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

Machine learning discoveries of TNF-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 Often, in biology, we are faced with the problem of exploring relevant unknown biological hypotheses in the form of myriads of combinations of factors/genes/proteins that might be affecting the pathway under certain conditions. In colorectal cancer (CRC) cells treated with ETC-1922159, many genes were found up and down regulated, individually. A recently developed search engine ranked combinations of Tumor necrosis factor (TNF)-X (X, a particular gene/protein) at 2nd order level after drug administration. These rankings reveal which TNF-X combinations might be working synergistically in CRC. If found true, oncologists can further test the combination of interest in wet lab and determine the mechanism of functioning between the TNF and X. In this research work, we cover combinations of TNF with Wnt, mucin (MUC), six transmembrane epithelial antigen of prostate 4 (STEAP4), ubiquitin conjugating enzyme E2 (UBE2) family and BCL family. Keywords: TNF, Porcupine inhibitor ETC-1922159, Sensitivity analysis, Colorectal cancer. 1. Introduction In the unpublished preprint Sinha [1], a frame work of a search engine was developed which can rank combinations of factors (genes/proteins) in a signaling pathway. Such combinations are of import due to the vast search space in which they exist and the difficulty to find them. The search engine facilitates in prioritizing the combinations5 as ranked biological hypotheses which the biologists might want to test in wet lab, to know if a synergistic combination is prevalent in a signaling pathway, in a direct or indirect manner. Interested readers are advised to go through unpublished preprints Sinha [1] and Sinha [2] for details regarding the search engine and the discoveries IML dicoveries of TNF-X synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at (1) the recently concluded first ever Wnt Gordon Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA. Preprint submitted to Preprint August 29, 2024 mentioned in there.10 2. Materials and Methods 2.1. Combinatorial search problem and a possible solution The issue of combinatorial search problem and a possible solution has been addressed in Sinha [3] and Sinha [2]. The details of the methodology of this manuscript have been explained in great detail in Sinha [3] & its application in Sinha [2]. Readers15 are requested to go through the same for gaining deeper insight into the working of the pipeline and its use of published data set generated after administration of ETC1922159. In order to understand the significance of the solution proposed to the problem of combinatorial search that the biologists face in revealing unknown biological search problem, these works are of importance.20 Briefly, from Sinha [2], the pipleline works by computing sensitivity indicies for each of these unique combinations and then vectorising these indices to connote and form discriminative feature vector for each combination. Since each combination is unique, the training and the test data are same. In the training data, the combinations are arranged and ranks from 1 to n are assigned. The ranking algorithm then learns25 the patterns from these combinations/sensitivity index vectors. Next the learned model is used to rank the test data by generating the ranking score for each of the unique combination. Sorting these shuffled scores of test data leads to prioritization of the combinations. Joachims [4] show an example of applying learned model to training data (same as the test data) in https://www.cs.cornell.edu/people/tj/svm_30 light/svm_rank.html. Note that these combinations are now ranked and give the biologists a chance to narrow down their focus on crucial biological hypotheses in the form of combinations which the biologists might want to test. Analogous to the webpage search engine, where the click of a button for a few key-words leads to a ranked list of web links, the pipeline uses sensitivity indices as an indicator of the strength of35 the influence of factors or their combinations, as a criteria to rank the combinations. 3. Results & Discussion 3.1. Tumor necrosis factor related synergies 3.1.1. TNF - WNT cross family analysis Brooks et al. [5] observed TNF-αinduced alterations in the Wnt signaling cascade as40 a potential mechanism for obesity-associated colorectal tumorigenesis. Effects of TNF inhibitors on parathyroid hormone and Wnt signaling antagonists in rheumatoid arthritis have been studies in Adami et al. [6]. A complex interaction between Wnt signaling and TNF-αin nucleus pulposus cells has been studied by Hiyama et al. [7]. Ma and Hottiger [8] study the crosstalk between Wnt/β-catenin and NF-κB signaling pathway45 during inflammation. Roubert et al. [9] study the influence of tumor necrosis factorαon the tumorigenic Wnt-signaling pathway in human mammary tissue from obese 2 women. Jang et al. [10] observe that WNT/β-catenin pathway modulates the TNFα-induced inflammatory response in bronchial epithelial cells. These studies suggest already existing synergistic roles of WNTs and TNFs. In CRC cells affected with50 ETC-1922159, members of TNF and WNT family were found to be up regulated. Our search engine alloted high numerical valued ranks to some of the combinations between WNTs and TNFs. Table 1 shows rankings of TNF w.r.t to WNTs on the left half and vice verse on the right half. On the left half, we found TNF-RSF1A/RSF10A/RSF10B/SF15 to be up regu-55 lated w.r.t WNT2B. These were reflected in rankings of 2170 (laplace) and 2127 (linear) for TNFRSF1A - WNT2B; 1861 (laplace), 2367 (linear) and 1800 (rbf) for TNFRSF10A - WNT2B; 2020 (laplace) and 1881 (rbf) for TNFRSF10B - WNT2B and 2476 (laplace) and 2073 (rbf) for TNFSF15 - WNT2B. TNF-RSF10A/RSF10D/RSF12A/SF10 were found to be up regulated w.r.t WNT4. These were reflected in rankings of 250960 (laplace) and 2460 (linear) for TNFRSF10A - WNT4; 2233 (linear) and 2126 (rbf) for TNFRSF10D - WNT4; 2294 (linear), 1775 (linear) and 2384 (rbf) for TNFRSF12A - WNT4 and 2451 (linear) and 1782 (rbf) for TNFSF10 - WNT4. TNF-RSF12A/SF10 were found to be up regulated w.r.t WNT7B. These were reflected in rankings of 2100 (laplace) and 1983 (rbf) for TNFRSF12A - WNT7B and 2462 (laplace) and 2179 (rbf)65 for TNFSF10 - WNT7B. TNFRSF21 were found to be up regulated w.r.t WNT9A. These were reflected in rankings of 1805 (laplace) and 1999 (linear) for TNFRSF21 - WNT9A. On the left half, we found WNT2B to be up regulated w.r.t TNF-RSF10B/RSF10D/RSF14. These were reflected in rankings of 1797 (laplace) and 2056 (rbf) for TNFRSF10B -70 WNT2B; 1989 (linear) and 2130 (rbf) for TNFRSF10D - WNT2B and 1932 (laplace) and 2399 (rbf) for TNFRSF14 - WNT2B. WNT4 was upregulated w.r.t TNF-AIP3/RSF10B. These are refliected in rankings of 2336 (laplace), 2511 (linear) and 2342 (rbf) for TNFAIP3 - WNT4 and 2105 (linear) and 2264 (rbf) for TNFRSF10B - WNT4. WNT7B was upregulated w.r.t TNF, TNF-RSF1A/RSF14. These are reflected in rankings of75 2511 (linear) and 2210 (rbf) for TNF - WNT7B; 2084 (laplace), 1975 (linear) and 2154 (rbf) for TNFRSF1A - WNT7B and 2079 (laplace) and 1928 (rbf) for TNFRSF14 - WNT7B. WNT9A was upregulated w.r.t TNF-AIP2/AIP3/RSF10A/RSF12A/SF10. These are reflected in rankings of 2125 (laplace) and 2437 (linear) for TNFAIP2 - WNT9A; 1764 (laplace) and 2460 (linear) for TNFAIP3 - WNT9A; 2259 (laplace)80 and 2413 (linear) for TNFRSF10A - WNT9A; 2345 (laplace) and 2466 (rbf) for TNFRSF12A - WNT9A and 2054 (laplace) and 2338 (linear) for TNFSF10 - WNT9A. Table 2 shows the derived influences which can be represented graphically, with the following influences - •TNF w.r.t WNT with TNF-RSF1A/RSF10A/RSF10B/SF15 < −WNT2B; TNF-RSF10A/RSF10D/RSF12A/SF10 <−WNT4; TNF-RSF12A/SF1085 <−WNT7B and TNF-RSF21 <−WNT9A; and •WNT w.r.t TNF with TNFRSF10B/RSF10D/RSF14 −>WNT2B; TNF-AIP3/RSF10B −>WNT4; TNF, TNFRSF1A/RSF14 −>WNT7B; and TNF-AIP2/AIP3/RSF10A/RSF12A/SF10 −>WNT9A. 3 RANKING TNF FAMILY VS WNT FAMILY RANKING OF TNF FAMILY W.R.TWNT2B RANKING OF WNT2B W.R.TIL FAMILY laplace linear rbf laplace linear rbf TNF - WNT2B 503 893 1656 TNF - WNT2B 1341 808 1366 TNFAIP1 - WNT2B 235 156 1811 TNFAIP1 - WNT2B 1671 1434 1404 TNFAIP2 - WNT2B 868 527 2439 TNFAIP2 - WNT2B 1130 218 1105 TNFAIP3 - WNT2B 1135 2381 1688 TNFAIP3 - WNT2B 997 1280 1902 TNFRSF1A - WNT2B 2170 2127 1628 TNFRSF1A - WNT2B 1747 1857 1550 TNFRSF10A - WNT2B 1861 2367 1800 TNFRSF10A - WNT2B 100 464 1162 TNFRSF10B - WNT2B 2020 615 1881 TNFRSF10B - WNT2B 1797 120 2056 TNFRSF10D - WNT2B 29 2515 1174 TNFRSF10D - WNT2B 1348 1989 2130 TNFRSF12A - WNT2B 1072 2061 1109 TNFRSF12A - WNT2B 1595 298 1432 TNFRSF14 - WNT2B 333 1585 1247 TNFRSF14 - WNT2B 1932 277 2399 TNFRSF21 - WNT2B 1275 648 1114 TNFRSF21 - WNT2B 1396 620 2136 TNFSF10 - WNT2B 1204 2287 1396 TNFSF10 - WNT2B 1732 738 1751 TNFSF15 - WNT2B 2476 359 2073 TNFSF15 - WNT2B 402 128 1875 RANKING OF TNF FAMILY W.R.TWNT4 RANKING OF WNT4 W.R.TIL FAMILY laplace linear rbf laplace linear rbf TNF - WNT4 1982 1301 928 TNF - WNT4 1021 420 864 TNFAIP1 - WNT4 1434 1078 804 TNFAIP1 - WNT4 1114 337 1015 TNFAIP2 - WNT4 1810 1047 330 TNFAIP2 - WNT4 1611 1341 423 TNFAIP3 - WNT4 646 1955 1534 TNFAIP3 - WNT4 2336 2511 2342 TNFRSF1A - WNT4 915 545 829 TNFRSF1A - WNT4 132 333 1321 TNFRSF10A - WNT4 2509 2460 897 TNFRSF10A - WNT4 535 202 582 TNFRSF10B - WNT4 517 875 1365 TNFRSF10B - WNT4 320 2105 2264 TNFRSF10D - WNT4 1719 2233 2126 TNFRSF10D - WNT4 660 49 341 TNFRSF12A - WNT4 2294 1775 2384 TNFRSF12A - WNT4 649 1756 780 TNFRSF14 - WNT4 1608 2284 1436 TNFRSF14 - WNT4 61 519 1542 TNFRSF21 - WNT4 1915 1596 93 TNFRSF21 - WNT4 201 533 657 TNFSF10 - WNT4 1747 2451 1782 TNFSF10 - WNT4 904 1511 2280 TNFSF15 - WNT4 1542 806 2439 TNFSF15 - WNT4 64 709 793 RANKING OF TNF FAMILY W.R.TWNT7 RANKING OF WNT7 W.R.TIL FAMILY laplace linear rbf laplace linear rbf TNF - WNT7B 815 381 47 TNF - WNT7B 1530 2511 2210 TNFAIP1 - WNT7B 313 1438 992 TNFAIP1 - WNT7B 2196 519 1058 TNFAIP2 - WNT7B 1897 85 631 TNFAIP2 - WNT7B 2121 599 1313 TNFAIP3 - WNT7B 577 1807 1251 TNFAIP3 - WNT7B 1901 1357 830 TNFRSF1A - WNT7B 165 844 353 TNFRSF1A - WNT7B 2084 1975 2154 TNFRSF10A - WNT7B 1084 1341 2119 TNFRSF10A - WNT7B 1301 1120 1663 TNFRSF10B - WNT7B 1274 1980 744 TNFRSF10B - WNT7B 1209 908 1075 TNFRSF10D - WNT7B 1314 774 1928 TNFRSF10D - WNT7B 1252 2301 1250 TNFRSF12A - WNT7B 2100 1332 1983 TNFRSF12A - WNT7B 1104 22 1879 TNFRSF14 - WNT7B 1576 981 1811 TNFRSF14 - WNT7B 2079 1028 1928 TNFRSF21 - WNT7B 1565 798 720 TNFRSF21 - WNT7B 2114 1219 737 TNFSF10 - WNT7B 1598 2462 2179 TNFSF10 - WNT7B 2129 763 204 TNFSF15 - WNT7B 1026 756 621 TNFSF15 - WNT7B 130 1599 2504 RANKING OF TNF FAMILY W.R.TWNT9A RANKING OF WNT9A W.R.TIL FAMILY laplace linear rbf laplace linear rbf TNF - WNT9A 1624 2025 799 TNF - WNT9A 1121 1930 1400 TNFAIP1 - WNT9A 1433 839 465 TNFAIP1 - WNT9A 1254 569 394 TNFAIP2 - WNT9A 40 2167 397 TNFAIP2 - WNT9A 2125 2437 1605 TNFAIP3 - WNT9A 1427 1109 2040 TNFAIP3 - WNT9A 1764 2460 1032 TNFRSF1A - WNT9A 1470 719 1933 TNFRSF1A - WNT9A 1645 58 1419 TNFRSF10A - WNT9A 2272 1234 918 TNFRSF10A - WNT9A 2259 2413 1204 TNFRSF10B - WNT9A 2249 1222 1071 TNFRSF10B - WNT9A 882 566 813 TNFRSF10D - WNT9A 410 2132 968 TNFRSF10D - WNT9A 1808 1055 568 TNFRSF12A - WNT9A 1080 373 1120 TNFRSF12A - WNT9A 2345 1211 2466 TNFRSF14 - WNT9A 1106 2166 198 TNFRSF14 - WNT9A 1127 1147 1191 TNFRSF21 - WNT9A 1805 1999 986 TNFRSF21 - WNT9A 1265 832 1098 TNFSF10 - WNT9A 1258 864 1839 TNFSF10 - WNT9A 2054 2338 1523 TNFSF15 - WNT9A 1621 1129 1139 TNFSF15 - WNT9A 37 1105 1076 Table 1: 2nd order combinatorial hypotheses between ABC and IL 4 UNEXPLORED COMBINATORIAL HYPOTHESES TNF w.r.t WNT TNF-RSF1A/RSF10A/RSF10B/SF15 WNT2B TNF-RSF10A/RSF10D/RSF12A/SF10 WNT4 TNF-RSF12A/SF10 WNT7B TNF-RSF21 WNT9A WNT w.r.t TNF TNF-RSF10B/RSF10D/RSF14 WNT2B TNF-AIP3/RSF10B WNT4 TNF, TNF-RSF1A/RSF14 WNT7B TNF-AIP2/AIP3/RSF10A/RSF12A/SF10 WNT9A Table 2: 2nd order combinatorial hypotheses between TNF and WNT family. 