Machine learning discoveries of TOP2A-X synergy in ETC-1922159 treated colorectal cancer cells

Machine learning discoveries of TOP2A-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 DNA topoisomerase II α(TOP2A) belongs to the family of topoisomerases, which regulates the (un)winding of the DNA due to its double helical structure. The main function of TOP2A is to relieve the topological stress during DNA transcription, assist in separation of chromatids and condensation of chromosomes. In colorectal cancer (CRC) cells treated with ETC-1922159, TOP2A was found to be down regulated along with other genes. A recently developed search engine ranked combinations of TOP2A-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 TOP2A-X combinations might be working synergistically in CRC. In this research work, I cover combinations of TOP2A with WNT, nucleolar and spindle associated protein (NUSAP), Wolf-Hirschhorn syndrome candidate (WHSC), rhophilin Rho GTPase binding protein antisense RNA (RHPN-AS1), AT-rich interaction domain (ARID), DNA topoisomerase II binding protein (TOPBP), ERCC excision repair (ERCC), enhancer of zeste polycomb repressive complex 2 subunit (EZH), cyclin dependent kinase (CDK), origin recognition complex subunit (ORC), interleukin (IL), ubiquitin specific peptidase (USP), RAD54, zinc finger protein (ZNF), high mobility group box (HMGB), E2F transcription factor (E2F), GINS complex subunit (GINS), minichromosome maintenance (MCM), budding uninhibited by benzimidazoles mitotic checkpoint (BUB), DEAD/H-box helicase (DDX), H2A histone family member (H2A) and structural maintenance of chromosomes (SMC) family. Keywords: TOP2A, Porcupine inhibitor ETC-1922159, Sensitivity analysis, Colorectal cancer. IML dicoveries of TOP2A-X synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at the first Wnt Gordon Research Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA.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 8, 2025 1. Introduction 1.1. Topoisomerases Becuase of the double-helical structure of the DNA, coils are often formed during relication, transcription and translation. This leads to building up of tension in the DNA structure. To maintain the relaxed topology of the DNA, topoisomerases act as essential proteins providing the required functionality. Wang [1] first discovered the existance of topoisomerases. In a review, McKie et al. [2] describe that topoisomerases are classified into different types depending on whether they catalyse the formation and religation of single-stranded (ss) or double-stranded (ds)DNA breaks. A detailed description of the structure and function of DNA topoisomerases can be found in Champoux [3]. 1.2. DNA topoisomerase II The ATP-dependent Type II which requires Mg2+, was identified in yeast by Goto and Wang [4]. Later, Adachi et al. [5] isolated the first conditional mutation in the mouse TOP2A. Watt and Hickson [6] particularly discuss the structure and function of type II. Of the two subtypes of type II, TOP2A has been found to be highly expressed in various malignancies, as reported by Zhou et al. [7]. In colon cancer, Zhang et al. [8] showed that the proliferation and invasion was suppressed by knockdown of TOP2A. Further, analysis by Carvalho et al. [9] revealed that TOP2A inhibitors were candidate drugs for rectal cancer treatment, based on drug repositioning. In colorectal cancer (CRC) cells treated with ETC-1922159, TOP2A was found to be down regulated along with other genes. TOP2A works in tandem with multiple components and some combinations of TOP2A 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 [10], 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 [11]. The work uses SVM package by Joachims [12] in https://www.cs.cornell.edu/people/tj/svm_light/ svm_rank.html. I use the adaptation to rank 2nd order gene combinations. 