Machine Learning Discoveries of GINS1-X Synergy in ETC-1922159 Treated Colorectal Cancer Cells

Machine learning discoveries of GINS1-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 GINS1 (PSF1 homolog) forms a part of complex called GINS, named after numbers 5-1-2-3 (go-ichi-ni-san in Japanese) apropos the 4 protein subunits of the complex, namely, SLD5, PSF1, PSF2, and PSF3. It is a subgroup of the CDC45-MCM-(2 to 7)- GINS (CMG) complex, the enzyme that unwinds double-stranded DNA at replication forks. GINS1 has been found to be overexpressed in multiple cancers. In colorectal cancer (CRC) cells treated with ETC-1922159, GISN1 was found to be down regulated along with other genes. A recently developed search engine ranked combinations of GISN1-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 GINS1-X combinations might be working synergistically in CRC. In this research work, I cover combinations of GINS1 with possible members of, origin recognition complex (ORC), minichromosome maintenance complex (MCM), DNAdirected DNA polymerases (POL), DNA-dependent RNA polymerase (POLR), topoisomerases (TOP), ubiquitin specific peptidase (USP), Fanconi anemia complementation group (FANC), forkhead box (FOX) and notch receptor (NOTCH) family. Keywords: GISN1, Porcupine inhibitor ETC-1922159, Sensitivity analysis, Machine learning, Colorectal cancer. 1. Introduction 1.1. Eukaryotic DNA replication machinery Eukaryotic DNA replication restricts DNA replication once per cell cycle. The replication machinery coordinates many proteins at the site of replication, forming the repliIML dicoveries of GINS1-X synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at the first Wnt Gordon Research Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA.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 11, 2025 some (Leman and Noguchi [1]). The replication process is a two step process in a given cell cycle - •during the G1-phase, there is loading of the replicative helicase, MCM-(2 to 7), onto chromatin by the ORC complex, where CDC6, CDT1, and MCM- (2 to 7) assemble at replication origins, and form pre-replicative complexes (pre-RCs). •In the subsequent S-phase, kinase activition allows fork establishment through CMG complex formation. Considering the architecture, Li and O’Donnell [2] and Parker et al. [3] elucidate that CMG complex surrounds the DNA at origins in two steps - •in the G1 phase, two MCM-(2 to 7) rings are assembled around duplex DNA at the origin, by the concerted actions of the ORC, CDC6, and CDT1, thus forming a double hexamer. •, in S phase, CDC45 and GINS are assembled onto each MCM-(2 to 7) ring for production of two CMGs that ultimately form two replication forks that travel in opposite directions. Further, EM studies have revealed a 20-subunit core replisome with the leading POLε and lagging POLα, primase on opposite faces of CMG, thus forming a fundamentally asymmetric replisome architecture. 1.2. GINS complex and cancer Via biochemical analysis it has been shown that GINS works at the heart of the replication apparatus as a component of the CMG complex which facilitates the unwinding of duplex DNA ahead of the moving replication fork (MacNeill [4] and MacNeill [5]). Xiang et al. [6] provide a comprehensive review regarding insight into the mechanisms and dynamics of how CMG is regulated during its assembly and activation in mammalian genomes, and how errors in CMG regulation due to oncogenic changes promote tumorigenesis. Seo and Kang [7] provide a review that emphasizes on molecular function of the CMG during replication and its relevance to cancers, based on published literature. They indicate that the CMG complex can be a potential target for a treatment of cancer and the feasibility of this replicative helicase as a therapeutic target has been tested recently. In breast cancer cells, Nakahara et al. [8] found that GINS1 (PSF1) was overexpressed and knockdown of PSF1 expression slowed the growth of breast cancer cell lines by delaying DNA replication. Further, reduced PSF1 expression also inhibited anchorage-independent growth in breast cancer cell lines. Similar results regarding role of PSF1 in hepatocellular carcinoma tissues, was reported by Zhou et al. [9]. Chromosomal