Machine learning discoveries of TIMELESS-X synergy in ETC-1922159 treated colorectal cancer cells

Machine learning discoveries of TIMELESS-X synergy in ETC-1922159 treated colorectal cancer cells shriprakash sinha Independent Researcher; Orcid ID : orcid.org/0000-0001-7027-5788 104-Madhurisha Heights Phase 1, Risali, Bhilai-490006, India Abstract TIMELESS plays a role in the cricadian rhythm of Drosophila, however in mammalian biology it is known to play its role as DNA replication fork component, promoter of fork progression and participator in cell cycle checkpoint signaling in response to DNA damage. In colorectal cancer (CRC) cells treated with ETC-1922159, TIMELESS was found to be down regulated along with other genes. A recently developed search engine ranked combinations of TIMELESS-X (X, a particular gene/protein) at 2nd order level after drug administration. Some of these combinations have been tested in wet lab, however many have been pointed out by the search engine that are yet to be explored/tested. These rankings reveal which TIMELESS-X combinations might be working synergistically in CRC. In this research work, I cover combinations of TIMELESS with members of ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3 related (ATR), matrix metallopeptidase (MMP), v-myc avian myelocytomatosis viral oncogene homolog (MYC), myosin (MYO), transcription termination factor (TTF), sirtuin (SIRT), TIMELESS interacting protein (TIPIN), UBX domain protein (UBXN), DEAD/H (Asp-Glu-Ala-Asp/His) box helicase (DDX), DNA polymerase (POL), DNA-dependent RNA polymerase (POLR), minichromosome maintenance complex (MCM), go-ichi-ni-san (GINS), F-box (FBX), ring finger protein (RNF), H2A histone family member (H2A), high mobility group (HMG), Wnt family member (WNT), microRNA non-coding host genes (MIR-x-HG), Forkhead box (FOX), poly(ADP-ribose) polymerase (PARP) and phosphodiesterase (PDE) family. Keywords: TIMELESS, Porcupine inhibitor ETC-1922159, Sensitivity analysis, Machine learning, Colorectal cancer. IML dicoveries of TIMELESS-X synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at the first Wnt Gordon Research Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA. Preprint submitted to Preprint January 26, 2025 1. Introduction 1.1. TIMELESS A circadian rhythm is a natural oscillation that repeats roughly every 24 hours and can refer to any process that originates within an organism and responds to the environment. They are regulated by a circadian clock, the primary function which is to co-ordinate biological processes so they occur at the correct time to maximize the fitness of an individual. Circadian rhythm consists of light/dark phases which coincide with the phases of the solar day. TIMELESS (TIM) is a gene in multiple species but widely known in Drosophila (dTIM) for regulating circadian rhythm, first reported by Sehgal et al. [1]. The mammalian TIM (mTIM) was identified by Gotter et al. [2] in mouse as a potential circadian clock component, however its role in clock regulation has been more controversial (Gotter [3]). Gotter et al. [2] show that mTIM is essential for embryonic development, but does not have substantiated circadian function. mTIM has now been characterized as a DNA replication fork component and has been shown to promote fork progression and participate in cell cycle checkpoint signaling in response to DNA damage (Cai and Chiu [4]). Cell cycle checkpoints ensure completion of biochemical reactions unique to G1, S, G2, and M in phase of proliferating mammalian cells, prior to emergence of subsequent phases. Gotter et al. [5] demonstrate that mTIM and its constitutive binding partner, TIMinteracting protein (TIPIN), are replisome-associated proteins, which associate with components of the endogenous replication fork complex, and are present at BrdUpositive DNA replication sites. Further, Cho