2 2. Results & Discussion 2.1. TOP2A related synergies 2.1.1. TOP2A - WNT10B / NUSAP1 / WHSC1 / RHPN1-AS1 / ARID5B / TOPBP1 In non-small cell lung cancer, Wu et al. [13]’s experimental findings showed tha WNT3A, c-MYC, and β-catenin, expression levels were elevated when TOP2A was overexpressed and vice versa during TOP2A knockdown. Hu et al. [14] revealed that NUSAP1 gene silencing induced apoptosis in human glioblastoma, through the downregulation of the downstream molecule TOP2A. In hepatocellular carcinoma, Bao et al. [15] showed that activating transcription factor 2 (ATF2), bound to and promoted the transcription of WHSC1, which further increased the expression of TOP2A by inducing the dimethylation of histone H3 lysine 36 (H3K36me2). Zhou et al. [16] demostrated that RHPN1-AS1 negatively regulated miR-485-5p which lead to promotion of the TOP2A expression in ovarian cancer cells. Tsai et al. [17] show that ARID1A loss causes DNA replication stress associated with R-loops and transcription-replication conflicts in human cells. They show a model in which deletion of ARID1A reduces BRG1/BRM-associated factor (BAF) binding, thus failing to recruit TOP2A, which leads to accumulation of R-loops. Broderick et al. [18] identify TOPBP1 as an interactor of TOP2A, and show that it is required for TOP2A recruitment to resolve ultra-fine anaphase bridges (UFBs) during mitosis. These combinations with TOP2A which have been experimentally tested show a combinatorial synergy in various cases. In colorectal cancer cells treated with ETC-1922159, these components taken individually and TOP2A, 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 TOP2A. 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 individual members w.r.t TOP2A. WNT10B - TOP2A shows low ranking of 181 (laplace), 153 (linear) and 213 (rbf). NUSAP1 - TOP2A shows low ranking of 87 (laplace), 174 (linear) and 126 (rbf). WHSC1 - TOP2A shows low ranking of 444 (laplace), 711 (linear) and 541 (rbf). RHPN1-AS1 - TOP2A shows low ranking of 339 (laplace), 1381 (linear) and 371 (rbf). ARID5B - TOP2A shows low ranking of 464 (laplace), 855 (linear) and 355 (rbf). TOPBP1 - TOP2A shows low ranking of 702 (laplace), 1430 (linear) and 1032 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. One can also interpret the results of the table 1 graphically, with the following influences - •individual members w.r.t TOP2A with TOP2A −>WNT10B / NUSAP1 / WHSC1 / RHPN1-AS1 / ARID5B / TOPBP1 . 2.1.2. TOP2A - ERCC Polo-like kinase 1-interacting checkpoint helicase (PICH), also known as excision repair cross-complementation group 6 like (ERCC6L), is a substrate if PLK1-interacting checkpoint helicase. Overexpression of PICH/ERCC6L is related to the proliferation 3 RANKING INDIVIDUAL MEMBERS VS TOP2A RANKING OF INDIVIDUAL MEMBERS W.R.TTOP2A laplace linear rbf WNT10B - TOP2A 181 153 213 NUSAP1 - TOP2A 87 174 126 WHSC1 - TOP2A 444 711 541 RHPN1-AS1 - TOP2A 339 1381 371 ARID5B - TOP2A 464 855 355 TOPBP1 - TOP2A 702 1430 1032 Table 1: 2nd order interaction ranking between TOP2A VS INDIVIDUAL members. UNEXPLORED COMBINATORIAL HYPOTHESES Individual members w.r.t TOP2A WNT10B / NUSAP1 / WHSC1 / RHPN1-AS1 / ARID5B / TOPBP1 TOP2A Table 2: 2nd order combinatorial hypotheses between TOP2A and individual members. of tumors and Li et al. [19] hypothesis that PICH can maintain genomic stability by regulating appropriate chromosome structure, ensuring proper chromosome segregation, and facilitating replication fork reversal, via PICH-PLK1-TOP2A axis. In colorectal cancer cells treated with ETC-1922159, ERCC family members and TOP2A, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these ERCC member along with TOP2A. 