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

Machine learning discoveries of Wnt-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 Wnt-X (X, a particular gene/protein) at 2nd order level after drug administration. These rankings reveal which Wnt-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 Wnt and X. In this research work, we cover combinations of Wnt with Achaete-scute complex homolog 2 (ASCL2), ATPbinding cassette (ABC) domain transporters, Interleukin (IL), ubiquitin conjugating enzyme E2 (UBE2) family, exosome (EXOSC), caspase (CASP), TP53 and B-cell lymphoma (BCL) family. Keywords: WNT, 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 Introduction In the unpublished preprint Sinha [1], a frame work of a search engine was developed which can rank combinations of factors (genes/proteins) in a signaling pathway.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 Interested readers are advised to go through unpublished preprints IML dicoveries of Wnt-X synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at (1) the recently concluded first ever Wnt Gordon Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA.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 Sinha [1] and Sinha [2] for details regarding the search engine and the discoveries 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. WNT related synergies 3.1.1. WNT10B-ASCL2 WNT10B has been found to be implicated in a range of cancers. In gastric cancer, the40 knockdown of WNT10B showed reduced expression of cell proliferation and migration as well as inhibition of epithelial-mesenchymal transition Wu et al. [5]. On the other hand, WNT10B is also involved in the formation of bone mass and progenitor maintenance of various kinds of tissue, while deletion of the same leads to loss of bone mass and mesenchymal progenitor cells Stevens et al. [6]. Their contribution is also reported45 in axonal regeneration in injured CNS Tassew et al. [7]. Furthermore, like WNT10B, 2 WNT10A and WNT6 have shown to play a major role in inhibiting adipogenesis and stimulates osteoblastogenesis while regulating the mesenchymal stem cells Cawthorn et al. [8] & Collins et al. [9]. Involvement in heptocellular carcinoma of WNT10B has been found wherein it is shown that stable silencing of WNT10B leads to significant50 reduction in proliferation, colony formation, migration and invasion in HepG2 HCC cell line Wu et al. [10]. Its implication in breast cancer Wend et al. [11] & Chen et al. [12] as well as endometrial cancer Chen et al. [13] has also been reported. In colorectal cancer, WNT10B has shown to play a dual function of both oncogenesis promotion via β-catenin/TCF pathway and the inhibition of cell growth, possibly55 via FGF family of proteins Yoshikawa et al. [14]. Methylation of WNT10B has been found in the some of the cancer cell lines while its reversal has lead to over-expression of the WNT10B. However, the over-expression of WNT10B has lead to reduced cell growth in cancer, indicating a β-catenin independent component to be behind such a phenomena. Methylation of over-expressed WNT10B and synergistic work with FGF60 family of proteins later indicate the promotion of oncogenesis, as has been demonstrated in Yoshikawa et al. [14]. In a more recent work, ASCL2 has been found to play a major role in stemness in colon crypts and is implicated in colon cancer Zhu et al. [15]. Switching off the ASCL2 leads to a literal blockage of the stemness process and vice versa. At the65 downstream level, ASCL2 is regulated