5 3.1.2. MUC - TNF cross family analysis In a recent development in Sheng et al. [11] MUC13 promoted tumor necrosis fac-90 tro (TNF)-induced NFkB activation by interacting with TNFR1 and the E3 ligase, cIAP1, to increase ubiquitination of RIPK1. Dharmani et al. [12] show that TNF-α and MUC2 (Mucin 2) play major roles in disease onset and progression in dextran sodium sulphate-induced colitis. TNF-αis also shown to induce mucin hypersecretion and MUC2 gene expression by human airway epithelial cells by Levine et al. [13].95 Also, inhibition of TNF-αinduced MUC5AC expression and production by wogonin through the inactivation of NF-κB signaling in airway epithelial cells, as shown by Sikder et al. [14]. Similarly, neutrophil elastase induces MUC5AC production in human airway epithelial cells via a cascade involving protein kinase-C, reactive oxygen species, and TNF-αconverting enzyme, as shown by Shao and Nadel [15]. TNF-αor100 transforming growth factor-αstimulation of human epithelial cells resulted in mucus secretion as measured by MUC5AC mRNA and protein (Lora et al. [16]). In earlier experiments by Fischer et al. [17], TNF-αwas found to stimulate mucin secretion and cyclic GMP production by guinea pig tracheal epithelial cells in vitro. Similar earlier experiments by Lin et al. [18], induction of mucin gene expression in middle ear of105 rats by TNF-αwas the potential cause for mucoid otitis media. Effects of TNF-αand IL-1βon mucin, lysozyme, IL-6 and IL-8 in passage-2 normal human nasal epithelial cells have been stuided by Yoon et al. [19]. Also, Mercogliano et al. [20] show that TNF-αinduced MUC4 expression elicits trastuzumab resistance in HER2-+ive breast cancer. These findings suggest deep synergy between Mucin family and TNF family110 members. However, not all synergies might have been explored till now. A set of family members of MUC and TNFs were found to be UP regulated after ETC-1922159 treatment in CRC cells. Tables 3 and 4 show the additional range of TNFs and MUCs that might be engaged in CRC through the NFkB pathway, in the light of the recent findings of MUC13 and115 TNFRSF1A in Sheng et al. [11]. Table 3 shows the rankings of the TNF family w.r.t to MUCIN family and table 4 shows the rankings of the MUCIN family w.r.t to TNF family. Followed by this are the derived influences from the majority votings of the rankings in the foregoing tables, which are depicted in table 5. Considering table 3, TNF family w.r.t MUC1, we find TNFAIP3, TNFRSF-10D/12A/14120 to be highly up regulated. These are reflected in the rankings of 2115 (laplace) and 1882 (rbf) for MUC1 - TNFAIP3; 2303 (laplace) and 2154 (linear) for MUC1 - TNFRSF10D; 2019 (laplace) and 2009 (linear) for MUC1 - TNFRSF12A; and 1955 (laplace) and 1899 (linear) for MUC1 - TNFRSF14. TNF family w.r.t MUC3A, we find TNFRSF10A/10D to be highly up regulated. These are reflected in the rankings of 2237 (laplace)125 and 1910 (linear) for MUC3 - TNFRSF10A; 1678 (laplace) and 2049 (linear) for MUC3 - TNFRSF10D. TNF family w.r.t MUC4 we find TNFRSF10D/TNFSF10 to be highly up regulated. These are reflected in the rankings of 2503 (laplace), 2403 (linear) and 2356 (rbf) for MUC4 - TNFRSF10D and 2134 (laplace) and 1957 (linear) for MUC4 - TNFSF10. TNF family w.r.t MUC12 we find TNFRSF21/TNFSF10 to130 be highly up regulated. These are reflected in the rankings of 1795 (laplace) and 2438 (linear) for MUC12 - TNFRSF21 and 1795 (linear) 2435 (rbf) for MUC12 - TNFSF10. TNF family w.rt MUC13 we find TNFRSF10A/TNFRSF10D to be highly up regu6 lated. These are reflected in the rankings of 2500 (laplace) and 1844 (rbf) for MUC13 - TNFRSF10A and 2263 (linear) and 2294 (rbf) for MUC13 - TNFRSF10D. TNF fam-135 ily w.r.t MUC17 we find TNFRSF-10A/10D/12A to be highly up regulated. These are reflected in the rankings of 2269 (laplace) 2364 (linear) and 2005 (rbf) for MUC17 - TNFRSF10A; 1798 (laplace) and 2302 (rbf) for MUC17 - TNFRSF10D and 2041 (laplace) and 2303 (linear) for MUC17 - TNFRSF12A. TNF family w.r.t MUC20 we find TNFAIP3/TNFSF15 to be highly up regulated. These are reflected in the rankings140 of 2205 (laplace) and 2136 (rbf) for MUC20 - TNFAIP3 and 2493 (laplace) and 2108 (rbf) for MUC20 - TNFSF15. Considering table 4, MUC1 w.r.t TNF family, we find TNFRSF1A to be highly up regulated. These are reflected in the rankings of 2344 (linear) and 2312 (rbf) for MUC1 - TNFRSF1A. MUC4 w.r.t TNF family, we find TNFAIP2 to be highly up regulated.145 These are reflected in the rankings of 1875 (laplace) and 1792 (linear) for MUC4 - TNFAIP2. MUC12 w.r.t TNF family we find TNFAIP1/TNFAIP2/TNFRSF21/TNFSF10 to be highly up regulated. These are reflected in the rankings of 2321 (laplace) and 2457 (rbf) for MUC12 - TNFAIP1; 1829 (linear) and 1913 (rbf) for MUC12 - TNFAIP2; 1975 (linear) and 1769 (rbf) for MUC12 - TNFRSF21; 2135 (linear) and 2255 (rbf)150 for MUC12 - TNFSF10. MUC12 w.r.t TNF family we find TNFRSF21/TNFSF10 to be highly up regulated. These are reflected in the rankings of 1795 (laplace) and 2438 (linear) for MUC12 - TNFRSF21 and 1795 (linear) and 2435 (rbf) for MUC12 - TNFSF10. MUC20 w.r.t TNF family we find TNFAIP1/TNFAIP2 to be highly up regulated. These are reflected in the rankings of 2266 (laplace) and 2057 (rbf) for MUC20155 - TNFAIP1 and 2404 (linear) and 2157 (rbf) for MUC20 - TNFAIP2. One can also interpret the results of the table 5 graphically, with the following influences - •TNF family w.r.t MUC family with MUC1 −>TNFAIP3/TNFRSF10D/12A/14; MUC3A −>TNFRSF-10A/10D; MUC4 −>TNFRSF10D/TNFSF10; MUC12 −>TNFRSF21/TNFSF10; MUC13 −>TNFRSF-10A/10D; MUC17 −>160 TNFRSF-10A/10D/12A; MUC20 −>TNFAIP3/TNFSF15 and •MUC family w.r.t TNF family with MUC1 <−TNFRSF1A; MUC4 <−TNFAIP2; MUC12 <−TNFAIP1/TNFAIP2/TNFRSF21/TNFSF10 and MUC13 <−TNFAIP1/TNFAIP2. 7 RANKING TNF FAMILY W.R.TMUC FAMILY RANKING OF TNF FAMILY W.R.TMUC1 RANKING OF TNF FAMILY W.R.TMUC3A laplace linear rbf laplace linear rbf MUC1 - TNF 112 72 88 MUC3A - TNF 1353 1659 1479 MUC1 - TNFAIP1 1193 1603 997 MUC3A - TNFAIP1 2178 1209 1347 MUC1 - TNFAIP2 716 405 2340 MUC3A - TNFAIP2 1075 1614 1158 MUC1 - TNFAIP3 2115 1636 1882 MUC3A - TNFAIP3 962 1020 2491 MUC1 - TNFRSF1A 1380 422 1390 MUC3A - TNFRSF1A 461 1708 189 MUC1 - TNFRSF10A 1009 2180 1095 MUC3A - TNFRSF10A 2237 1910 335 MUC1 - TNFRSF10B 1923 732 88 MUC3A - TNFRSF10B 450 1443 2040 MUC1 - TNFRSF10D 2303 2154 376 MUC3A - TNFRSF10D 1678 2049 102 MUC1 - TNFRSF12A 2019 2009 1700 MUC3A - TNFRSF12A 2349 1315 382 MUC1 - TNFRSF14 1955 1899 1429 MUC3A - TNFRSF14 956 1442 1953 MUC1 - TNFRSF21 337 477 968 MUC3A - TNFRSF21 1297 1492 1959 MUC1 - TNFSF10 1111 1592 1198 MUC3A - TNFSF10 891 257 798 MUC1 - TNFSF15 936 986 2391 MUC3A - TNFSF15 2285 795 1164 RANKING OF TNF FAMILY W.R.TMUC4 RANKING OF TNF FAMILY W.R.TMUC12 laplace linear rbf laplace linear rbf MUC4 - TNF 1896 231 1355 MUC12 - TNF 1862 102 135 MUC4 - TNFAIP1 864 397 987 MUC12 - TNFAIP1 1386 479 942 MUC4 - TNFAIP2 73 1011 1087 MUC12 - TNFAIP2 1056 303 1587 MUC4 - TNFAIP3 1159 1751 179 MUC12 - TNFAIP3 2493 1259 1330 MUC4 - TNFRSF1A 179 71 16 MUC12 - TNFRSF1A 1709 1440 837 MUC4 - TNFRSF10A 1668 1892 1652 MUC12 - TNFRSF10A 598 531 363 MUC4 - TNFRSF10B 2024 1396 331 MUC12 - TNFRSF10B 409 1572 1297 MUC4 - TNFRSF10D 2503 2403 2356 MUC12 - TNFRSF10D 30 102 149 MUC4 - TNFRSF12A 1684 700 745 MUC12 - TNFRSF12A 298 882 153 MUC4 - TNFRSF14 1675 2029 1146 MUC12 - TNFRSF14 1749 2237 135 MUC4 - TNFRSF21 647 326 323 MUC12 - TNFRSF21 1795 607 2438 MUC4 - TNFSF10 936 2134 1957 MUC12 - TNFSF10 801 1795 2435 MUC4 - TNFSF15 1440 1180 1627 MUC12 - TNFSF15 1741 889 1098 RANKING OF TNF FAMILY W.R.TMUC13 RANKING OF TNF FAMILY W.R.TMUC17 laplace linear rbf laplace linear rbf MUC13 - TNF 2282 220 127 MUC17 - TNF 683 362 515 MUC13 - TNFAIP1 378 230 1935 MUC17 - TNFAIP1 117 188 272 MUC13 - TNFAIP2 2464 220 697 MUC17 - TNFAIP2 1311 414 351 MUC13 - TNFAIP3 2274 1233 1446 MUC17 - TNFAIP3 1589 1547 1539 MUC13 - TNFRSF1A 274 2152 514 MUC17 - TNFRSF1A 428 205 329 MUC13 - TNFRSF10A 2500 938 1844 MUC17 - TNFRSF10A 2269 2364 2005 MUC13 - TNFRSF10B 1891 1497 225 MUC17 - TNFRSF10B 1199 1323 2120 MUC13 - TNFRSF10D 1191 2263 2294 MUC17 - TNFRSF10D 1798 1378 2302 MUC13 - TNFRSF12A 460 1753 1704 MUC17 - TNFRSF12A 2041 2303 1049 MUC13 - TNFRSF14 2220 1602 1359 MUC17 - TNFRSF14 2043 825 1700 MUC13 - TNFRSF21 1612 1673 127 MUC17 - TNFRSF21 2013 393 119 MUC13 - TNFSF10 2236 1598 1495 MUC17 - TNFSF10 280 1025 817 MUC13 - TNFSF15 2423 1488 1292 MUC17 - TNFSF15 833 967 950 RANKING OF TNF FAMILY W.R.TMUC20 laplace linear rbf MUC20 - TNF 2267 262 145 MUC20 - TNFAIP1 1273 2296 178 MUC20 - TNFAIP2 1062 598 339 MUC20 - TNFAIP3 2205 435 2136 MUC20 - TNFRSF1A 483 2346 145 MUC20 - TNFRSF10A 100 2305 917 MUC20 - TNFRSF10B 775 1578 1556 MUC20 - TNFRSF10D 200 1487 799 MUC20 - TNFRSF12A 318 1607 2258 MUC20 - TNFRSF14 410 1832 745 MUC20 - TNFRSF21 1686 2259 164 MUC20 - TNFSF10 1005 2139 1548 MUC20 - TNFSF15 2493 387 2108 Table 3: 2nd order interaction ranking between TNF w.r.t MUC family members. 8 RANKING MUC FAMILY W.R.TTNF FAMILY RANKING OF MUC1 W.R.TTNF FAMILY RANKING OF MUC3A W.R.TTNF FAMILY laplace linear rbf laplace linear rbf MUC1 - TNF 368 142 21 MUC3A - TNF 1478 985 2373 MUC1 - TNFAIP1 692 91 1591 MUC3A - TNFAIP1 1485 536 1698 MUC1 - TNFAIP2 2290 476 398 MUC3A - TNFAIP2 1254 1265 75 MUC1 - TNFAIP3 810 492 748 MUC3A - TNFAIP3 1844 960 243 MUC1 - TNFRSF1A 1089 2344 2312 MUC3A - TNFRSF1A 496 574 792 MUC1 - TNFRSF10A 1263 351 826 MUC3A - TNFRSF10A 1315 1525 1815 MUC1 - TNFRSF10B 1630 1604 2103 MUC3A - TNFRSF10B 351 1920 1489 MUC1 - TNFRSF10D 975 1026 984 MUC3A - TNFRSF10D 596 950 1016 MUC1 - TNFRSF12A 1597 1811 1078 MUC3A - TNFRSF12A 436 595 2124 MUC1 - TNFRSF14 739 2119 938 MUC3A - TNFRSF14 1612 1383 329 MUC1 - TNFRSF21 766 1495 2322 MUC3A - TNFRSF21 1254 1357 1162 MUC1 - TNFSF10 1360 1969 477 MUC3A - TNFSF10 774 980 2053 MUC1 - TNFSF15 424 1183 542 MUC3A - TNFSF15 75 1261 624 RANKING OF MUC4 W.R.TTNF FAMILY RANKING OF MUC12 W.R.TTNF FAMILY laplace linear rbf laplace linear rbf MUC4 - TNF 1656 777 565 MUC12 - TNF 266 1223 628 MUC4 - TNFAIP1 2483 895 390 MUC12 - TNFAIP1 2321 668 2457 MUC4 - TNFAIP2 1875 1792 180 MUC12 - TNFAIP2 281 1829 1913 MUC4 - TNFAIP3 54 498 464 MUC12 - TNFAIP3 2353 153 576 MUC4 - TNFRSF1A 1074 753 68 MUC12 - TNFRSF1A 1481 1952 1406 MUC4 - TNFRSF10A 683 311 997 MUC12 - TNFRSF10A 445 337 888 MUC4 - TNFRSF10B 98 1413 704 MUC12 - TNFRSF10B 792 164 133 MUC4 - TNFRSF10D 1916 230 80 MUC12 - TNFRSF10D 167 193 521 MUC4 - TNFRSF12A 1321 2190 150 MUC12 - TNFRSF12A 216 2093 302 MUC4 - TNFRSF14 606 704 1493 MUC12 - TNFRSF14 105 59 69 MUC4 - TNFRSF21 1225 1967 1093 MUC12 - TNFRSF21 1471 1975 1769 MUC4 - TNFSF10 815 1108 1906 MUC12 - TNFSF10 662 2135 2255 MUC4 - TNFSF15 1141 1841 920 MUC12 - TNFSF15 1619 2204 1257 RANKING OF MUC13 W.R.TTNF FAMILY RANKING OF MUC17 W.R.TTNF FAMILY laplace linear rbf laplace linear rbf MUC13 - TNF 623 292 295 MUC17 - TNF 203 811 57 MUC13 - TNFAIP1 823 755 81 MUC17 - TNFAIP1 381 193 118 MUC13 - TNFAIP2 1118 2464 116 MUC17 - TNFAIP2 1069 822 136 MUC13 - TNFAIP3 1189 546 541 MUC17 - TNFAIP3 2132 47 937 MUC13 - TNFRSF1A 978 1506 490 MUC17 - TNFRSF1A 120 497 864 MUC13 - TNFRSF10A 1180 540 1926 MUC17 - TNFRSF10A 852 218 346 MUC13 - TNFRSF10B 280 1105 190 MUC17 - TNFRSF10B 933 1667 1166 MUC13 - TNFRSF10D 655 725 1668 MUC17 - TNFRSF10D 546 133 304 MUC13 - TNFRSF12A 401 1242 999 MUC17 - TNFRSF12A 18 1675 86 MUC13 - TNFRSF14 1324 374 389 MUC17 - TNFRSF14 819 296 1014 MUC13 - TNFRSF21 690 2337 107 MUC17 - TNFRSF21 1659 814 889 MUC13 - TNFSF10 1146 1208 2159 MUC17 - TNFSF10 387 1542 156 MUC13 - TNFSF15 1633 314 155 MUC17 - TNFSF15 1207 1040 522 RANKING OF MUC20 W.R.TTNF FAMILY laplace linear rbf MUC20 - TNF 57 216 903 MUC20 - TNFAIP1 1265 2266 2057 MUC20 - TNFAIP2 241 2404 2157 MUC20 - TNFAIP3 484 1012 513 MUC20 - TNFRSF1A 748 173 2193 MUC20 - TNFRSF10A 620 427 1054 MUC20 - TNFRSF10B 765 1563 790 MUC20 - TNFRSF10D 509 2185 794 MUC20 - TNFRSF12A 216 2093 302 MUC20 - TNFRSF14 2298 651 1368 MUC20 - TNFRSF21 2374 1611 1140 MUC20 - TNFSF10 1257 1088 1031 MUC20 - TNFSF15 142 2159 7 Table 4: 2nd order interaction ranking between MUC w.r.t TNF family members. 9 UNEXPLORED COMBINATORIAL HYPOTHESES UBE2 w.r.t TNF TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF10D/RSF12A/RSF14/RSF21/SF15 UBE2A TNF-RSF10A/RSF10B/RSF10D/RSF14/RSF21 UBE2B TNF, TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF12A/SF15 UBE2F TNF-RSF12A/RSF14/RSF21 UBE2H TNF, TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF12A/RSF14/RSF21/SF15 UBE2J1 TNF, TNF-AIP1/AIP2/RSF1A/RSF10A/RSF10D/RSF12A/SF10/SF15 UBE2Z TNF w.r.t UBE2 TNF-RSF10A UBE2A TNF-RSF10A/RSF12A/SF10/SF15 UBE2B TNF-RSF14 UBE2F TNF-SF15 UBE2H TNF-RSF10A/RSF10D/RSF14 UBE2J1 TNF-RSF10A/RSF14 UBE2Z Table 10: 2nd order combinatorial hypotheses between TNF and UBE2 family. (rbf) for TNFAIP2 - UBE2Z; 2473 (laplace), 2194 (linear) and 2405 (rbf) for TNFRSF1A - UBE2Z; 2234 (laplace) and 2152 (linear) for TNFRSF10A - UBE2Z; 2264 (laplace), 2360 (linear) and 2278 (rbf) for TNFRSF10D - UBE2Z; 2207 (laplace) and260 2149 (linear) for TNFRSF12A - UBE2Z; 2374 (linear) and 2235 (rbf) for TNFSF10 - UBE2Z; and 2081 (laplace) and 2102 (rbf) for TNFSF15 - UBE2Z; One the right side is the ranking of TNF family w.r.t UBE2 family. We found TNFRSF10A to be up regulated w.r.t