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 individual members w.r.t TOP2A. ERCC6L - TOP2A shows low ranking of 40 (laplace), 24 (linear) and 24 (rbf). ERCC8 - TOP2A shows low ranking of 870 (laplace) and 903 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. One can also interpret the results of the table 3 graphically, with the following influences - •ERCC family w.r.t TOP2A with TOP2A −>ERCC-6L/8. 2.1.3. TOP2A - EZH/CDK In glioblastoma, Freitag et al. [20] show that EZH2 is overexpressed and combined EZH2-CDK4/6 inhibition increases antitumor activity against glioblastoma, by boosting cell death and cell stress, reverses stemness characteristics, disrupts endoplasmatic reticulum-mitochondrial homeostasis and reduces the invasion capability in GBM spheroids. Mechanistically, this was due to transcriptional changes in several genes, of which 4 RANKING ERCC FAMILY VS TOP2A RANKING OF ERCC FAMILY W.R.TTOP2A laplace linear rbf ERCC6L - TOP2A 40 24 24 ERCC8 - TOP2A 870 1714 903 Table 3: 2nd order interaction ranking between TOP2A VS ERCC family. UNEXPLORED COMBINATORIAL HYPOTHESES ERCC family w.r.t TOP2A ERCC-6L/8 TOP2A Table 4: 2nd order combinatorial hypotheses between TOP2A and ERCC family. TOP2A is one of them. Slightly unrelated, but during retinal development in zebrafish Jin et al. [21] suggest that to regulate S phase entry, CDK1 interacts with cyclin A2 through phosphorylation of TOP2A. In colorectal cancer cells treated with ETC1922159, EZH/CDK family members and TOP2A, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these EZH/CDK member along with TOP2A. 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 individual members w.r.t TOP2A. EZH2 - TOP2A shows low ranking of 1328 (laplace), 1463 (linear) and 1106 (rbf). CDK1 - TOP2A shows low ranking of 57 (laplace), 51 (linear) and 70 (rbf). CDK5RAP1 - TOP2A shows low ranking of 789 (laplace), 865 (linear) and 996 (rbf). CDK4 - TOP2A shows low ranking of 1277 (laplace), 948 (linear) and 1476 (rbf). CDK20 - TOP2A shows low ranking of 1370 (laplace), 1021 (linear) and 1421 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, CDK5RAP2 and CDK6 showed high ranking with TOP2A, thus indicating that they might not be working synergistically with TOP2A, before the drug treatment. One can also interpret the results of the table 5 graphically, with the following influences - •EZH-CDK family w.r.t TOP2A with TOP2A −>EZH2 and TOP2A −>CDK-1/5RAP1/4/20. 5 RANKING EZH-CDK FAMILY VS TOP2A RANKING OF EZH-CDK FAMILY W.R.TTOP2A laplace linear rbf EZH2 - TOP2A 1328 1463 1106 CDK1 - TOP2A 57 51 70 CDK5RAP1 - TOP2A 789 865 996 CDK4 - TOP2A 1277 948 1476 CDK20 - TOP2A 1370 1021 1421 CDK5RAP2 - TOP2A 1935 1717 2130 CDK6 - TOP2A 2544 1511 2151 Table 5: 2nd order interaction ranking between TOP2A VS EZH-CDK family. UNEXPLORED COMBINATORIAL HYPOTHESES EZH/CDK family w.r.t TOP2A EZH2 TOP2A CDK-1/5RAP1/4/20 TOP2A Table 6: 2nd order combinatorial hypotheses between TOP2A and EZH-CDK family. 2.1.4. TOP2A - ORC In primary and immortalized glioma cells, Yang et al. [22] showed that depleting/knockout of ORC6 decreased cell viability and proliferation, disrupted cell cycle progression and mobility, and triggered apoptosis. Further, via in vivo experiments, they demonstrated that ORC6 depletion decreased expression of Cyclin A2/B2/TOP2A. In colorectal cancer cells treated with ETC-1922159, ORC family members and TOP2A, 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 TOP2A. 