activation of the mitotic Cell Biol. Scholar). the of FADD in APC/C-Cdh1 inactivation in system we cells to a from its APC/C motif A. H. T. A. H. H. of cell-cycle 2008; Scholar). APC/C and cell show that cells with FADD APC/C-Cdh1 and G1 with APC/C to the control cells cells in G1, further that FADD the G1 to S further a for FADD in APC/C-Cdh1 inactivation in we a of the FADD two of cells a in the of accumulation of mitotic cyclins cyclin and upon G1 to S transition in cells from a arrest The FADD the G1 to S transition in cells to and to the that cells and which to of Rb and activation of the E2F transcription E2F leads to the expression of S-phase cyclins E) and Emi1 and APC/C-Cdh1 promoting the entry of cells into S We that due to cells to FADD to cell in We the of FADD and Emi1 in and and that cells Emi1 protein to and which Emi1 and FADD in that FADD a of G1 to S transition in a function that is by Emi1 FADD in an inability to S phase cyclins due to APC/C-Cdh1 control and FADD cells at G1 and S cells with a proteasomal inhibitor APC/C inhibitor an inhibitor of for cells of cyclin in S phase which not in the of that and proteasomal degradation of cyclin not in control cells at point to APC/C-Cdh1 in S cells of cyclin identical in FADD while as as but not treatment the of cyclin to cells of APC/C-Cdh1 upon inhibition of APC/C the proteasomal machinery but not in cells the that the APC/C-Cdh1 not in the absence of FADD the G1 to S to demonstrate APC/C-Cdh1 hyperactivity in APC/C from G1 APC/C-Cdh1 from control and FADD cells and for in cyclin as a prior to of G1 cells and a which of APC/C but in FADD as as cyclin in FADD in cyclin of the enhanced in cells to the control cells in that APC/C-Cdh1 is in the absence of with and that of APC/C-Cdh1 including cyclin cyclin cyclin and to in and in a in the G1 to S further that the enhanced in cells to the we Cdh1 with FADD cells required in G1 prior to a transition into S while a of cells a G1 arrest the imaging in D and of Cdh1 in cells in an G1 to S transition wherein the G1 in cells to in Cdh1 in cells a of FADD and Cdh1 required to a G1 to S transition in wherein of the cells a G1 arrest despite the absence of FADD expression for the that the G1 arrest a of APC/C-Cdh1 in to FADD requires Cdh1, the of the APC/C-Cdh1 and that the in the G1 to S transition in FADD cells is the of Cdh1 and thus APC/C-Cdh1 The is that APC/C-Cdh1 is upon phosphorylation by CDKs as as through and inhibition of its by Emi1 through a mechanism (3Cappell S.D. Chung M. Jaimovich A. Spencer S.L. Meyer T. Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry.Cell. 2016; 166: 167-180Google Scholar, M. protein degradation by ubiquitin 2014; Scholar). a consensus motif as as a at the we that function to we the of FADD to interact with Cdh1 of Cdh1 in of FADDWT in cells FADD and of FADD with Cdh1 further by of which in a of Cdh1 of the KEN-box the of FADD with Cdh1, while of the to not and that FADD its KEN-box domain to interact with further that FADD interacts with the co-activator of the APC/C in to the of the KEN-box for FADD’s with Cdh1, that the of FADD with the of the wherein the mutant of FADD with to FADDWT we of of FADD G1 to S transition of the cell cycle in We for expression of a FADD in a cell imaging of FADDWT cells not show in the G1 to S wherein APC/C-Cdh1 inactivation as by accumulation at in of the from to control cells in APC/C-Cdh1 inactivation as as a of cells to the S phase the imaging A in APC/C-Cdh1 inactivation in cells through the of the of its from a cells as as FADDWT for APC/C-Cdh1 inactivation, as by accumulation of cyclin cyclin and at into for cell in to wherein cyclin cyclin and to upon arrest in and a to of the APC/C-Cdh1 at the of that the accumulation of cyclin at in control cells not upon of the We of mitotic cells and that of control and cells at of of cells further that leads to a G1 to S A in accumulation as as cyclin cyclin A2 and in and cell cycle to a of the mutant that to to Cdh1, FADD to function as an APC/C-Cdh1 thus APC/C-Cdh1 to is We the that to the APC/C complex by the APC/C-Cdh1 complex from cells wherein expression of FADDWT interacts with Cdh1, not that with APC/C from cells in G1 FADDKEN, a mutant that to with Cdh1 to the APC/C and to with the APC/C further we a that FADDWT to demonstrate that FADD to the APC/C-Cdh1 complex to with the APC/C despite its inability to interact with Cdh1 that FADD interact with of the APC/C-Cdh1 which provides a for the is in The of the G1 to S transition is that Emi1 and cyclin