instability (CIN) is a trademark of cancer and comprises structural (S-CIN) and whole chromosomal (W-CIN). In human cancers, recent works have indicated that replication stress (RS), known to contribute to S-CIN, also affects mitotic chromosome segregation. B¨ ohly et al. [10] showed that RS-induced increased origin firing was sufficient to trigger W-CIN and discovered that overexpression of these origin firing genes, like GINS1 and CDC45, correlated with W-CIN. In another study regarding hepatocellular carcinoma, Liang et al. [11] found that ZEB1 was a regulator of GINS1 induced epithelial mesenchymal transition (EMT), and GINS1 promoted EMT and tumor metastasis through β-catenin signaling. Finally, through immunohistochemistry studies in bladder cancer (BC) tissues, Fu et al. [12] demonstrate that increase in GINS1 expression. They show that GINS1 silencing arrested the cell cycle at the phase of G0/G1 phase, which inhibited BC cell growth both in vitro and in vivo, 2 and GINS1 knockdown also hindered the AKT/mTOR pathway. In colorectal cancer (CRC) cells treated with ETC-1922159, GISN1 was found to be down regulated along with other genes. GISN1 works in tandem with multiple components and some combinations of GISN1 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. 1.3. Combinatorial search problem and a possible solution In a recently published work Sinha [13], 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 [14]. The work uses SVM package by Joachims [15] 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. GISN1 related synergies 2.1.1. GINS1 - ORC Coulombe et al. [16] analysed the pre-RC proteomic interactome in human cells and found ORC-ubiquitin-ligase-1 (OBI1) to be associated with the ORC complex. OBI1 silencing resulted in defective origin firing, as shown by reduced CMG formation and OBI1 catalysed the multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase. Further, to determine which step of replication origin licensing or firing wass regulated by OBI1, they analysed the expression and chromatin association of key replication factors. Depletion of ORC1 lead to impaired recruitment of the MCM-(2 to 7) complex to chromatin, which confirmed defective origin licensing and as a consequence of this, chromatin binding of factors involved in the subsequent firing step, such as PSF1/GINS1 was reduced. Also, OBI1 knockdown impaired chromatin recruitment of replisome component PSF1/GINS1 by approximately 50%. These experiments confirm a possible synergy between ORC and GINS1. In colorectal cancer cells treated with ETC-1922159, ORC family members and GINS1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these ORC members along with GINS1. 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 ORC members w.r.t GINS1. ORC1 - GINS1 shows low ranking of 89 (laplace) and 224 (linear). ORC6 - GINS1 shows low ranking of 715 (laplace) and 419 (linear). ORC5 - GINS1 shows low ranking of 1451 (laplace) and 3 670 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, ORC2 and ORC3 showed high ranking with GINS1, thus indicating that they might not be working synergistically with GINS1, before the drug treatment. RANKING ORC FAMILY VS GINS1 RANKING OF ORC FAMILY W.R.TGINS1 laplace linear rbf ORC1 - GINS1 89 224 2514 ORC6 - GINS1 715 419 2320 ORC5 - GINS1 1451 2345 670 ORC2 - GINS1 2009 2147 178 ORC3 - GINS1 2338 1795 2154 Table 1: 2nd order interaction ranking between GINS1 VS ORC members. One can also interpret the results of the table 1 graphically, with the following influences - •ORC members w.r.t GINS1 with GINS1 −>ORC-1/6/5. UNEXPLORED COMBINATORIAL HYPOTHESES ORC members w.r.t GINS1 ORC-1/6/5 GINS1 Table 2: 2nd order combinatorial hypotheses between GINS1 and ORC members. 2.1.2. GINS1 - MCM As mentioned in one of the previous paragraphs, MCM family interacts with GINS in the CMG-complex formation. Brosh Jr and Trakselis [17] review the role of MCM10 in regulation of fork progression and regression and state that MCM10 crosslinks significantly with two members (PSF-1/2) i.e GINS-1/2, CDC45, and MCM-2/5/6 on the N-face. In colorectal cancer cells treated with ETC-1922159, MCM family members and GINS1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these MCM members along with GINS1. 