by TCF4/β-catenin via non-coding RNA target named WiNTRLINC1 Giakountis et al. [16]. Activation of ASCL2 leads to feedforward transcription of the non-coding RNA and thus a loop is formed which helps in the stemness and is highly effective in colon cancer. At the upstream level, ASCL2 is known act as a WNT/RSPONDIN switch that controls the stemness Schuijers et al.70 [17]. It has been shown that removal of RSPO1 lead to decrease in the Wnt signaling due to removal of the FZD receptors that led to reduced expression of ASCL2. Also, low levels of LGR5 were observed due to this phenomena. The opposite happened by increasing the RSPO1 levels. After the drug treatment, it was found that ASCL2 was highly suppressed pointing to the inhibition of stemness in the colorectal75 cancer cells. Also, Schuijers et al. [17] show that by genetically disrupting PORCN or inducing a PORCN inhibitor (like IWP-2), there is loss of stem cell markers like LGR5 and RNF43, which lead to disappearance of stem cells and moribund state of mice. A similar affect can be found with ETC-1922159, where there is suppression of RNF43 and LGR5 that lead to inhibition of the Wnt pathway and thus the ASCL280 regulation. These wet lab evidences are confirmed in the relatively low ranking of the combination ASCL2-RNF43 via the inhibition of PORCN-WNT that leads to blocking of the stemness that is induced by ASCL2. Since ASCL2 is directly mediated by the WNT proteins, the recorded ASCL2-WNT10B combination showed low priority ranking of 488, 497 and 321 for rbf, laplace and linear kernels, respectively, thus indicating85 a possible connection between WNT10B and ASCL2 activation. WNT10B might be playing a crucial role in stemness. This is further confirmed by wet lab experiments in Reddy et al. [18], which show BVES deletion results in amplified stem cell activity and Wnt signaling after radiation. WNT10B has been implicated in colorectal cancer Yoshikawa et al. [14].90 3 RANKING ABC FAMILY W.R.TWNT FAMILY RANKING OF ABC FAMILY W.R.TWNT-2B RANKING OF ABC FAMILY W.R.TWNT4 laplace linear rbf laplace linear rbf WNT2B - ABC-A5 2108 310 72 ABC-A5 - WNT4 359 1285 433 ABC-B11 - WNT2B 319 2132 18 ABC-B11 - WNT4 872 1284 867 WNT2B - ABC-C3 1853 262 2498 ABC-C3 - WNT4 10 617 296 WNT2B - ABC-C5 2213 1685 840 WNT4 - ABC-C5 1383 2119 215 WNT2B - ABC-C13 1149 1191 2175 WNT4 - ABC-C13 1649 1814 542 WNT2B - ABC-D1 1119 177 2163 ABC-D1 - WNT4 1041 1171 1740 WNT2B - ABC-G1 1068 1583 214 ABC-G1 - WNT4 1020 1146 2025 WNT2B - ABC-G2 1500 1533 172 ABC-G2 - WNT4 784 1431 435 RANKING OF ABC FAMILY W.R.TWNT-7B RANKING OF ABC FAMILY W.R.TWNT-9A laplace linear rbf laplace linear rbf ABC-A5 - WNT7B 1550 516 995 ABC-A5 - WNT9A 735 349 1479 ABC-B11 - WNT7B 968 599 324 ABC-B11 - WNT9A 843 1647 689 ABC-C3 - WNT7B 694 1668 695 ABC-C3 - WNT9A 1590 359 2136 WNT7B - ABC-C5 979 1715 2268 ABC-C5 - WNT9A 1295 368 2265 WNT7B - ABC-C13 950 2245 2298 ABC-C13 - WNT9A 1394 2294 1134 ABC-D1 - WNT7B 252 850 1215 ABC-D1 - WNT9A 910 2367 675 ABC-G1 - WNT7B 269 733 1160 ABC-G1 - WNT9A 426 2457 1074 ABC-G2 - WNT7B 1717 224 264 ABC-G2 - WNT9A 1108 2350 960 Table 1: 2nd order interaction ranking between ABC w.r.t WNT family members. 