required for the inactivation of APC/C-Cdh1. through its inhibitor function and cyclin by that FADD function as an inhibitor of APC/C-Cdh1 by to Cdh1 through its KEN-box the of FADD to APC/C-Cdh1 of Emi1 and cyclin we cells with a cyclin kinase inhibitor the absence of CDK4/6 kinase phosphorylation of Rb is in a G1 arrest due to E2F inhibition by Rb cells in by treatment in a for wherein of the cells at in to at in cells with of FADD in cells in of cells an G1, by the accumulation of at while of cells a to APC/C-Cdh1 the imaging FADD in with treatment the of cells to APC/C-Cdh1 as by accumulation in of cells of FADDWT in cells in of cells accumulation of APC/C-Cdh1 at to of APC/C-Cdh1 inactivation CDK4/6 by in the of to APC/C-Cdh1 inactivation as by a to The that to as a that in to of control cells FADD cells not show accumulation the imaging APC/C-Cdh1 with a and biochemical of cells from a arrest into the that FADDWT and APC/C-Cdh1 inactivation as by the accumulation of cyclin cyclin and and while in the absence of FADD S phase to in control cells treatment but absence of FADD of in of APC/C-Cdh1 to in the of E and the single-cell results that of FADDWT results in the inactivation of APC/C-Cdh1 the absence of CDK4/6 and promotes the G1 to S a of FADDKEN, APC/C-Cdh1 at in cells to the control cells E and inactivation of APC/C-Cdh1 Rb we cells and single-cell of cells that in G1 that the of to to cells A and which robust at of the as at of the A and the inactivation of APC/C-Cdh1 in FADDWT and cells D and E) not to Rb-E2F CDK4/6 phosphorylation of Rb releases E2F to Emi1 and cyclin which in with leads to inactivation of transition into S CDK4/6 is by to Rb maintains APC/C-Cdh1 in an due to the absence of Emi1 and Cyclin leading to a G1 identical FADD leads to APC/C-Cdh1 inactivation despite the absence of Emi1 and cyclin E of Rb in Overall, results demonstrate that FADD as an APC/C-Cdh1 inhibitor of Rb phosphorylation Emi1 and cyclin an role in G1 to S we in FADD cells a (3Cappell S.D. Chung M. Jaimovich A. Spencer S.L. Meyer T. Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry.Cell. 2016; 166: 167-180Google Scholar, S.L. S.D. Meyer T. The decision is by a in at mitotic Scholar). that in activation in with the that APC/C-Cdh1 leading to cyclin E and the in G1 to S G. FADD cell cycle, and is with in S. A. Scholar, B.A. of FADD by the kinase promotes Scholar, G. A. FADD cell and is with and of J. A. A Fas-associated protein with to the protein is for Cell. Biol. Scholar, J. M. of FADD at by its Cell. Scholar, J. A. FADD-deficient FADD is required for early cell S. A. Scholar, J. A. A. and in lacking that phosphorylation of FADD at and its nuclear is in the of FADD in cell division and the role of Ser-194 FADD in the APC/C-Cdh1 the G1 to S we wherein FADD a targeted of FADD by expression of a of of the of FADD in cells in a of to FADD cells expression of the with FADD the of to a in identical wherein to the not of the G1 the that in to control cells cells an in G1 in expression of FADDWT in FADD cells in a of the in G1 in FADD to in FADD while of FADD to the G1 in FADD provides a of the of of a cells a of cell at and the of cells in G1 S and in cells the of cells and and a of cells arrested in G1 at imaging in cells wherein FADDWT to the FADD a in cell the and the of cells in G1, S, and to control the A the mutant of FADD to the of cells of with an accumulation in G1 We to the of the FADD in of a that to the of of the FADD FADDWT to in the as as the while the mutant from the and the the is in with J. M. of FADD at by its Cell. Scholar, G. FADD cell cycle, and is with in S. A. that phosphorylation leads to nuclear of we cell and cells a FADD in A and demonstrate that the of FADD is in S and cells and while of in G1 cells in and FADD in cell in a G1 arrest FADD A and and the of FADD of FADD in cell as as cells of FADD in cells and in cells APC/C-Cdh1 activation to mitotic and its is required for the of cells in G1 through proteasomal degradation of S cyclins and APC/C-Cdh1 maintains the G1 state, and its inactivation is a for cells to to the cell commitment is the G1 to S transition to robust and tightly regulated and is often dysregulated in (8Pines J. Cubism and the cell cycle: the many faces of the APC/C.Nat. Rev. Mol. Cell Biol. 2011; 12: 427-438Google Scholar, 9Watson E.R. Brown N.G. Peters J.M. Stark H. Schulman B.A. Posing the APC/C E3 ubiquitin ligase to orchestrate cell division.Trends Cell Biol. 2019; 29: 117-134Google Scholar). two to APC/C-Cdh1 inactivation prior to S-phase