4 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 MCM members w.r.t GINS1. MCM4 - GINS1 shows low ranking of 17 (laplace) and 63 (linear). MCM3 - GINS1 shows low ranking of 35 (laplace) and 90 (linear). MCM2 - GINS1 shows low ranking of 41 (laplace) and 143 (linear). MCM10 - GINS1 shows low ranking of 114 (laplace) and 139 (linear). MCM6 - GINS1 shows low ranking of 156 (laplace) and 231 (linear). MCM5 - GINS1 shows low ranking of 210 (laplace) and 509 (linear). MCM8 - GINS1 shows low ranking of 243 (laplace) and 849 (linear). MCM7 - GINS1 shows low ranking of 1383 (laplace), 184 (linear) and 34 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, MCMDC2 showed high ranking with GINS1, thus indicating that they might not be working synergistically with GINS1, before the drug treatment. RANKING MCM FAMILY VS GINS1 RANKING OF MCM FAMILY W.R.TGINS1 laplace linear rbf MCM4 - GINS1 17 63 2677 MCM3 - GINS1 35 90 2629 MCM2 - GINS1 41 143 2701 MCM10 - GINS1 114 139 2742 MCM6 - GINS1 156 231 2427 MCM5 - GINS1 210 509 2212 MCM8 - GINS1 243 849 2024 MCM7 - GINS1 1383 184 34 MCMDC2 - GINS1 1707 1454 2380 Table 3: 2nd order interaction ranking between GINS1 VS MCM members. One can also interpret the results of the table 3 graphically, with the following influences - •MCM members w.r.t GINS1 with GINS1 −>MCM-4/3/2/10/6/5/8/7. 2.1.3. GINS1 - POL/POLR Bell and Labib [18] elucidate that DNA synthesis is carried out by three multisubunit DNA-directed DNA polymerases, POLα, POLδ), and POLε). Further, POLεand 5 UNEXPLORED COMBINATORIAL HYPOTHESES MCM members w.r.t GINS1 MCM-4/3/2/10/6/5/8/7 GINS1 Table 4: 2nd order combinatorial hypotheses between GINS1 and MCM members. POLαare connected to the CMG helicase via GINS. Later, Denkiewicz-Kruk et al. [19] concluded that the impaired interaction between GINS and POLεrequired involvement of error-prone POLζ, and increased participation of recombination as a rescue mechanism for recovery of impaired replication forks. In colorectal cancer cells treated with ETC-1922159, POL family members and GINS1, were found to be down regulated and their regulation was recorded independently. Additionally, DNA-dependent RNA polymerase (POLR) family members were also observed as down regulated. I was able to rank 2nd order combination of these POL/POLR members along with GINS1. 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 members w.r.t GINS1. POLε2 (or E2) - GINS1 shows low ranking of 87 (laplace) and 393 (linear). POLθ(or Q) - GINS1 shows low ranking of 203 (laplace) and 102 (linear). POLα1 (or A1) - GINS1 shows low ranking of 377 (laplace), 412 (linear) and 1100 (rbf). POLδ1 (or D1) - GINS1 shows low ranking of 407 (laplace), 691 (linear) and 1255 (rbf). POLδ2 (or D2) - GINS1 shows low ranking of 417 (laplace) and 323 (rbf). POLγ2 (or G2) - GINS1 shows low ranking of 918 (laplace) and 1129 (linear). POLε3 (or E3) - GINS1 shows low ranking of 1429 (laplace), 1265 (linear) and 810 (rbf). POLα2 (or A2) - GINS1 shows low ranking of 1295 (linear) and 811 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, POLβ(or B) showed high ranking with GINS1, thus indicating that they might not be working synergistically with GINS1, before the drug treatment. The table 5 also shows rankings of POLR members w.r.t GINS1. POLR-3K - GINS1 shows low ranking of 459 (laplace), 993 (linear) and 222 (rbf). POLR-1C - GINS1 shows low ranking of 529 (laplace) and 806 (linear). POLR-1E - GINS1 shows low ranking of 670 (laplace), 588 (linear) and 888 (rbf). POLR-1B - GINS1 shows low ranking of 889 (laplace), 1019 (linear) and 280 (rbf). POLR-3E - GINS1 shows low ranking of 1209 (laplace) and 720 (rbf). POLR-2G - GINS1 shows low ranking of 1312 (laplace) and 1000 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, POLR-2K, POLR-3A, POLR-2F, POLR-1A, POLR-2D and POLR-1D showed high ranking with GINS1, thus indicating that they might not be working synergistically with GINS1, before the drug treatment. One can also interpret the results of the table 5 graphically, with the following influences - •POL members w.r.t GINS1 with GINS1 −>POL-ε2 (or E2) / θ(or Q) / α1 (or A1) / δ1 (or D1) / δ2 (or D2) / γ2 (or G2) / ε3 (or E3) / α2 (or A2), and GINS1 −>POLR-3K/1C/1E/1B/3E/2G. 