et al. [6] show that mTIM and TIMinteracting protein (TIPIN) complex couple replicative DNA helicase CMG (CDC45, MCM2-7, GINS) and stimulate the activities of DNA polymerase (POL) α,δ, and ε, in progressing the replication fork. mTIM is essential for ATR-dependent CHK1 activation and S-phase arrest (Yang et al. [7]). ATR and ataxia telangiectasia mutated (ATM) work at the heart of sensing DNA damage and phosphorylate checkpoint kinase 1/2 (CHK1/2), as observed by Blackford and Jackson [8]. In response to DNA damage and efficient homologous recombination repair, Xie et al. [9] show that mTIM physically interacts with and recruits poly [ADP-ribose] polymerase 1 (PARP1), independent of poly(ADP-ribosyl)ation, to damaged sites. Rao and Lin [10] review that abnormal expression of certain clock genes has been found in patients with colorectal cancer (CRC) and their correlation with clinicopathological features has also been explored. In CRC cells treated with ETC-1922159, TIMELESS was found to be down regulated along with other genes. Some combinations of TIMELESS have been confirmed in wet lab, however, many of the combinations have not been explored/tested or are known. To reveal these combinations, I use a modification of a recently published machine learning based search engine, details of which are given in the next section. 2 1.2. Combinatorial search problem and a possible solution In a recently published work Sinha [11], a frame work of a search engine was developed which can rank combinations of factors (genes/proteins) in a signaling pathway. Readers are requested to go through the adaptation of the above mentioned work for gaining deeper insight into the working of the pipeline and its use of published data set generated after administration of ETC-1922159, Sinha [12]. The work uses SVM package by Joachims [13] in https://www.cs.cornell.edu/people/tj/svm_light/ svm_rank.html. I use the adaptation to rank 2nd order gene combinations. 2. Results & Discussion 2.1. TIMELESS related synergies 2.1.1. TIMELESS - Individual members mTIM is essential for ATR-dependent CHK1 activation and S-phase arrest and Yang et al. [7] report that TIM is likewise essential for ATM-dependent CHK2-mediated signaling of doxorubicin-induced DNA double strand breaks. Further, they show that TIM depletion attenuates doxorubicin-induced G2/M cell cycle arrest and sensitizes cancer cells to doxorubicin-induced cytotoxicity. ATR and ataxia telangiectasia mutated (ATM) work at the heart of sensing DNA damage and phosphorylate checkpoint kinase 1/2 (CHK1/2), as observed by Blackford and Jackson [8]. Liu et al. [14] found that the hippocampal MMP9 expression was correlated with the expression levels of circadian genes PER2, PER3, TIMELESS, FBXL3, and NFIL3. PER2 and TIMELESS were upregulated in mouse hippocampus after sleep deprivation (SD). Chi et al. [15] found that overexpression of TIM dramatically enhanced, while knockdown of TIM suppressed the self-renewal of cancer stem cells (CSCs), cell invasion and migration abilities of breast cancer cells in vitro. Their mechanism studies revealed that TIM upregulated the expression and activity of MYC. In CRC tissues, Cao et al. [16] found that TIMELESS was upregulated and mechanistic investigations showed that it activated the β-catenin pathway by binding to Myosin-9 (MYO9). During S phase, the replication and transcription of genomic DNA need to accommodate each other, otherwise their machineries might collide leading to chromosomal instability. Akamatsu and Kobayashi [17] characterized the human replication fork barrier (RFB) that is present downstream from the 47S pre-rRNA gene (ribosomal DNA [rDNA]). They found that the most proximal transcription terminator, SAL box T1, acts as a polar RFB, while the other, SAL box T4/T5, arrests replication forks bidirectionally. The fork-arresting