entry. which is by to APC/C-Cdh1 and its as a and as a with the Brown N.G. G. E.R. A. of mechanism of E3 ligase Mol. Biol. Scholar). Additionally, the of Cdh1 from APC/C upon cyclin phosphorylation of Cdh1 inactivates APC/C-Cdh1 entry into (3Cappell S.D. Chung M. Jaimovich A. Spencer S.L. Meyer T. Irreversible APC(Cdh1) inactivation underlies the point of no return for cell-cycle entry.Cell. 2016; 166: 167-180Google Scholar, 4Robbins J.A. Cross F.R. Requirements and reasons for effective inhibition of the anaphase promoting complex activator CDH1.Mol. Biol. Cell. 2010; 21: 914-925Google Scholar). The novel into an mechanism that promotes the G1 to S by that FADD as an APC/C-Cdh1 inhibitor and is required for the G1 to S transition in to The of FADD’s role in APC/C-Cdh1 inhibition and the G1 to S transition by that in the absence of Emi1 and cyclin transcription a of of FADD the inactivation of APC/C-Cdh1 despite the absence of Rb phosphorylation. of the FADD at and its enhanced expression is in and and cancer, which with G. FADD cell cycle, and is with in S. A. Scholar, P. K. The with in cell J. Scholar, A. L. A. M. cyclin and in and 2008; Scholar, F. and in to expression two of and J. Cancer. 2006; Scholar, P. J.A. A. L. of in an of Scholar), that of the G1 to S by FADD to FADD is for its function as an for the activation of the cell death A.M. K. M. a novel death interacts with the death domain of and Scholar, A.M. K. S. is a of and tumor factor Biol. Scholar, J. A. A Fas-associated protein with to the protein is for Cell. Biol. Scholar). wherein FADD is to the cell cycle in to in that a in cell death and cell of FADD upon phosphorylation by CK1α at by G. FADD cell cycle, and is with in S. A. Scholar, B.A. of FADD by the kinase promotes and J. M. of FADD at by its Cell. Scholar, J. Cell cycle by its phosphorylation Biol. Scholar), which a that the two of the nuclear Ser-194 of FADD is with and its of B.A. of FADD by the kinase promotes Scholar, G. A. FADD cell and is with Scholar, S. K. M. FADD phosphorylation is critical for cell cycle in J. Cancer. 2006; Scholar). Additionally, results as as that expression of a mutant as as a mutant in cells and leads to in cell J. A. A Fas-associated protein with to the protein is for Cell. Biol. Scholar, J. M. of FADD at by its Cell. Scholar, B.A. of FADD by the kinase promotes Scholar, G. A. FADD cell and is with Scholar, S.L. A. phosphorylation in Fas-associated death domain (FADD) results in early cell cycle Biol. due to of the APC/C that phosphorylation of FADD is in the S and phase of the cell cycle and in G1, that and its nuclear required for the transition of cells from G1 to S. The for APC/C-Cdh1 inhibition by FADD in to Emi1 and two APC/C-Cdh1 in and S.D. M. Meyer T. from a to an inhibitor of to the cell Scholar, J.M. D and of and inhibition of the Biol. 2008; Scholar), its function as a inhibitor of APC/C-Cdh1 by the of Cdh1 the and the KEN-box at the of of the that the enhanced to S-phase cyclins and thus a G1 in cells required the of Cdh1, cells of Cdh1 and FADD to demonstrate a G1 a in G1 as a of Cdh1 with S.D. M. Meyer T. from a to an inhibitor of to the cell Scholar, J. T. Lee J.R. the role of in the of S-phase Biol. Cell. 2014; Scholar). that expression of a FADDWT cells of while expression of the mutant to nuclear the mutant to interact with not the G1 arrest in FADD provides that FADD’s role in the G1 to S transition requires its nuclear with and with The that the to interact with Cdh1, to APC/C-Cdh1 in G1, and a that to FADD that its with and inhibition of the the of FADD’s as an APC/C-Cdh1 inhibitor to of which as inhibitor of cell cycle entry in cells for the and and from cells a from of in and and cells in and cell at and with early cells that no by The in cyclin from FADD cyclin A2 cyclin cyclin A Rb from Cell Cdh1 from and and from and from The in with as Cell The for the a from M. L. protein of the anaphase promoting complex Cdh1 is in and in and Cell Biol. 2008; 10: Scholar), and by FADD into and restriction of the a from of cell cycle phase in cells with Scholar). to the FADD for FADDKEN, and to cell and cells from the FADD of and FADD of control at a of FADD and FADD not the in of the by the to the and of the not Cdh1 from and at a of the cells in the imaging FADD by the of to the expression of FADD the the cells and by by cells with and and a from and Cell and and cells with 1 with 1 in The the and with a to FADDKEN, and cell to to the protein expression as by in by into of cells the the The and cells The cells