UBE2A. This is reflected in rankings of 2116 (laplace) and 2376 (rbf) for TNFRSF10A - UBE2A. TNF-RSF10A/RSF12A/SF10/SF15 were265 up regulated w.r.t UBE2B. These are reflected in rankings of 2318 (linear) and 2265 (linear) for TNFRSF10A - UBE2B; 1940 (laplace); 1868 (linear) and 1758 (linear) for TNFRSF12A - UBE2B; 2208 (laplace); 2326 (linear) and 2470 (linear) for TNFSF10 - UBE2B; and 2055 (laplace) and 1964 (linear) for TNFSF15 - UBE2B. TNF-RSF14 were up regulated w.r.t UBE2F. These is reflected in rankings of 2324 (laplace) and270 1924 (linear) for TNF-RSF14 - UBE2F. TNF-SF15 were up regulated w.r.t UBE2H. These is reflected in rankings of 2416 (linear) and 1960 (rbf) for TNF-SF15 - UBE2H. TNF-RSF10A/RSF10D/RSF14 were up regulated w.r.t UBE2J1. These are reflected in rankings of 2379 (laplace), 2213 (linear) and 2135 (rbf) for TNFRSF10A - UBE2J1; 2393 (laplace) and 2102 (rbf) for TNFRSF10D - UBE2J1; and 2133 (linear) and 2313275 (rbf) for TNFRSF14 - UBE2J1. TNF-RSF10A/RSF14 were up regulated w.r.t UBE2Z. These are reflected in rankings of 2410 (laplace) and 2103 (laplace) for TNFRSF10A - UBE2Z and 1779 (laplace) and 2100 (rbf) for TNFRSF14 - UBE2Z. One can also interpret the results of the table 10 graphically, with the following influences - •UBE2 w.r.t TNF with TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF10D/RSF12A/RSF14/280 RSF21/SF15 −>UBE2A; TNF-RSF10A/RSF10B/RSF10D/RSF14/RSF21 −>UBE2B; TNF, TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF12A/SF15 −>UBE2F; TNF-RSF12A/RSF14/RSF21 −>UBE2H; TNF, TNF-AIP1/RSF1A/RSF10A/RSF10B/RSF12A/RSF14/RSF21/SF15 −>UBE2J1; and TNF, TNF-AIP1/AIP2/RSF1A/RSF10A/RSF10D/RSF12A/SF10/SF15 16 −>UBE2Z •TNF w.r.t UBE2 with TNF-RSF10A <−UBE2A; TNF-RSF10A/RSF12A/SF10/SF15285 <−UBE2B; TNF-RSF14 <−UBE2F; TNF-SF15 <−UBE2H; TNF-RSF10A/RSF10D/RSF14 <−UBE2J1; TNF-RSF10A/RSF14 <−UBE2Z. 17 3.1.5. TNF - BCL cross family analysis Tamatani et al. [33] observe that tumor necrosis factor induces BCL-2 and BCL-x expression through NFκB activation in primary hippocampal neurons. The role of Bcl-2290 Expression in EGF Inhibition of TNF-α/IFN-γ-induced Villous Trophoblast Apoptosis has been studied by Ho et al. [34]. Genestier et al. [35] show that tumor necrosis factor-αup-regulates BCL-2 expression and decreases calcium-dependent apoptosis in human B cell lines. In breast carcinoma cells, Bcl-x and Bcl-2 inhibit TNF and FAS-induced apoptosis and activation of phospholipase A2 (J¨ a¨ attel¨ a et al. [36]). Kim295 et al. [37] show that TNF-α-induced ROS production triggering apoptosis is directly linked to Romo1 and BCL-XL. Kuwata et al. [38] showed that IL-10-inducible BCL3 negatively regulates LPS-induced TNF-αproduction in macrophages. Esche et al. [39] showed that tumor necrosis factor-α-promoted expression of BCL-2 and inhibition of mitochondrial cytochrome c release mediate resistance of mature dendritic cells300 to melanoma-induced apoptosis. These studies show a definite synergy between BCL family and TNFs. In CRC cells treated with ETC-1922159, both TNF members and BCL were found to be up regulated. Our search engine alloted the dual combinations with numerically high ranked values thus pointing to the possible synergies that might be existing in the cells and may not have been explored. Table 11 and 12 show the305 rankings of each with the other. On the left side is the ranking of BCL family w.r.t TNF family. We found BCL2L2 to be up regulated w.r.t TNF, TNF-AIP1/RSF1A/RSF10B/RSF10D/RSF12A/RSF14/RSF21/ SF10/SF15. These are reflected in rankings of 1822 (laplace), 1926 (linear) and 2359 (rbf) for TNF - BCL2L2; 2266 (laplace), 2478 (linear) and 1847 (rbf) for TNFAIP1310 - BCL2L2; 2311 (linear) and 1920 (rbf) for TNFRSF1A - BCL2L2; 2478 (laplace) and 2239 (rbf) for TNFRSF10B - BCL2L2; 2278 (linear) and 2237 (rbf) for TNFRSF10D - BCL2L2; 1945 (laplace) and 2484 (rbf) for TNFRSF12A - BCL2L2; 2358 (laplace) and 2310 (rbf) for TNFRSF14 - BCL2L2; 2292 (laplace) and 1850 (linear) for TNFRSF21 - BCL2L2; 2438 (laplace) and 2013 (rbf) for TNFSF10 - BCL2L2315 and 2443 (linear) and 2350 (rbf) for TNFSF15 - BCL2L2; BCL2L3 was up regulated w.r.t TNF, TNF-AIP1/RSF1A/RSF10A/RSF10D/RSF12A/RSF14/RSF21/ SF10/SF15. These are reflected in rankings of 2437 (laplace), 2482 (linear) and 2482 (rbf) for TNF - BCL2L13; 1863 (laplace) and 2386 (linear) for TNFAIP1 - BCL2L13; 1962 (linear) and 2489 (rbf) for TNFRSF1A - BCL2L13; 2055 (linear) and 2499 (rbf) for TN-320 FRSF10A - BCL2L13; 2204 (laplace), 2159 (linear) and 2343 (rbf) for TNFRSF10D - BCL2L13; 2183 (laplace), 2509 (linear) for TNFRSF12A - BCL2L13; 1852 (laplace), 1974 (linear) and 2339 (rbf) for TNFRSF14 - BCL2L13; 2280 (laplace), 2424 (linear) and 2301 (rbf) for TNFRSF21 - BCL2L13; 2429 (linear) and 1803 (rbf) for TNFSF10 - BCL2L13; and 2438 (linear) and 2252 (rbf) for TNFSF15 - BCL2L13; BCL3 was325 up regulated w.r.t TNFRSF10B. This is reflected in rankings of 2427 (laplace) and 1868 (rbf). BCL6 was up regulated w.r.t TNF, TNF-AIP1/AIP2/RSF1A/RSF10A/RSF10D/RSF21. These are reflected in rankings of 2271 (laplace), 2071 (linear) and 1810 (rbf) for TNF - BCL6; 2135 (laplace) and 2158 (linear) for TNFAIP1 - BCL6; 2340 (laplace) 1808 (rbf) for TNFAIP2 - BCL6; 1771 (linear) and 2503 (rbf) for TNFRSF1A - BCL6;330 and 1831 (linear) and 2096 (rbf) for TNFRSF10A - BCL6; and 2213 (laplace) and 2188 (rbf) for TNFRSF10D - BCL6; and 2071 (linear) and 2335 (rbf) for TNFRSF21 18 RANKING TNF FAMILY VS BCL FAMILY RANKING OF BCL2L1 W.R.TTNF FAMILY RANKING OF TNF FAMILY W.R.TBCL2L1 laplace linear rbf laplace linear rbf TNF - BCL2L1 174 14 235 TNF - BCL2L1 56 1101 294 TNFAIP1 - BCL2L1 1527 435 791 TNFAIP1 - BCL2L1 1497 1150 74 TNFAIP2 - BCL2L1 2142 1735 798 TNFAIP2 - BCL2L1 1485 1735 400 TNFAIP3 - BCL2L1 2467 842 867 TNFAIP3 - BCL2L1 1109 1939 1553 TNFRSF1A - BCL2L1 1004 1558 383 TNFRSF1A - BCL2L1 492 376 1016 TNFRSF10A - BCL2L1 1906 1270 1222 TNFRSF10A - BCL2L1 2273 1928 508 TNFRSF10B - BCL2L1 1506 2235 589 TNFRSF10B - BCL2L1 1003 2252 2217 TNFRSF10D - BCL2L1 1920 1555 1787 TNFRSF10D - BCL2L1 1868 2420 2392 TNFRSF12A - BCL2L1 1254 1388 1319 TNFRSF12A - BCL2L1 1923 53 1936 TNFRSF14 - BCL2L1 688 237 2009 TNFRSF14 - BCL2L1 340 2350 2414 TNFRSF21 - BCL2L1 1465 1269 100 TNFRSF21 - BCL2L1 2139 718 289 TNFSF10 - BCL2L1 532 560 2332 TNFSF10 - BCL2L1 2115 2299 1307 TNFSF15 - BCL2L1 1026 