activity at these sites depends on polymerase I (Pol I) transcription termination factor 1 (TTF1) and TIMELESS. In oral squamous cell carcinoma (OSCC), Chen et al. [18] found elevated expression of TIMELESS which enhanced cell proliferation, increased glycolytic activity (glucose uptake and lactate production), and suppressed oxidative phosphorylation (evidenced by reduced oxygen consumption and altered pH levels). SIRT1 was positively associated with TIMELESS expression and expression of SIRT1 increased with the overexpression of TIMELESS levels and decreased with the knockdown of TIMELESS. Gotter et al. [5] demonstrate that mTIM and TIPIN 3 are replisome-associated proteins. Both proteins associate with components of the endogenous replication fork complex, and are present at BrdU-positive DNA replication sites. A direct binding of the TIM-TIPIN complex to the 34 kDa subunit of replication protein A provides a biochemical explanation for the potential coupling role of these proteins. The eukaryotic replisome is rapidly disassembled during DNA replication termination. In metazoa, the cullin-RING ubiquitin ligase CUL-2LRR-1 drives ubiquitylation of the CMG helicase, leading to replisome disassembly by the p97/CDC-48 ”unfoldase”. Xia et al. [19] show that the TIMELESS-TIPIN complex is required for CUL-2LRR-1 recruitment and CMG helicase ubiquitylation. Aided by this complex, CUL-2LRR-1 directs ubiquitylation enzymes to ubiquitylate the MCM7 subunit of CMG. Subsequently, the UBXN3 directly stimulates the disassembly of ubiquitylated CMG by CDC-48 UFD-1 NPL-4. These experimental studies confirm the existence of synergy between the above involved factors with TIMELESS. In colorectal cancer cells treated with ETC-1922159, these inidividual members, and TIMELESS, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these individual members along with TIMELESS. Table 1 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 2 generated from analysis of the ranks in table 1. The table 1 shows rankings of these individual members w.r.t TIMELESS. ATR - TIMELESS shows low ranking of 1390 (laplace) and 890 (rbf). ATRIP - TIMELESS shows low ranking of 7 (linear) and 1429 (rbf). MMP11 - TIMELESS shows low ranking of 591 (laplace) and 581 (rbf). MYO19 - TIMELESS shows low ranking of 1359 (laplace) and 648 (linear). TIPIN - TIMELESS shows low ranking of 368 (laplace), 491 (linear) and 436 (rbf). UBXN8 - TIMELESS shows low ranking of 885 (laplace) and 1361 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, ATM, MYC, TTF2 and SIRT3 showed high ranking and might not be synergistically working with TIMELESS, before treatment. One can also interpret the results of the table 1 graphically, with the following influences - •individual members w.r.t TIMELESS with TIMELESS −>ATR / ATRIP/ MMP11 / MYO19 / TIPIN / UBXN8. 2.1.2. TIMELESS - DDX Lerner et al. [20] found that TIMELESS contributes to the ability of the replisome to sense replication-hindering G-quadruplex (G4) formation and ensures resolution of these structures by DDX11 to maintain processive DNA synthesis. These experimental studies confirm the existence of synergy between the above involved factors with TIMELESS. In colorectal cancer cells treated with ETC-1922159, these DDX members, and TIMELESS, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these DDX members along with TIMELESS. Table 3 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 4 generated from analysis of the ranks in table 3. The table 3 shows rankings of DDX members w.r.t TIMELESS. DDX12P - TIMELESS shows low ranking of 131 (laplace) and 8 (rbf). DDX28 - TIMELESS shows 4 RANKING INDIVIDUAL MEMBERS VS TIMELESS RANKING OF INDIVIDUAL MEMBERS W.R.TTIMELESS laplace linear rbf ATM - TIMELESS 1183 2721 1705 ATR - TIMELESS 1390 2352 890 ATRIP - TIMELESS 2278 7 1429 MMP11 - TIMELESS 591 2682 581 MYC - TIMELESS 1886 1392 2336 MYO19 - TIMELESS 1359 648 2092 TTF2 - TIMELESS 1930 2393 1729 SIRT3 - TIMELESS 1080 1709 2189 TIPIN - TIMELESS 368 491 436 UBXN8 - TIMELESS 885 2478 1361 Table 1: 2nd order interaction ranking between TIMELESS VS Individual members. UNEXPLORED COMBINATORIAL HYPOTHESES Individual members w.r.t TIMELESS ATR TIMELESS ATRIP TIMELESS MMP11 TIMELESS MYO19 TIMELESS TIPIN TIMELESS UBXN8 TIMELESS Table 2: 2nd order combinatorial hypotheses between TIMELESS and individual members. low ranking of 386 (laplace) and 408 (rbf). DDX18 - TIMELESS shows low ranking of 492 (laplace) and 1265 (rbf). DDX11 - TIMELESS shows low ranking of 605 (laplace), 1452 (linear) and 653 (rbf). DDX27 - TIMELESS shows low ranking of 705 (laplace), 843 (linear) and 2279 DDX51 - TIMELESS shows low ranking of 963 (laplace) and 545 (rbf). DDX20 - TIMELESS shows low ranking of 1314 (laplace) and 756 (rbf). DDX55 - TIMELESS shows low ranking of 1438 (laplace) and 746 (rbf). These rankings point to the synergy existing between the two components, which have 5 been down regulated after the drug treatment. Further, DDX54, DDX19A, DDX10, DDX21, DDX31, DDX46 and DDX56 showed high ranking and might not be synergistically working with TIMELESS, before treatment. RANKING DDX MEMBERS VS TIMELESS RANKING OF DDX MEMBERS W.R.TTIMELESS laplace linear rbf laplace linear rbf DDX12P - TIMELESS 131 2039 8 DDX28 - TIMELESS 386 2297 408 DDX18 - TIMELESS 492 1610 1265 DDX11 - TIMELESS 605 1452 653 DDX54 - TIMELESS 697 1798 1747 DDX27 - TIMELESS 705 843 2279 DDX51 - TIMELESS 963 2458 545 DDX20 - TIMELESS 1314 1715 756 DDX55 - TIMELESS 1438 2115 746 DDX19A - TIMELESS 1566 436 1947 DDX10 - TIMELESS 1721 2550 1540 DDX21 - TIMELESS 2141 187 1980 DDX31 - TIMELESS 2217 2370 2295 DDX46 - TIMELESS 2453 1672 2495 DDX56 - TIMELESS 2516 1615 2683 Table 3: 2nd order interaction ranking between TIMELESS VS DDX members. One can also interpret the results of the table 3 graphically, with the following influences - •DDX members w.r.t TIMELESS with TIMELESS −>DDX-12P/28/18/11/27/51/20/55. UNEXPLORED COMBINATORIAL HYPOTHESES DDX members w.r.t TIMELESS DDX-12P/28/18/11/27/51/20/55 TIMELESS Table 4: 2nd order combinatorial hypotheses between TIMELESS and DDX members. 2.1.3. TIMELESS - POL/POLR Cho et al. [6] show that mTIM and TIM-interacting protein (TIPIN) complex couple replicative DNA helicase CMG (CDC45, MCM2-7, GINS) and stimulate the activities of DNA polymerase (POL) α,δ, and ε, in progressing the replication fork. These experimental studies confirm the existence of synergy between the above involved factors with TIMELESS. In colorectal cancer cells treated with ETC-1922159, these POL members, and TIMELESS, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these POL members along with TIMELESS. Additionally I ranked POLR (DNA-dependent RNA polymerase) also along with TIMELESS to see if there exists any synergy. Table 5 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 6 generated from analysis of the ranks in table 5. The table 5 shows rankings of POL/POLR members w.r.t TIMELESS. POLA1 - TIMELESS shows low ranking of 334 (laplace) and 145 (rbf). POLD1 - TIMELESS shows 6 low ranking of 399 (laplace) and 942 (rbf). POLE2 - TIMELESS shows low ranking of 479 (laplace) and 89 (rbf). POLE3 - TIMELESS shows low ranking of 1111 (laplace) and 1487 (rbf). POLG2 - TIMELESS shows low ranking of 1220 (laplace) and 1251 (rbf). POLR3K - TIMELESS shows low ranking of 96 (laplace) and 678 (rbf). POLR1A - TIMELESS shows low ranking of 515 (laplace), 737 (linear) and 1093 (rbf). POLR1E - TIMELESS shows low ranking of 820 (laplace) and 665 (rbf). POLR1B - TIMELESS shows low ranking of 1077 (laplace), 694 (linear) and 817 (rbf). POLR3E - TIMELESS shows low ranking of 1249 (laplace) and 1459 (linear). POLR2D - TIMELESS shows low ranking of 1264 (laplace) and 1106 (linear). POLR2G - TIMELESS shows low ranking of 1299 (laplace) and 1059 (rbf). POLR1C - TIMELESS shows low ranking of 1122 (linear) and 1313 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, POLA2, POLD2, POLB, POLQ, POLR1D, POLR3A, POLR2K and POLR2F showed high ranking and might not be synergistically working with TIMELESS, before treatment. RANKING POL MEMBERS VS TIMELESS RANKING OF POL MEMBERS W.R.TTIMELESS laplace linear rbf laplace linear rbf POLA1 - TIMELESS 334 2106 145 POLD1 - TIMELESS 399 2699 942 POLE2 - TIMELESS 479 2206 89 POLE3 - TIMELESS 1111 2242 1487 POLG2 - TIMELESS 1220 2197 1251 POLQ - TIMELESS 1561 617 2432 POLA2 - TIMELESS 2454 83 1717 POLD2 - TIMELESS 2467 2216 1091 POLB - TIMELESS 2706 1515 2585 RANKING POLR MEMBERS VS TIMELESS RANKING OF POLR MEMBERS W.R.TTIMELESS laplace linear rbf laplace linear rbf POLR3K - TIMELESS 96 1591 678 POLR1A - TIMELESS 515 737 1093 POLR1E - TIMELESS 820 2734 665 POLR1B - TIMELESS 1077 694 817 POLR3E - TIMELESS 1249 1459 1715 POLR2D - TIMELESS 1264 1106 1932 POLR2G - TIMELESS 1299 2486 1059 POLR1C - TIMELESS 1551 1122 1313 POLR1D - TIMELESS 1692 1374 2448 POLR3A - TIMELESS 2405 1021 2534 POLR2K - TIMELESS 2476 1567 2612 POLR2F - TIMELESS 2694 1632 2635 Table 5: 2nd order interaction ranking between TIMELESS VS POL/POLR members. One can also interpret the results of the table 5 graphically, with the following influences - •POL members w.r.t TIMELESS with TIMELESS −>POL-A1/D1/E2/E3/G2 and •POLR members w.r.t TIMELESS with TIMELESS −>POLR-3K/1A/1E/1B/3E/2D/2G/1C. 2.1.4. TIMELESS - MCM/GINS Cho et al. [6] show that mTIM and TIM-interacting protein (TIPIN) complex couple replicative DNA helicase CMG (CDC45, MCM2-7, GINS) and stimulate the activities of DNA polymerase (POL) α,δ, and ε, in progressing the replication fork. These experimental studies confirm the existence of synergy between the above involved factors with TIMELESS. In colorectal cancer cells treated with ETC-1922159, 7 UNEXPLORED COMBINATORIAL HYPOTHESES POL members w.r.t TIMELESS POL-A1/D1/E2/E3/G2 TIMELESS POLR members w.r.t TIMELESS POLR-3K/1A/1E/1B/3E/2D/2G/1C TIMELESS Table 6: 2nd order combinatorial hypotheses between TIMELESS and POL/POLR members. these MCM/GINS members, and TIMELESS, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these MCM/GINS members along with TIMELESS. Table 7 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 8 generated from analysis of the ranks in table 7. The table 7 shows rankings of MCM/GINS members w.r.t TIMELESS. MCM5 - TIMELESS shows low ranking of 147 (laplace), 770 (linear) and 926 (rbf). MCM3 - TIMELESS shows low ranking of 166 (laplace), 741 (linear) amd 1033 (rbf). MCM2 - TIMELESS shows low ranking of 311 (laplace), 130 (linear) and 1438 (rbf). MCM4 - TIMELESS shows low ranking of 668 (laplace) and 305 (linear). MCMDC2 - TIMELESS shows low ranking of 718 (laplace), 1476 (linear) and 1508 (rbf). MCM8 - TIMELESS shows low ranking of 1085 (laplace), 13 (linear) and 1323 (rbf). MCM7 - TIMELESS shows low ranking of 1363 (laplace) and 461 (rbf). GINS4 - TIMELESS shows low ranking of 343 (laplace) and 186 (rbf). GINS1 - TIMELESS shows low ranking of 893 (laplace) and 865 (linear). GINS3 - TIMELESS shows low ranking of 939 (laplace), 401 (linear) and 274 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, MCM6, MCM10 and GINS2 showed high ranking and might not be synergistically working with TIMELESS, before treatment. One can also interpret the results of the table 7 graphically, with the following influences - •MCM members w.r.t TIMELESS with TIMELESS −>MCM-5/3/2/4/DC2/8/7 and •GINS members w.r.t TIMELESS with TIMELESS −>GINS-4/1/3. 