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 individual members w.r.t TOP2A. ORC1 - TOP2A shows low ranking of 258 (laplace), 145 (linear) and 279 (rbf). ORC6 - TOP2A shows low ranking of 1296 (laplace), 490 (linear) and 1449 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. 6 Further, ORC5, ORC3 and ORC2 showed high ranking with TOP2A, thus indicating that they might not be working synergistically with TOP2A, before the drug treatment. RANKING ORC FAMILY VS TOP2A RANKING OF ORC FAMILY W.R.TTOP2A laplace linear rbf ORC1 - TOP2A 258 145 279 ORC6 - TOP2A 1296 490 1449 ORC5 - TOP2A 1583 2340 1380 ORC3 - TOP2A 2341 2220 2215 ORC2 - TOP2A 2464 2530 2429 Table 7: 2nd order interaction ranking between TOP2A VS ORC family. One can also interpret the results of the table 7 graphically, with the following influences - •ORC family w.r.t TOP2A with TOP2A −>ORC-1/6. UNEXPLORED COMBINATORIAL HYPOTHESES ORC family w.r.t TOP2A ORC-1/6 TOP2A Table 8: 2nd order combinatorial hypotheses between TOP2A and ORC family. 2.1.5. TOP2A - IL Through in vivo and in vitro experiments Li et al. [23] showed that knockdown of TOP2A inhibited inflammation and IL17 signaling pathway, and promoted proliferation of ulcerative colitis (an inflammatory disease of the colonic mucosa). In colorectal cancer cells treated with ETC-1922159, IL family members and TOP2A, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these IL members along with TOP2A. Table 9 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 10 generated from analysis of the ranks in table 9. The table 9 shows rankings of individual members w.r.t TOP2A. IL33 - TOP2A shows low ranking of 591 (laplace), 152 (linear) and 818 (rbf). IL17D - TOP2A shows low 7 ranking of 1054 (laplace), 1194 (linear) and 853 (rbf). IL17RD - TOP2A shows low ranking of 1193 (laplace) and 1260 (rbf). IL1RL2 - TOP2A shows low ranking of 1237 (laplace) and 1328 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. RANKING IL FAMILY VS TOP2A RANKING OF IL FAMILY W.R.TTOP2A laplace linear rbf IL33 - TOP2A 591 152 818 IL17D - TOP2A 1054 1194 853 IL17RD - TOP2A 1193 2235 1260 IL1RL2 - TOP2A 1237 2123 1328 Table 9: 2nd order interaction ranking between TOP2A VS IL family. One can also interpret the results of the table 9 graphically, with the following influences - •IL family w.r.t TOP2A with TOP2A −>IL-33/17D/17RD/1RL2. UNEXPLORED COMBINATORIAL HYPOTHESES IL family w.r.t TOP2A IL-33/17D/17RD/1RL2 TOP2A Table 10: 2nd order combinatorial hypotheses between TOP2A and IL family. 2.1.6. TOP2A - USP Fielding et al. [24] show that deubiquitylase USP15 is required for TOP2A accumulation during G2, and USP15 depletion causes formation of anaphase chromosome bridges. In colorectal cancer cells treated with ETC-1922159, USP family members and TOP2A, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these USP members along with TOP2A. Table 11 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 12 generated from analysis of the ranks in table 11. The table 11 shows rankings of individual members w.r.t TOP2A. USP13 - TOP2A shows low ranking of 49 (laplace), 63 (linear) and 43 (rbf). USP36 - TOP2A shows 8 low ranking of 964 (laplace) and 1077 (rbf). USP28 - TOP2A shows low ranking of 1147 (laplace), 1546 (linear) and 1004 (rbf). USP1 - TOP2A shows low ranking of 1375 (linear) and 1241 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, USP39 and USP10 showed high ranking with TOP2A, thus indicating that they might not be working synergistically with TOP2A, before the drug treatment. RANKING USP FAMILY VS TOP2A RANKING OF USP FAMILY W.R.TTOP2A laplace linear rbf USP13 - TOP2A 49 63 43 USP36 - TOP2A 964 1738 1077 USP28 - TOP2A 1147 1546 1004 USP1 - TOP2A 1586 1375 1241 USP39 - TOP2A 2238 938 2224 USP10 - TOP2A 2695 2717 2707 Table 11: 2nd order interaction ranking between TOP2A VS USP family. One can also interpret the results of the table 11 graphically, with the following influences - •USP family w.r.t TOP2A with TOP2A −>USP-13/36/28/1. UNEXPLORED COMBINATORIAL HYPOTHESES USP family w.r.t TOP2A USP-13/36/28/1 TOP2A Table 12: 2nd order combinatorial hypotheses between TOP2A and USP family. 