1551 1134 TNFSF15 - BCL2L1 453 423 25 RANKING OF BCL2L2 W.R.TTNF FAMILY RANKING OF TNF FAMILY W.R.TBCL2L2 laplace linear rbf laplace linear rbf TNF - BCL2L2 1822 1926 2359 TNF - BCL2L2 2140 109 1062 TNFAIP1 - BCL2L2 2266 2478 1847 TNFAIP1 - BCL2L2 2235 1607 712 TNFAIP2 - BCL2L2 823 535 1117 TNFAIP2 - BCL2L2 109 1002 54 TNFAIP3 - BCL2L2 1201 1103 1511 TNFAIP3 - BCL2L2 1470 1696 1276 TNFRSF1A - BCL2L2 1124 2311 1920 TNFRSF1A - BCL2L2 1912 169 1531 TNFRSF10A - BCL2L2 1063 1532 2458 TNFRSF10A - BCL2L2 1643 1095 953 TNFRSF10B - BCL2L2 2478 739 2239 TNFRSF10B - BCL2L2 2153 1164 1983 TNFRSF10D - BCL2L2 910 2278 2237 TNFRSF10D - BCL2L2 35 1012 1905 TNFRSF12A - BCL2L2 1945 240 2484 TNFRSF12A - BCL2L2 1971 1633 975 TNFRSF14 - BCL2L2 2358 1648 2310 TNFRSF14 - BCL2L2 1027 825 1228 TNFRSF21 - BCL2L2 2292 1850 1014 TNFRSF21 - BCL2L2 1138 486 554 TNFSF10 - BCL2L2 2438 547 2013 TNFSF10 - BCL2L2 2212 902 169 TNFSF15 - BCL2L2 1196 2443 2350 TNFSF15 - BCL2L2 2285 165 1330 RANKING OF BCL2L13 W.R.TTNF FAMILY RANKING OF TNF FAMILY W.R.TBCL2L13 laplace linear rbf laplace linear rbf TNF - BCL2L13 2437 2482 2482 TNF - BCL2L13 1162 103 462 TNFAIP1 - BCL2L13 1863 2386 989 TNFAIP1 - BCL2L13 852 606 787 TNFAIP2 - BCL2L13 793 293 1846 TNFAIP2 - BCL2L13 438 479 742 TNFAIP3 - BCL2L13 1350 1030 2129 TNFAIP3 - BCL2L13 1804 879 626 TNFRSF1A - BCL2L13 1173 1962 2489 TNFRSF1A - BCL2L13 1577 1512 476 TNFRSF10A - BCL2L13 737 2055 2499 TNFRSF10A - BCL2L13 1534 2360 1105 TNFRSF10B - BCL2L13 1992 885 906 TNFRSF10B - BCL2L13 2177 960 1053 TNFRSF10D - BCL2L13 2204 2159 2343 TNFRSF10D - BCL2L13 171 1983 960 TNFRSF12A - BCL2L13 2183 2509 241 TNFRSF12A - BCL2L13 59 1706 2046 TNFRSF14 - BCL2L13 1852 1974 2339 TNFRSF14 - BCL2L13 2459 2381 1187 TNFRSF21 - BCL2L13 2280 2424 2301 TNFRSF21 - BCL2L13 52 1054 394 TNFSF10 - BCL2L13 1088 2429 1803 TNFSF10 - BCL2L13 1764 1186 1227 TNFSF15 - BCL2L13 1286 2438 2252 TNFSF15 - BCL2L13 638 1962 814 Table 11: 2nd order combinatorial hypotheses between BCL and TNF - BCL6; BCL10 was up regulated w.r.t TNF-RSF10D/RSF12A. These are reflected in rankings of 1831 (laplace) and 2040 (rbf) for TNFRSF10D - BCL10; and 2015 (laplace) and 1883 (rbf) for TNFRSF12A - BCL10;335 On the right side is the ranking of TNF family w.r.t BCL family. We found TNFRSF10A/RSF10B/RSF10D/RSF12A/RSF14/SF10 to be up regulated w.r.t BCL2L1. 19 These are reflected in rankings of 2273 (laplace) and 1928 (linear) for TNFRSF10A - BCL2L1; 2252 (linear) and 2217 (rbf) for TNFRSF10B - BCL2L1; 1868 (laplace), 2420 (linear) and 2392 (rbf) for TNFRSF10D - BCL2L1; 1923 (laplace) and 1936 (rbf)340 for TNFRSF12A - BCL2L1; 2350 (linear) and 2414 (rbf) for TNFRSF14 - BCL2L1 and 2115 (laplace) and 2299 (linear) for TNFSF10 - BCL2L1; TNFRSF10B was up regulated w.r.t BCL2L2. This is reflected in rankings of 2153 (laplace) and 1983 (rbf) for TNFRSF10B - BCL2L2; TNFRSF14 was up regulated w.r.t BCL2L13. This is reflected in rankings of 2459 (laplace) and 2381 (linear) for TNFRSF14 - BCL2L13;345 TNF-RSF10A/SF10D/SF10 were up regulated w.r.t BCL3. These are reflected in rankings of 2388 (laplace) and 2493 (linear) for TNFRSF10A - BCL3; 2213 (laplace) and 1972 (rbf) for TNFRSF10D - BCL3 and 1926 (laplace) and 2107 (rbf) for TNFSF10 - BCL3; TNFRSF12A was up regulated w.r.t BCL6. This is reflected in rankings of 2200 (linear) and 1910 (rbf) for TNFRSF12A - BCL6; TNF-AIP1/SF10/SF15350 was up regulated w.r.t BCL9L. This is reflected in rankings of 2470 (linear) and 1802 (rbf) for TNFAIP1 - BCL9L; 1812 (laplace), 1796 (linear) and 2095 (rbf) for TNFSF10 - BCL9L; and 1939 (laplace), 2114 (linear) and 2405 (rbf) for TNFSF15 - BCL9L; TNFRSF14 was up regulated w.r.t BCL10. This is reflected in rankings of 1894 (linear) and 2227 (rbf) for TNFRSF14 - BCL10;355 One can also interpret the results of the table 13 graphically, with the following influences - •BCL w.r.t TNF with TNF, TNF-AIP1/RSF1A/RSF10B/RSF10D/RSF12A/RSF14/RSF21/SF10/SF15 −>BCL2L2; TNF, TNF-AIP1/RSF1A/RSF10A/RSF10D/RSF12A/ RSF14/RSF21/SF10/SF15 −>BCL2L13; TNFRSF10B −>BCL3; TNF, TNF-AIP1/AIP2/RSF1A/RSF10A/RSF10D/RSF21 −>BCL6; TNF-RSF10D/RSF12A −>BCL10; •TNF w.r.t BCL with TNF-RSF10A/RSF10B/RSF10D/RSF12A/360 RSF14/SF10 <−BCL2L1; TNF-RSF10B <−BCL2L2; TNF-RSF14 <−BCL2L13; TNF-RSF10A/SF10D/SF10 <−BCL3; TNF-RSF12A <−BCL6; TNF-AIP1 <− BCL9L; TNF-SF10/SF15 <−BCL9L and TNF-RSF14 <−BCL10. 20 RANKING TNF FAMILY VS BCL FAMILY RANKING OF BCL3 W.R.TTNF FAMILY RANKING OF TNF FAMILY W.R.TBCL3 laplace linear rbf laplace linear rbf TNF - BCL3 652 370 642 TNF - BCL3 598 311 2473 TNFAIP1 - BCL3 168 723 124 TNFAIP1 - BCL3 596 500 158 TNFAIP2 - BCL3 642 856 1098 TNFAIP2 - BCL3 59 776 323 TNFAIP3 - BCL3 2377 534 567 TNFAIP3 - BCL3 300 940 1527 TNFRSF1A - BCL3 163 206 740 TNFRSF1A - BCL3 83 476 1355 TNFRSF10A - BCL3 799 865 1044 TNFRSF10A - BCL3 2388 2493 88 TNFRSF10B - BCL3 1632 2427 1868 TNFRSF10B - BCL3 757 1508 1062 TNFRSF10D - BCL3 1110 858 714 TNFRSF10D - BCL3 2213 1091 1972 TNFRSF12A - BCL3 273 931 623 TNFRSF12A - BCL3 671 1869 1286 TNFRSF14 - BCL3 232 85 1422 TNFRSF14 - BCL3 2149 1311 1650 TNFRSF21 - BCL3 340 1384 2474 TNFRSF21 - BCL3 411 729 998 TNFSF10 - BCL3 1537 1753 1638 TNFSF10 - BCL3 1926 1523 2107 TNFSF15 - BCL3 129 284 729 TNFSF15 - BCL3 1649 1032 2122 RANKING OF BCL6 W.R.TTNF FAMILY RANKING OF TNF FAMILY W.R.TBCL6 laplace linear rbf laplace linear rbf TNF - BCL6 2271 2071 1810 TNF - BCL6 806 437 1411 TNFAIP1 - BCL6 2135 2158 1330 TNFAIP1 - BCL6 1089 850 372 TNFAIP2 - BCL6 2340 1428 1808 TNFAIP2 - BCL6 152 334 703 TNFAIP3 - BCL6 267 1336 1219 TNFAIP3 - BCL6 1884 1935 855 TNFRSF1A - BCL6 1598 1771 2503 TNFRSF1A - BCL6 788 741 1130 TNFRSF10A - BCL6 1327 1831 2096 TNFRSF10A - BCL6 607 1249 2360 TNFRSF10B - BCL6 1373 1873 1264 TNFRSF10B - BCL6 1746 1282 361 TNFRSF10D - BCL6 2213 2188 788 TNFRSF10D - BCL6 1540 1301 2008 TNFRSF12A - BCL6 1867 99 2261 TNFRSF12A - BCL6 545 2200 1910 TNFRSF14 - BCL6 1409 1337 2028 TNFRSF14 - BCL6 731 1302 1902 TNFRSF21 - BCL6 645 2071 2335 TNFRSF21 - BCL6 40 1850 50 TNFSF10 - BCL6 919 445 99 TNFSF10 - BCL6 2119 1102 1626 TNFSF15 - BCL6 2106 1692 1451 TNFSF15 - BCL6 969 1475 226 RANKING OF BCL9L W.R.TTNF FAMILY RANKING OF TNF FAMILY W.R.TBCL9L laplace linear rbf laplace linear rbf TNF - BCL9L 1964 1172 1478 TNF - BCL9L 2218 98 425 TNFAIP1 - BCL9L 439 1445 264 TNFAIP1 - BCL9L 766 2470 1802 TNFAIP2 - BCL9L 1250 1473 696 TNFAIP2 - BCL9L 646 567 85 TNFAIP3 - BCL9L 534 630 618 TNFAIP3 - BCL9L 1046 1223 2296 TNFRSF1A - BCL9L 2050 1096 978 TNFRSF1A - BCL9L 863 500 825 TNFRSF10A - BCL9L 212 1682 980 TNFRSF10A - BCL9L 2140 241 1547 TNFRSF10B - BCL9L 952 698 685 TNFRSF10B - BCL9L 286 414 2046 TNFRSF10D - BCL9L 1315 181 1423 TNFRSF10D - BCL9L 1956 112 990 TNFRSF12A - BCL9L 430 1167 1470 TNFRSF12A - BCL9L 1797 1280 1699 TNFRSF14 - BCL9L 1433 635 1497 TNFRSF14 - BCL9L 670 1055 1540 TNFRSF21 - BCL9L 495 2326 468 TNFRSF21 - BCL9L 1291 1378 246 TNFSF10 - BCL9L 1889 974 183 TNFSF10 - BCL9L 1812 1796 2095 TNFSF15 - BCL9L 878 2389 71 TNFSF15 - BCL9L 1939 2114 2405 RANKING OF BCL10 W.R.TTNF FAMILY RANKING OF TNF FAMILY W.R.TBCL10 laplace linear rbf laplace linear rbf TNF - BCL10 708 79 979 TNF - BCL10 1931 114 1573 TNFAIP1 - BCL10 1657 805 1298 TNFAIP1 - BCL10 690 1941 7 TNFAIP2 - BCL10 1101 2197 312 TNFAIP2 - BCL10 523 2099 339 TNFAIP3 - BCL10 985 813 767 TNFAIP3 - BCL10 935 595 1870 TNFRSF1A - BCL10 745 1191 1288 TNFRSF1A - BCL10 362 173 448 TNFRSF10A - BCL10 451 819 954 TNFRSF10A - BCL10 1547 415 2426 TNFRSF10B - BCL10 791 537 1446 TNFRSF10B - BCL10 582 658 1464 TNFRSF10D - BCL10 1831 1694 2040 TNFRSF10D - BCL10 302 19 2497 TNFRSF12A - BCL10 2015 1072 1883 TNFRSF12A - BCL10 1865 1234 1540 TNFRSF14 - BCL10 254 1400 847 TNFRSF14 - BCL10 1175 1894 2227 TNFRSF21 - BCL10 1912 571 958 TNFRSF21 - BCL10 848 1943 418 TNFSF10 - BCL10 1743 931 1657 TNFSF10 - BCL10 2020 1522 1054 TNFSF15 - BCL10 254 1469 577 TNFSF15 - BCL10 1256 188 1074 Table 12: 2nd order combinatorial hypotheses between BCL and TNF 21 UNEXPLORED COMBINATORIAL HYPOTHESES BCL w.r.t TNF TNF, TNF-AIP1/RSF1A/RSF10B/RSF10D/RSF12A/RSF14/RSF21/SF10/SF15 BCL2L2 TNF, TNF-AIP1/RSF1A/RSF10A/RSF10D/RSF12A/RSF14/RSF21/SF10/SF15 BCL2L13 TNFRSF10B BCL3 TNF, TNF-AIP1/AIP2/RSF1A/RSF10A/RSF10D/RSF21 BCL6 TNF-RSF10D/RSF12A BCL10 TNF w.r.t BCL TNF-RSF10A/RSF10B/RSF10D/RSF12A/RSF14/SF10 BCL2L1 TNF-RSF10B BCL2L2 TNF-RSF14 BCL2L13 TNF-RSF10A/SF10D/SF10 BCL3 TNF-RSF12A BCL6 TNF-AIP1 BCL9L TNF-SF10/SF15 BCL9L TNF-RSF14 BCL10 Table 13: 2nd order combinatorial hypotheses between TNF and BCL family. 22 Conclusion Presented here are a range of multiple synergistic TNF 2nd order combinations that365 were ranked via a search engine. Later, two way cross family analysis between components of these combinations were conducted. Via majority voting across the ranking methods, it was possible to find plausible unexplored synergistic combinations that might be prevalent in CRC cells after treatment with ETC-1922159 drug. The two-way cross family analysis also assists in deriving influences between components370 which serve as hypotheses for further tests. If found true, it paves way for biologists/oncologists to further investigate and understand the mechanism behind the synergy through wet experiments. Conflict of interest There are no conflicts to declare.375 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 author380 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. [40]. The ETC-1922159 was released in Singapore in July 2015 under the flagship of the Agency for Science, Technology and Research (A*STAR) and Duke-National University of385 Singapore Graduate Medical School (Duke-NUS). 4. References [1] S. Sinha, Inchoative discovery of plausible (un)explored synergistic combinatorial biological hypotheses for static/time series wnt measurements via ranking search engine : Biosearch engine design, Preprints (2018). [2] S. Sinha, Sensitivity analysis based ranking reveals unknown biological hypotheses for down regulated genes in390 time buffer during administration of porcn-wnt inhibitor etc-1922159 in crc, bioRxiv (2017) 180927. [3] S. Sinha, Prioritizing 2nd order interactions via support vector ranking using sensitivity indices on time series wnt measurements, bioRxiv (2017) 060228. [4] 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. 217–226.395 23 [5] R. S. Brooks, E. D. Ciappio, G. Bennett, J. W. Crott, J. B. Mason, Z. Liu, Tnf-αinduced alterations in the wnt signaling cascade: a potential mechanism for obesity-associated colorectal tumorigenesis, 2010. [6] G. Adami, G. Orsolini, S. Adami, O. Viapiana, L. Idolazzi, D. Gatti, M. Rossini, Effects of tnf inhibitors on parathyroid hormone and wnt signaling antagonists in rheumatoid arthritis, Calcified tissue international 99 (2016) 360–364.400 [7] A. Hiyama, K. Yokoyama, T. Nukaga, D. Sakai, J. Mochida, A complex interaction between wnt signaling and tnf-α in nucleus pulposus cells, Arthritis research & therapy 15 (2013) R189. [8] B. Ma, M. O. Hottiger, Crosstalk between wnt/β-catenin and nf-κb signaling pathway during inflammation, Frontiers in immunology 7 (2016) 378. [9] A. Roubert, K. Gregory, Y. Li, A. C. Pfalzer, J. Li, S. S. Schneider, R. J. Wood, Z. Liu, The influence of tumor necro-405 sis factor-αon the tumorigenic wnt-signaling pathway in human mammary tissue from obese women, Oncotarget 8 (2017) 36127. [10] J. Jang, Y. Jung, S. Chae, S.-I. Chung, S.-M. Kim, Y. Yoon, Wnt/β-catenin pathway modulates the tnf-α-induced inflammatory response in bronchial epithelial cells, Biochemical and biophysical research communications 484 (2017) 442–449.410 [11] Y. H. Sheng, Y. He, S. Z. Hasnain, R. Wang, H. Tong, D. T. Clarke, R. Lourie, I. Oancea, K. Wong, J. W. Lumley, et al., Muc13 protects colorectal cancer cells from death by activating the nf-κb pathway and is a potential therapeutic target, Oncogene 36 (2017) 700. [12] P. Dharmani, P. Leung, K. Chadee, Tumor necrosis factor-αand muc2 mucin play major roles in disease onset and progression in dextran sodium sulphate-induced colitis, PloS one 6 (2011) e25058.415 [13] S. J. Levine, P. Larivee, C. Logun, C. W. Angus, F. P. Ognibene, J. H. Shelhamer, Tumor necrosis factor-alpha induces mucin hypersecretion and muc-2 gene expression by human airway epithelial cells., American Journal of Respiratory Cell and Molecular Biology 12 (1995) 196–204. [14] M. A. Sikder, H. J. Lee, M. Z. Mia, S. H. Park, J. Ryu, J.-H. Kim, S. Y. Min, J.