2.1.5. TIMELESS - FBX/RNF/H2A The mammalian FBXL10-RNF68-RNF2 ubiquitin ligase complex (FRRUC) monoubiquitylates H2A to repress transcription in unstressed cells. Rona et al. [21] found that the FRRUC was recruited to sites of DNA damage in a PARP1and TIMELESSdependent manner to promote mono-ubiquitylation of H2A, a local decrease of H2A levels, and an increase of H2A.Z incorporation. Both the FRRUC and H2A.Z promoted transcriptional repression, double strand break signaling, and homologous recombination repair (HRR). These experimental studies confirm the existence of synergy be8 RANKING MCM MEMBERS VS TIMELESS RANKING OF MCM MEMBERS W.R.TTIMELESS laplace linear rbf MCM5 - TIMELESS 147 770 926 MCM3 - TIMELESS 166 741 1033 MCM2 - TIMELESS 311 130 1438 MCM4 - TIMELESS 668 305 1598 MCMDC2 - TIMELESS 718 1476 1508 MCM8 - TIMELESS 1085 13 1323 MCM7 - TIMELESS 1363 1839 461 MCM6 - TIMELESS 1570 2448 1404 MCM10 - TIMELESS 2636 1238 2008 RANKING GINS MEMBERS VS TIMELESS RANKING OF GINS MEMBERS W.R.TTIMELESS laplace linear rbf GINS4 - TIMELESS 343 2088 186 GINS1 - TIMELESS 893 865 2160 GINS3 - TIMELESS 939 401 274 GINS2 - TIMELESS 2338 165 2699 Table 7: 2nd order interaction ranking between TIMELESS VS POL/POLR members. UNEXPLORED COMBINATORIAL HYPOTHESES MCM members w.r.t TIMELESS MCM-5/3/2/4/DC2/8/7 TIMELESS GINS members w.r.t TIMELESS GINS-4/1/3 TIMELESS Table 8: 2nd order combinatorial hypotheses between TIMELESS and POL/POLR members. 9 UNEXPLORED COMBINATORIAL HYPOTHESES PDE members w.r.t TIMELESS PDE-7A TIMELESS Table 18: 2nd order combinatorial hypotheses between TIMELESS and PDE members. 3. Conclusion Presented here are a range of multiple synergistic TIMELESS 2nd order combinations that were ranked via a machine learning based search engine. Via majority voting across the ranking methods, it was possible to find plausible unexplored synergistic combinations of TIMELESS-X that might be prevalent in CRC cells after treatment with ETC-1922159 drug. Conflict of interest There are no conflicts to declare. Author’s contributions Concept, design, in silico implementation - SS. Analysis and interpretation of results - SS. Manuscript writing - SS. Manuscript revision - SS. Approval of manuscript - SS Acknowledgements Special thanks to Mrs. Rita Sinha and late Mr. Prabhat Sinha for supporting the author financially, without which this work could not have been made possible. Source of Data Data used in this research work was released in a publication in Madan et al. [26]. 4. References References [1] A. Sehgal, J. L. Price, B. Man, M. W. Young, Loss of circadian behavioral rhythms and per rna oscillations in the drosophila mutant timeless, Science 263 (1994) 1603–1606. 16 [2] A. L. Gotter, T. Manganaro, D. R. Weaver, L. F. Kolakowski, B. Possidente, S. Sriram, D. T. MacLaughlin, S. M. Reppert, A time-less function for mouse timeless, Nature neuroscience 3 (2000) 755–756. [3] A. L. Gotter, A timeless debate: resolving tim’s noncircadian roles with possible clock function, Neuroreport 17 (2006) 1229–1233. [4] Y. D. Cai, J. C. Chiu, Timeless in animal circadian clocks and beyond, The FEBS journal 289 (2022) 6559–6575. [5] A. L. Gotter, C. Suppa, B. S. Emanuel, Mammalian timeless and tipin are evolutionarily conserved replication fork-associated factors, Journal of molecular biology 366 (2007) 36–52. [6] W.-H. Cho, Y.-H. Kang, Y.-Y. An, I. Tappin, J. Hurwitz, J.-K. Lee, Human tim-tipin complex affects the biochemical properties of the replicative dna helicase and dna polymerases, Proceedings of the National Academy of Sciences 110 (2013) 2523–2527. [7] X. Yang, P. A. Wood, W. J. Hrushesky, Mammalian timeless is required for atm-dependent chk2 activation and g2/m checkpoint control, Journal of Biological Chemistry 285 (2010) 3030–3034. [8] A. N. Blackford, S. P. Jackson, Atm, atr, and dna-pk: The trinity at the heart of the dna damage response, Molecular cell 66 (2017) 801–817. [9] S. Xie, O. Mortusewicz, H. T. Ma, P. Herr, R. Y. Poon, T. Helleday, C. Qian, Timeless interacts with parp-1 to promote homologous recombination repair, Molecular