6 RANKING POL FAMILY VS GINS1 RANKING OF POL FAMILY W.R.TGINS1 laplace linear rbf POLε2 (or E2) - GINS1 87 393 1829 POLθ(or Q) - GINS1 203 102 2743 POLα1 (or A1) - GINS1 377 412 1100 POLδ1 (or D1) - GINS1 407 691 1255 POLδ2 (or D2) - GINS1 417 1766 323 POLγ2 (or G2) - GINS1 918 1129 1955 POLε3 (or E3) - GINS1 1429 1265 810 POLα2 (or A2) - GINS1 2207 1295 811 POLβ(or B) - GINS1 2367 2046 1838 RANKING POLR FAMILY VS GINS1 RANKING OF POLR FAMILY W.R.TGINS1 laplace linear rbf POLR-3K - GINS1 459 993 222 POLR-1C - GINS1 529 806 2252 POLR-1E - GINS1 670 588 888 POLR-1B - GINS1 889 1019 280 POLR-3E - GINS1 1209 2203 720 POLR-2G - GINS1 1312 1699 1000 POLR-2K - GINS1 2028 2559 1521 POLR-3A - GINS1 2230 1772 1573 POLR-2F - GINS1 2309 2468 1396 POLR-1A - GINS1 2317 1157 1625 POLR-2D - GINS1 2425 1719 792 POLR-1D - GINS1 2612 2732 957 Table 5: 2nd order interaction ranking between GINS1 VS POL members. 7 UNEXPLORED COMBINATORIAL HYPOTHESES POL members w.r.t GINS1 POL-ε2 (or E2) / θ(or Q) / α1 (or A1) / δ1 (or D1) / δ2 (or D2) / γ2 (or G2) / ε3 (or E3) / α2 (or A2) GINS1 POLR members w.r.t GINS1 POLR-3K/1C/1E/1B/3E/2G GINS1 Table 6: 2nd order combinatorial hypotheses between GINS1 and POL members. 2.1.4. GINS1 - TOP Yang et al. [20] revealed through functional assays that GINS1 aggravated glioma malignant phenotypes in vitro and in vivo. Their study identified that GINS1 interacted with TOP2A and promoted glioma cell proliferation and migration through USP15mediated deubiquitination of TOP2A protein. Day et al. [21] demostrated through biochemical and structural studies that GINS interacted with TOPBP1 through two separate binding surfaces, 1.involving a conserved amino acids in the TOPBP1-GINI region and 2.a surface on TOPBP1-BRCT4. These two surfaces bind to opposite ends of the A domain of the GINS subunit PSF1/GINS1. In colorectal cancer cells treated with ETC-1922159, TOP family members and GINS1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these TOP members along with GINS1. 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 TOP members w.r.t GINS1. TOP2A - GINS1 shows low ranking of 25 (laplace) and 896 (linear). TOP1MT - GINS1 shows low ranking of 366 (laplace) and 489 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, TOPBP1 and TOPB2 showed high ranking with GINS1, thus indicating that they might not be working synergistically with GINS1, before the drug treatment. Also, as a synergy between TOP2A and GINS1, through USP15 was demonstrated in one of the forgoing literature, a ranking of the recorded members of USP family with GINS1 was also tabulated. The table 7 shows rankings of USP members w.r.t GINS1, also. USP13 - GINS1 shows low ranking of 328 (laplace) and 75 (linear). USP28 - GINS1 shows low ranking of 1283 (laplace), 750 (linear) and 443 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, USP36, USP1, USP39 and USP10 showed high ranking with GINS1, thus indicating that they might not be working synergistically with GINS1, before the drug treatment. One can also interpret the results of the table 7 graphically, with the following influences - •TOP members w.r.t GINS1 with GINS1 −>TOP-2A/1MT, and •USP 8 RANKING TOP FAMILY VS GINS1 RANKING OF TOP FAMILY W.R.TGINS1 laplace linear rbf TOP2A - GINS1 25 896 2393 TOP1MT - GINS1 366 489 1827 TOPBP1 - GINS1 1833 2405 1433 TOP2B - GINS1 1885 2177 1009 RANKING USP FAMILY VS GINS1 RANKING OF USP FAMILY W.R.TGINS1 laplace linear rbf USP13 - GINS1 328 75 2737 USP28 - GINS1 1283 750 443 USP36 - GINS1 1588 2080 1360 USP1 - GINS1 1682 1887 1929 USP39 - GINS1 2454 2122 175 USP10 - GINS1 2500 2573 1636 Table 7: 2nd order interaction ranking between GINS1 VS TOP members. members w.r.t GINS1 with GINS1 −>USP-13/28. 2.1.5. GINS1 - FANC Although the CMG complex implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Huang et al. [22] report that this restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM. Following this, FANCM binds the replisome complex, with associated release of the GINS proteins. The results indicated that, there was a FANCM/FANCD2-dependent absence of GINS proteins from replisomes. Thus there might be a synergy between the FANC family and GINS. In colorectal cancer cells treated with ETC-1922159, FANC family mem9