3.1.2. ABC transporters - WNT cross family analysis Hlavata et al. [19] have shown the role of ABC transporters in progression and clinical outcome of colorectal cancer. Work by Kobayashi et al. [20] show that Wnt-β catenin signaling regulates ABCC3 (MRP3) transporter expression in colorectal cancer. ABCA2 belongs to the category of ABC transporters that play an essential role95 in the development of resistance by the efflux of anticancer agents outside of cancer cells Hlavata et al. [19]. Hlavata et al. [19] observed that ABCA2 had no significant change/affect in colorectal cancer cases. Kobayashi et al. [20] found ABCA2 to be downregulated in colorectal cancer case. In ETC-1922159 affected CRC cells, down regulation of ABCA2 was observed, after the inhibition of proliferation in respective100 cells. Multiple members of ABC transporters and WNTs were found to be UP regulated after ETC-159 in CRC cells and WNTs are known to regulate ABCs. Below, we show a range of up regulated, possible unknown and unexplored synergistic 2nd order combinations that were ranked by the search engine. Note that the high numerical valued ranks (i.e nearing to 1800/2000 and above) indicate high potential of synergy105 that might be existing in CRC cells after the drug administration. Majority voting of rankings across the three different kernels point to the potential of the synergistic discovery. Wet labs investigations will assist in confirmation of these discoveries and if proven true, might lead to understanding of further mechanism between the components.110 Tables 1 and 2 show the rankings of ABC family w.r.t to WNT family members and WNT family w.r.t to ABC family members, respectively. From these two tables, 4 RANKING WNT FAMILY W.R.TABC FAMILY RANKING OF WNT FAMILY W.R.TABC-A5 RANKING OF WNT FAMILY W.R.TABC-B11 laplace linear rbf laplace linear rbf ABC-A5 - WNT2B 1549 2018 2132 WNT2B - ABC-B11 1083 703 1887 ABC-A5 - WNT4 1375 2436 2449 WNT4 - ABC-B11 156 298 1517 ABC-A5 - WNT7B 2420 1527 460 WNT7B - ABC-B11 1134 204 2323 ABC-A5 - WNT9A 1989 2209 2365 WNT9A - ABC-B11 226 2134 1480 RANKING OF WNT FAMILY W.R.TABC-C3 RANKING OF WNT FAMILY W.R.TABC-C5 laplace linear rbf laplace linear rbf ABC-C3 - WNT2B 1127 1482 1905 WNT2B - ABC-C5 1970 2309 2248 ABC-C3 - WNT4 897 1454 489 WNT4 - ABC-C5 2129 229 230 ABC-C3 - WNT7B 656 2080 772 WNT7B - ABC-C5 1539 756 1258 ABC-C3 - WNT9A 2339 1616 814 ABC-C5 - WNT9A 213 2183 2480 RANKING OF WNT FAMILY W.R.TABC-C13 RANKING OF WNT FAMILY W.R.TABC-D1 laplace linear rbf laplace linear rbf WNT2B - ABC-C13 950 2150 2048 WNT2B - ABC-D1 1751 1370 1174 WNT4 - ABC-C13 538 326 2242 WNT4 - ABC-D1 45 1784 101 WNT7B - ABC-C13 2508 1830 1219 WNT7B - ABC-D1 2238 2021 1121 WNT9A - ABC-C13 738 2501 634 WNT9A - ABC-D1 732 1526 1759 RANKING OF WNT FAMILY W.R.TABC-G1 RANKING OF WNT FAMILY W.R.TABC-G2 laplace linear rbf laplace linear rbf WNT2B - ABC-G1 318 775 2040 WNT2B - ABC-G2 1342 1987 1230 WNT4 - ABC-G1 2169 157 39 WNT4 - ABC-G2 862 1352 1985 WNT7B - ABC-G1 587 1808 1866 WNT7B - ABC-G2 2334 2145 1526 WNT9A - ABC-G1 856 2350 920 WNT9A - ABC-G2 1919 1284 2003 Table 2: 2nd order interaction ranking between WNT w.r.t ABC family members. we derive the plausible influences that might be existing in a two way format that is depicted in table 3. In table 1, WNT2B - ABC-C3 combination shows a majority voting of 1853 (laplace) and 2498 (rbf). Similarly, WNT7B - ABC-C13 shows a majority115 voting of 2245 (linear) and 2298 (rbf). These two combinations are depicted in table 3 as ABC members influenced by WNT members (see under ABC w.r.t WNT). Reversibily, in table 2 ABC-A5 - WNT2B shows a majority voting of 2018 (linear) and 2132 (rbf), ABC-A5 - WNT4 shows a majority voting of 2436 (linear) and 2449 (rbf), ABC-A5 - WNT9A shows a majority voting of 1989 (laplace), 2209 (linear) and120 2365 (rbf), WNT2B - ABC-C5 shows a majority voting of 1970 (laplace), 2309 (linear) and 2248 (rbf), ABC-C5 - WNT9A shows a majority voting of 2183 (linear) and 2480 (rbf), WNT2B - ABC-C13 shows a majority voting of 2150 (linear) and 2048 (rbf), WNT7B - ABC-C13 shows a majority voting of 2508 (laplace) and 1830 (linear), WNT7B - ABC-D1 shows a majority voting of 2238 (laplace) and 2021 (linear),125 WNT7B - ABC-G1 shows a majority voting of 1808 (linear) and 1866 (rbf), WNT7B - ABC-G2 shows a majority voting of 2334 (linear) and 2145 (rbf) and WNT9A - ABCG2 shows a majority voting of 1919 (laplace) and 2003 (rbf). These point to WNT members influenced by ABC members (see under WNT w.r.t ABC). Hypothetically, what we find is that the synergies can be bi-directional also and might contain various130 intermitent factors through which the factors might be working synergistically. These hypothese form present themselves as important combinations that might be of interest 5 UNEXPLORED COMBINATORIAL HYPOTHESES ABC w.r.t WNT WNT-2B ABC-C3 WNT-7B ABC-C13 WNT w.r.t ABC ABC-A5 WNT-2B/4/9A WNT-2B/9A ABC-C5 WNT-2B/7B ABC-C13 WNT-7B ABC-D1 WNT-7B ABC-G1 WNT-7B/9A ABC-G2 Table 3: 2nd order combinatorial hypotheses between ABC and WNT family members. to biologists/oncologists. One can also interpret the results of the table 3 graphically, with the following influences - •ABC w.r.t WNT with WNT-2B −>ABC-C3; WNT-7B −>ABC-C13;135 and •WNT w.r.t ABC with ABC-A5 <−WNT-2B/4/9A; WNT-2B/9A <−ABC-C5; WNT-2B/7B <−ABC-C13; WNT-7B <−ABC-D1; WNT-7B <−ABC-G1; WNT7B/9A <−ABC-G2. Thus, in this way, we can utilize the search engine to derive the various probable combinations between the factors of interest and their interdependent influences through the two-way cross family analysis.140 3.1.3. IL - WNT cross family analysis Interleukin (IL) has been found in cross talk with WNT pathway. Kaler et al. [21] show that NFκB induced WNT signaling in colorectal cancer via interleukin-1βIL1B. Further, Zhong et al. [22] have shown that nitric oxide mediates crosstalk between interleukin 1βand Wnt signaling in primary human chondrocytes by reducing DKK1145 and FRZB expression. The role of IL-17 (Interleukin-17) family is known to be controversial in CRC, however there are cases were it has been reported to be a prognostic marker for colorectal cancer Lin et al. [23] & Housseau et al. [24]. A homologue of the family, IL-17D a novel cytokine has been discovered Starnes et al. [25] and found to play a role in many of the cancers. In cells treated with ETC-1922159, IL-150 17D was found to be down regulated and reversibly it must have been regulated in the colorectal cancer cases. Recently, crosstalk between WNT/β-Catenin and NF-κB signaling pathway during inflammation has been reported by Ma and Hottiger [26]. 6 Ma et al. [27] also show WNT/β-catenin negative feedback loop inhibits IL-1 induced matrix metalloproteinase expression in human articular chondrocytes. Masckauch´ an155 et al. [28] conclude that WNT/β-catenin signaling promotes angiogenesis possibly via the induction of known angiogenic regulators such as Interleukin-8. In mouse colon, Interleukin-1 signaling is shown to mediate obesity-promoted elevations in inflammatory cytokines, WNT activation, and epithelial proliferation by Pfalzer et al. [29]. In pulmonary fibrosis, Aumiller et al. [30] show that WNT/β-Catenin signaling induces160 IL-1βexpression by alveolar epithelial cells. Chen et al. [31] show that IL-23 promotes the epithelial-mesenchymal transition of oesophageal carcinoma cells via the WNT/βcatenin pathway. Finally, Malysheva