cell 60 (2015) 163–176. [10] X. Rao, L. Lin, Circadian clock as a possible control point in colorectal cancer progression, International journal of oncology 61 (2022) 1–12. [11] S. Sinha, Machine learning ranking of plausible (un) explored synergistic gene combinations using sensitivity indices of time series measurements of wnt signaling pathway, Integrative Biology 16 (2024) zyae020. [12] S. Sinha, Sensitivity analysis based ranking reveals unknown biological hypotheses for down regulated genes in time buffer during administration of porcn-wnt inhibitor etc-1922159 in crc, bioRxiv (2017) 180927. [13] T. Joachims, Training linear svms in linear time, in: Proceedings of the 12th ACM SIGKDD international conference on Knowledge discovery and data mining, ACM, 2006, pp. 217–226. [14] X. Liu, J. Sun, Z. Ling, T. Dong, Relationship between circadian rhythm-related genes and extracellular matrix: implications for sleep deprivation, Sleep and Breathing 28 (2024) 697–705. [15] L. Chi, Y. Zou, L. Qin, W. Ma, Y. Hao, Y. Tang, R. Luo, Z. Wu, Timeless contributes to the progression of breast cancer through activation of myc, Breast Cancer Research 19 (2017) 1–10. [16] M. Cao, Y. Wang, Y. Xiao, D. Zheng, C. Zhi, X. Xia, X. Yuan, Activation of the clock gene timeless by h3k27 acetylation promotes colorectal cancer tumorigenesis by binding to myosin-9, Journal of Experimental & Clinical Cancer Research 40 (2021) 162. [17] Y. Akamatsu, T. Kobayashi, The human rna polymerase i transcription terminator complex acts as a replication fork barrier that coordinates the progress of replication with rrna transcription activity, Molecular and cellular biology (2015). [18] Y. Chen, Z. Han, L. Zhang, C. Gao, J. Wei, X. Yang, Y. Han, Y. Li, C. Zhang, Y. Wei, et al., Timeless promotes reprogramming of glucose metabolism in oral squamous cell carcinoma, Journal of Translational Medicine 22 (2024) 21. [19] Y. Xia, R. Fujisawa, T. D. Deegan, R. Sonneville, K. P. Labib, Timeless-tipin and ubxn-3 promote replisome disassembly during dna replication termination in caenorhabditis elegans, The EMBO Journal 40 (2021) e108053. [20] L. K. Lerner, S. Holzer, M. L. Kilkenny, S. ˇ Svikovi´ c, P. Murat, D. Schiavone, C. B. Eldridge, A. Bittleston, J. D. Maman, D. Branzei, et al., Timeless couples g-quadruplex detection with processing by ddx 11 helicase during dna replication, The EMBO journal 39 (2020) e104185. [21] G. Rona, D. Roberti, Y. Yin, J. K. Pagan, H. Homer, E. Sassani, A. Zeke, L. Busino, E. Rothenberg, M. Pagano, Parp1-dependent recruitment of the fbxl10-rnf68-rnf2 ubiquitin ligase to sites of dna damage controls h2a. z loading, Elife 7 (2018) e38771. 17 [22] Z. Wang, S. He, L. Xin, Y. Zhou, L. Zhao, F. Wang, Hmgb1-mediated transcriptional activation of circadian gene timeless contributes to endometrial cancer progression through wnt-β-catenin pathway, Cellular Signalling 116 (2024) 111045. [23] Y. Zou, X. Lin, J. Bu, Z. Lin, Y. Chen, Y. Qiu, H. Mo, Y. Tang, W. Fang, Z. Wu, Timeless-stimulated mir-5188foxo1/β-catenin-c-jun feedback loop promotes stemness via ubiquitination of β-catenin in breast cancer, Molecular Therapy 28 (2020) 313–327. [24] J. Saldanha, J. Rageul, J. A. Patel, A. L. Phi, N. Lo, J. J. Park, H. Kim, The timeless and parp1 interaction suppresses replication-associated dna gap accumulation, Nucleic Acids Research (2024) gkae445. [25] E. Barrio-Alonso, P. J. Lituma, M. J. Notaras, R. Albero, Y. Bouchekioua, N. Wayland, I. N. Stankovic, T. Jain, S. Gao, D. P. Calderon, et al., Circadian protein timeless regulates synaptic function and memory by modulating camp signaling, Cell reports 42 (2023). [26] B. Madan, Z. Ke, N. Harmston, S. Y. Ho, A. Frois, J. Alam, D. A. Jeyaraj, V. Pendharkar, K. Ghosh, I. H. Virshup, et al., Wnt addiction of genetically defined cancers reversed by porcn inhibition, Oncogene 35 (2016) 2197. 18