2.1.7. TOP2A - RAD54-ZNF TOP2 form cleavage complexes (TOP2ccs) during their catalytic cycle to relieve topological stress, however they can be trapped by TOP2 poisons. Trapped TOP2ccs by action of TOP2 poisons, block transactions on DNA and generate genotoxic stress. Zhang et al. [25] uncovered RAD54L2 which mediates a TOP2-specific DNA damage 9 low ranking of 211 (laplace), 154 (linear) and 99 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, BUB3 showed high ranking with TOP2A, thus indicating that they might not be working synergistically with TOP2A, before the drug treatment. RANKING BUB FAMILY VS TOP2A RANKING OF BUB FAMILY W.R.TTOP2A laplace linear rbf BUB1 - TOP2A 19 41 44 BUB1B - TOP2A 211 154 99 BUB3 - TOP2A 2203 1521 2167 Table 21: 2nd order interaction ranking between TOP2A VS BUB family. One can also interpret the results of the table 21 graphically, with the following influences - •BUB family w.r.t TOP2A with TOP2A −>BUB-1/1B. UNEXPLORED COMBINATORIAL HYPOTHESES BUB family w.r.t TOP2A BUB-1/1B TOP2A Table 22: 2nd order combinatorial hypotheses between TOP2A and BUB family. 2.1.12. TOP2A - DDX Zhang et al. [32] suggest that DDX11-AS1 knockdown resulted in reduced resistance of esophageal cancer cells to paclitaxel by inhibiting TOP2A transcription via TAF1. In colorectal cancer cells treated with ETC-1922159, DDX family members and TOP2A, 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 TOP2A. Table 23 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 24 generated from analysis of the ranks in table 23. The table 23 shows rankings of individual members w.r.t TOP2A. DDX11-AS1 - TOP2A shows low ranking of 310 (laplace), 365 (linear) and 406 (rbf). DDX12P - TOP2A shows low ranking of 435 (laplace), 76 (linear) and 450 (rbf). DDX55 - TOP2A shows low ranking of 806 (laplace), 1389 (linear) and 919 (rbf). DDX11 - 16 TOP2A shows low ranking of 829 (laplace) and 813 (rbf). DDX28 - TOP2A shows low ranking of 965 (laplace), 1108 (linear) and 871 (rbf). DDX18 - TOP2A shows low ranking of 1084 (laplace) and 1273 (rbf). DDX20 - TOP2A shows low ranking of 1369 (laplace), 393 (linear) and 1401 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, DDX54, DDX10, DDX31, DDX21, DDX51, DDX19A, DDX56, DDX46 and DDX27 showed high ranking with TOP2A, thus indicating that they might not be working synergistically with TOP2A, before the drug treatment. RANKING DDX FAMILY VS TOP2A RANKING OF DDX FAMILY W.R.TTOP2A laplace linear rbf laplace linear rbf DDX11-AS1 - TOP2A 310 365 406 DDX12P - TOP2A 435 76 450 DDX55 - TOP2A 806 1389 919 DDX11 - TOP2A 829 1730 813 DDX28 - TOP2A 965 1108 871 DDX18 - TOP2A 1084 2242 1273 DDX20 - TOP2A 1369 393 1401 DDX54 - TOP2A 1759 2524 1974 DDX10 - TOP2A 1933 857 1829 DDX31 - TOP2A 1951 2386 1515 DDX21 - TOP2A 1979 606 2054 DDX51 - TOP2A 1987 1289 1949 DDX19A - TOP2A 2144 1726 2063 DDX56 - TOP2A 2324 1537 2326 DDX46 - TOP2A 2338 2584 2579 DDX27 - TOP2A 2580 1170 2591 Table 23: 2nd order interaction ranking between TOP2A VS DDX family. One can also interpret the results of the table 23 graphically, with the following influences - •DDX family w.r.t TOP2A with TOP2A −>DDX-11-AS1/12P/55/11/28/18/20. UNEXPLORED COMBINATORIAL HYPOTHESES DDX family w.r.t TOP2A DDX-11-AS1/12P/55/11/28/18/20 TOP2A Table 24: 2nd order combinatorial hypotheses between TOP2A and DDX family. 