-H. Hong, J. H. Seok, C. J. Lee, Inhibition of tnf-α-induced muc5ac mucin gene expression and production by wogonin through the inactivation of420 nf-κb signaling in airway epithelial cells, Phytotherapy Research 28 (2014) 62–68. [15] M. X. Shao, J. A. Nadel, Neutrophil elastase induces muc5ac mucin production in human airway epithelial cells via a cascade involving protein kinase c, reactive oxygen species, and tnf-α-converting enzyme, The Journal of Immunology 175 (2005) 4009–4016. [16] J. M. Lora, D. M. Zhang, S. M. Liao, T. Burwell, A. M. King, P. A. Barker, L. Singh, M. Keaveney, J. Morgenstern,425 J. C. Guti´ errez-Ramos, et al., Tumor necrosis factor-αtriggers mucus production in airway epithelium through an iκb kinase β-dependent mechanism, Journal of Biological Chemistry 280 (2005) 36510–36517. [17] B. M. Fischer, L. G. Rochelle, J. A. Voynow, N. J. Akley, K. B. Adler, Tumor necrosis factor-αstimulates mucin secretion and cyclic gmp production by guinea pig tracheal epithelial cells in vitro, American Journal of Respiratory Cell and Molecular Biology 20 (1999) 413–422.430 [18] J. Lin, A. Haruta, H. Kawano, S. B. Ho, G. L. Adams, S. K. Juhn, Y. Kim, Induction of mucin gene expression in middle ear of rats by tumor necrosis factorα: Potential cause for mucoid otitis media, The Journal of infectious diseases 182 (2000) 882–887. [19] J.-H. Yoon, K.-S. Kim, H. U. Kim, J. A. Linton, J.-G. Lee, Effects of tnf-a and il-1 ß on mucin, lysozyme, il-6 and il-8 in passage-2 normal human nasal epithelial cells, Acta oto-laryngologica 119 (1999) 905–910.435 [20] M. F. Mercogliano, M. De Martino, L. Venturutti, M. A. Rivas, C. J. Proietti, G. Inurrigarro, I. Frahm, D. H. Allemand, E. G. Deza, S. Ares, et al., Tnfα-induced mucin 4 expression elicits trastuzumab resistance in her2positive breast cancer, Clinical Cancer Research 23 (2017) 636–648. [21] R. T. Scarl, C. M. Lawrence, H. M. Gordon, C. S. Nunemaker, Steap4: its emerging role in metabolism and homeostasis of cellular iron and copper, Journal of Endocrinology 234 (2017) R123–R134.440 24 [22] M. Moldes, F. Lasnier, X. Gauthereau, C. Klein, J. Pairault, B. F` eve, A.-M. Chambaut-Gu´ erin, Tumor necrosis factor-α-induced adipose-related protein (tiarp), a cell-surface protein that is highly induced by tumor necrosis factor-αand adipose conversion, Journal of Biological Chemistry 276 (2001) 33938–33946. [23] F. Zhang, Y. Tao, Z. Zhang, X. Guo, P. An, Y. Shen, Q. Wu, Y. Yu, F. Wang, Metalloreductase steap3 coordinates the regulation of iron homeostasis and inflammatory responses, Haematologica 97 (2012) 1826–1835.445 [24] Y. Tanaka, I. Matsumoto, K. Iwanami, A. Inoue, N. Umeda, M. Sugihara, T. Hayashi, S. Ito, T. Sumida, Sixtransmembrane epithelial antigen of prostate 4 (steap4) is expressed on monocytes/neutrophils, and is regulated by tnf antagonist in patients with rheumatoid arthritis., Clinical and experimental rheumatology 30 (2012) 99–102. [25] Y. Tanaka, I. Matsumoto, K. Iwanami, A. Inoue, R. Minami, N. Umeda, A. Kanamori, N. Ochiai, K. Miyazawa, M. Sugihara, Six-transmembrane epithelial antigen of prostate4 (steap4) is a tumor necrosis factor alpha-induced450 protein that regulates il-6, il-8, and cell proliferation in synovium from patients with rheumatoid arthritis, Modern rheumatology 22 (2012) 128–136. [26] G. H. Gauss, M. D. Kleven, A. K. Sendamarai, M. D. Fleming, C. M. Lawrence, The crystal structure of sixtransmembrane epithelial antigen of the prostate 4 (steap4), a ferri/cuprireductase, suggests a novel interdomain flavin-binding site, Journal of Biological Chemistry 288 (????) 20668–20682.455 [27] C.-m. ZHANG, X. Chi, B. Wang, M. Zhang, Y.-h. NI, R.-h. CHEN, X.-n. LI, X.-r. GUO, Downregulation of steap4, a highly-expressed tnf-α-inducible gene in adipose tissue, is associated with obesity in humans 1, Acta Pharmacologica Sinica 29 (2008) 587–592. [28] Y. Liang, X. Xing, M. A. Beamer, W. R. Swindell, M. K. Sarkar, L. W. Roberts, J. J. Voorhees, J. M. Kahlenberg, P. W. Harms, A. Johnston, et al., Six-transmembrane epithelial antigens of the prostate comprise a novel inflam-460 matory nexus in patients with pustular skin disorders, Journal of Allergy and Clinical Immunology 139 (2017) 1217–1227. [29] I. M. Gomes, C. J. Maia, C. R. Santos, Steap proteins: from structure to applications in cancer therapy, Molecular Cancer Research 10 (2012) 573–587. [30] B. Fu, S. Li, L. Wang, M. A. Berman, M. E. Dorf, The ubiquitin conjugating enzyme ube2l3 regulates tnfα-induced465 linear ubiquitination, Cell research 24 (2014) 376. [31] Y.-P. Li, S. H. Lecker, Y. Chen, I. D. Waddell, A. L. Goldberg, M. B. Reid, Tnf-αincreases ubiquitin-conjugating activity in skeletal muscle by up-regulating ubch2/e220k, The FASEB Journal 17 (2003) 1048–1057. [32] N. Shembade, A. Ma, E. W. Harhaj, Inhibition of nf-κb signaling by a20 through disruption of ubiquitin enzyme complexes, Science 327 (2010) 1135–1139.470 [33] M. Tamatani, Y. H. Che, H. Matsuzaki, S. Ogawa, H. Okado, S.-i. Miyake, T. Mizuno, M. Tohyama, Tumor necrosis factor induces bcl-2 and bcl-x expression through nfκb activation in primary hippocampal neurons, Journal of Biological Chemistry 274 (1999) 8531–8538. [34] S. Ho, B. Winkler-Lowen, D. Morrish, J. Dakour, H. Li, L. Guilbert, The role of bcl-2 expression in egf inhibition of tnf-α/ifn-γ-induced villous trophoblast apoptosis, Placenta 20 (1999) 423–430.475 [35] L. Genestier, N. Bonnefoy-Berard, J.-P. Rouault, M. Flacher, J.-P. Revillard, Tumor necrosis factor-αup-regulates bcl-2 expression and decreases calcium-dependent apoptosis in human b cell lines, International immunology 7 (1995) 533–540. [36] M. J¨ a¨ attel¨ a, M. Benedict, M. Tewari, J. Shayman, V. Dixit, Bcl-x and bcl-2 inhibit tnf and fas-induced apoptosis and activation of phospholipase a2 in breast carcinoma cells., Oncogene 10 (1995) 2297–2305.480 [37] J. Kim, S. Lee, J. Park, Y. Yoo, Tnf-α-induced ros production triggering apoptosis is directly linked to romo1 and bcl-x l, Cell death and differentiation 17 (2010) 1420. [38] H. Kuwata, Y. Watanabe, H. Miyoshi, M. Yamamoto, T. Kaisho, K. Takeda, S. Akira, Il-10-inducible bcl-3 negatively regulates lps-induced tnf-αproduction in macrophages, Blood 102 (2003) 4123–4129. [39] C. Esche, G. V. Shurin, J. M. Kirkwood, G.-Q. Wang, H. Rabinowich, G. Pirtskhalaishvili, M. R. Shurin, Tu-485 mor necrosis factor-α-promoted expression of bcl-2 and inhibition of mitochondrial cytochrome c release mediate resistance of mature dendritic cells to melanoma-induced apoptosis, Clinical cancer research 7 (2001) 974s–979s. [40] 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. 25