2.1.13. TOP2A - H2A Zhang et al. [33] show that histone H2A phosphorylation modification generated by the mitotic kinase BUB1, is necessary and sufficient for the centromeric localization of TOP2A. In colorectal cancer cells treated with ETC-1922159, H2A family members and TOP2A, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these H2A members along with TOP2A. Table 25 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 26 generated from analysis of the ranks in table 25. 17 The table 25 shows rankings of individual members w.r.t TOP2A. H2AFV - TOP2A shows low ranking of 409 (laplace) and 798 (rbf). H2AFZ - TOP2A shows low ranking of 1178 (laplace) and 1108 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, H2AFX showed high ranking with TOP2A, thus indicating that they might not be working synergistically with TOP2A, before the drug treatment. RANKING H2A FAMILY VS TOP2A RANKING OF H2A FAMILY W.R.TTOP2A laplace linear rbf H2AFV - TOP2A 409 1916 798 H2AFZ - TOP2A 1178 1962 1108 H2AFX - TOP2A 2602 1080 2458 Table 25: 2nd order interaction ranking between TOP2A VS H2A family. One can also interpret the results of the table 25 graphically, with the following influences - •H2A family w.r.t TOP2A with TOP2A −>H2A-FV/FZ. UNEXPLORED COMBINATORIAL HYPOTHESES H2A family w.r.t TOP2A H2A-FV/FZ TOP2A Table 26: 2nd order combinatorial hypotheses between TOP2A and H2A family. 2.1.14. TOP2A - SMC The TOP2A-dependent arrest is responsible ofr segregation of sister chromatids and has been identified as dysfunctional in various tumour cell lines. Deiss et al. [34] show that the SMC5/6 complex regulates the TOP2A-dependent G2 arrest and sister chromatid disjunction via NSE2-mediated SUMOylation of TOP2A. In colorectal cancer cells treated with ETC-1922159, SMC family members and TOP2A, were found to be down regulated and their regulation was recorded independently. I was able to rank 2nd order combination of these SMC members along with TOP2A. Table 27 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 28 generated from analysis of the ranks in table 27. The table 27 shows rankings of individual members w.r.t TOP2A. SMC2 - TOP2A 18 shows low ranking of 335(laplace), 970 (linear) and 340 (rbf). SMC4 - TOP2A shows low ranking of 915(laplace), 446 (linear) and 986 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, SMC1A showed high ranking with TOP2A, thus indicating that they might not be working synergistically with TOP2A, before the drug treatment. RANKING SMC FAMILY VS TOP2A RANKING OF SMC FAMILY W.R.TTOP2A laplace linear rbf SMC2 - TOP2A 335 970 340 SMC4 - TOP2A 915 446 986 SMC1A - TOP2A 2461 898 2353 Table 27: 2nd order interaction ranking between TOP2A VS SMC family. One can also interpret the results of the table 27 graphically, with the following influences - •SMC family w.r.t TOP2A with TOP2A −>SMC-2/4. UNEXPLORED COMBINATORIAL HYPOTHESES SMC family w.r.t TOP2A SMC-2/4 TOP2A Table 28: 2nd order combinatorial hypotheses between TOP2A and SMC family. 3. Conclusion Presented here are a range of multiple synergistic TOP2A 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 TOP2A-X that might be prevalent in CRC cells after treatment with ETC-1922159 drug. Conflict of interest There are no conflicts to declare. 19 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 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. [35]. 4. References References [1] J. C. Wang, Interaction between dna and an escherichia coli protein ω, Journal of molecular biology 55 (1971) 523–IN16. [2] S. J. McKie, K. C. Neuman, A. 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