et al. [32] show that IL-6/WNT interactions in rheumatoid arthritis. Family members belonging to each of the factors like WNT, IL etc, might be in-165 volved synergistically in pathological case or otherwise. IL and WNT members were found to be up regulated after the treatment of ETC-1922159 in colorectal cancer cells. We present here, multiple plausible and alternative synergistic combinatorial biological hypotheses for IL-WNT combination, which emerge after a cross family member analysis of the in silico revelations pertaining to the components under investigation.170 Table 4 shows IL-WNT two way cross family analysis. The left side of the table contains rankings of IL family with respect to WNTs and the right side of the table contains rankings of WNT family with respect to ILs. Depicted in table are the plausible combinatorial hypotheses derived from majority voting of the rankings in table 4. On the left half, w.r.t WNT2B, IL-6ST/8/17REL show a synergy with175 WNT2B. These are reflected with rankings of 1797 (linear) and 2088 (rbf) for IL-6ST - WNT2B; rankings of 2107 (laplace), 1817 (linear) and 2088 (rbf) for IL-8 - WNT2B and rankings of 1824 (laplace) and 2241 (rbf) for IL-17REL - WNT2B, respectively. W.r.t WNT4, IL-1B/1RAP/15RA/17C show a synergy with WNT4. These are reflected with rankings of 1867 (laplace) and 1976 (linear) for IL-1B - WNT4; rankings180 of 2302 (laplace) and 1826 (linear) for IL-1RAP - WNT4; rankings of 1987 (laplace) and 2265 (linear) for IL-15RA - WNT4 and rankings of 2018 (laplace) and 1881 (linear) for IL-17C - WNT4, respectively. W.r.t WNT7B, IL-1RN/17REL show a synergy with WNT7B. These are reflected with rankings of 1882 (laplace) and 1796 (linear) for IL-1RN - WNT7B and rankings of 2053 (laplace), 2445 (linear) and 2489 (rbf)185 for IL-17REL - WNT4, respectively. W.r.t WNT9A, IL-1RAP/15RA show a synergy with WNT9A. These are reflected with rankings of 2273 (linear) and 2159 (rbf) for IL-1RAP - WNT9A and rankings of 1776 (laplace) and 2380 (linear) for IL-15RA - WNT9A, respectively. On the right half, WNT2B w.r.t IL family, IL-1A/1RAP/8 show a synergy with190 WNT2B. These are reflected with rankings of 2290 (laplace) and 2427 (rbf) for IL-1A - WNT2B; rankings of 2488 (laplace) and 1892 (rbf) for IL-1RAP - WNT2B and rankings of 2157 1824 (laplace) and 2025 (linear) for IL-8 - WNT2B, respectively. WNT4 w.r.t IL family, IL-8/10RB show a synergy with WNT4. These are reflected with rankings of 1980 (laplace) and 2144 (linear) for IL-8 - WNT4 and rankings of 1828195 (laplace), 2259 (linear) and 1993 (rbf) for IL-10RB - WNT4; respectively. WNT7B w.r.t IL family, IL-1A/1RN/6ST/17C show a synergy with WNT7B. These are reflected with rankings of 2134 (linear) and 2312 (rbf) for IL-1A - WNT7B; rankings of 1907 (laplace) and 2162 (linear) for IL-1RN - WNT7B; rankings of 1881 (lin7 ear) and 2020 (rbf) for IL-ST - WNT7B; and rankings of 1956 (laplace), 2388 (lin-200 ear) and 1982 (rbf) for IL-17C - WNT7B, respectively. WNT9A w.r.t IL family, IL-1RAP/15RA/17REL show a synergy with WNT9A. These are reflected with rankings of 2003 (laplace) and 2179 (linear) for IL-1RAP - WNT9A; rankings of 2149 (laplace) and 2362 (linear) for IL-15RA - WNT9A; and rankings of 2101 (laplace) and 1940 (linear) for IL-17REL - WNT9A, respectively. One can also interpret the results205 of the table 5 graphically, with the following influences - •IL w.r.t WNT with IL6ST/8/17REL <−WNT-2B; IL-1B/1RAP/15RA/17C <−WNT-4; IL-1RN/17REL <−WNT-7B; IL-1RAP/15RA <−WNT-9A and •WNT w.r.t IL with IL-1A/1RAP/8 −>WNT-2B; IL-8/10RB −>WNT-4; IL-1A/1RN/6ST/17C −>WNT-7B and IL1RAP/15RA/17REL −>WNT-9A.210 8 RANKING IL FAMILY VS WNT FAMILY RANKING OF IL FAMILY W.R.TWNT-2B RANKING OF WNT-2B W.R.TIL FAMILY laplace linear rbf laplace linear rbf IL1A - WNT2B 6 2363 924 IL1A - WNT2B 2290 1360 2427 IL1B - WNT2B 1015 1278 794 IL1B - WNT2B 847 2168 1369 IL1RAP - WNT2B 1481 1391 799 IL1RAP - WNT2B 2488 35 1892 IL1RN - WNT2B 1229 1967 1582 IL1RN - WNT2B 1307 43 2514 IL2RG - WNT2B 1434 1100 2335 IL2RG - WNT2B 1384 1255 1283 IL6ST - WNT2B 1157 1797 2088 IL6ST - WNT2B 776 242 1481 IL8 - WNT2B 2107 1817 2251 IL8 - WNT2B 2157 2025 593 IL10RB - WNT2B 961 2494 512 IL10RB - WNT2B 2419 856 1419 IL15 - WNT2B 1008 1214 1714 IL15 - WNT2B 1171 625 1215 IL15RA - WNT2B 728 1782 1382 IL15RA - WNT2B 2262 1021 657 IL17C - WNT2B 477 2357 1483 IL17C - WNT2B 1947 1304 1331 IL17REL - WNT2B 1824 12 2241 IL17REL - WNT2B 1980 919 1617 RANKING OF IL FAMILY W.R.TWNT-4 RANKING OF WNT-4 W.R.TIL FAMILY laplace linear rbf laplace linear rbf IL1A - WNT4 2500 1346 955 IL1A - WNT4 507 221 91 IL1B - WNT4 1867 1976 1682 IL1B - WNT4 129 250 291 IL1RAP - WNT4 2302 1826 803 IL1RAP - WNT4 74 19 1553 IL1RN - WNT4 1314 856 104 IL1RN - WNT4 851 1218 2029 IL2RG - WNT4 1289 590 319 IL2RG - WNT4 520 920 424 IL6ST - WNT4 1315 273 2422 IL6ST - WNT4 991 1443 2454 IL8 - WNT4 1722 549 11 IL8 - WNT4 1980 2144 1267 IL10RB - WNT4 1700 153 1055 IL10RB - WNT4 1828 2259 1993 IL15 - WNT4 1012 871 1658 IL15 - WNT4 959 553 448 IL15RA - WNT4 1987 2265 819 IL15RA - WNT4 788 139 645 IL17C - WNT4 2018 1639 1881 IL17C - WNT4 406 276 232 IL17REL - WNT4 1019 425 893 IL17REL - WNT4 955 595 1689 RANKING OF IL FAMILY W.R.TWNT-7B RANKING OF WNT-7B W.R.TIL FAMILY laplace linear rbf laplace linear rbf IL1A - WNT7B 662 950 149 IL1A - WNT7B 1058 2134 2312 IL1B - WNT7B 290 167 502 IL1B - WNT7B 1683 1871 1575 IL1RAP - WNT7B 872 1976 789 IL1RAP - WNT7B 381 1728 1517 IL1RN - WNT7B 1882 1796 503 IL1RN - WNT7B 1907 2162 1605 IL2RG - WNT7B 1381 446 482 IL2RG - WNT7B 1070 1695 2245 IL6ST - WNT7B 819 1284 1528 IL6ST - WNT7B 1268 1881 2020 IL8 - WNT7B 2232 220 701 IL8 - WNT7B 1551 58 2149 IL10RB - WNT7B 1318 1198 656 IL10RB - WNT7B 375 2145 803 IL15 - WNT7B 1000 290 245 IL15 - WNT7B 2307 1524 1687 IL15RA - WNT7B 1535 1054 2204 IL15RA - WNT7B 1575 191 1949 IL17C - WNT7B 515 263 113 IL17C - WNT7B 1956 2388 1982 IL17REL - WNT7B 2053 2445 2489 IL17REL - WNT7B 322 859 1631 RANKING OF IL FAMILY W.R.TWNT-9A RANKING OF WNT-9A W.R.TIL FAMILY laplace linear rbf laplace linear rbf IL1A - WNT9A 199 2228 1270 IL1A - WNT9A 597 1322 469 IL1B - WNT9A 305 2266 466 IL1B - WNT9A 776 652 1010 IL1RAP - WNT9A 1773 2273 2159 IL1RAP - WNT9A 2003 2179 964 IL1RN - WNT9A 2479 1506 1503 IL1RN - WNT9A 1363 1829 1632 IL2RG - WNT9A 1489 598 865 IL2RG - WNT9A 186 260 1276 IL6ST - WNT9A 2229 761 1103 IL6ST - WNT9A 2099 1416 1674 IL8 - WNT9A 346 1103 1910 IL8 - WNT9A 589 1751 1529 IL10RB - WNT9A 1836 1556 1006 IL10RB - WNT9A 1021 2127 1534 IL15 - WNT9A 168 1445 855 IL15 - WNT9A 1357 1025 1709 IL15RA - WNT9A 1776 206 2380 IL15RA - WNT9A 2149 2362 737 IL17C - WNT9A 72 2442 569 IL17C - WNT9A 1532 2465 1607 IL17REL - WNT9A 2512 24 580 IL17REL - WNT9A 2101 1940 313 Table 4: 2nd order interaction ranking between ABC w.r.t IL family members. 9 ings of 2479 (linear) and 1739 (rbf) for WNT7B - CASP4 and rankings of 2278 (lin-320 ear) and 1939 (rbf) for WNT9A - CASP4, respectively. W.r.t CASP5, WNT-7B shows promise of up regulation. This is reflected with rankings of 2112 (laplace), 1919 (linear) and 2440 (rbf) for WNT7B - CASP5. W.r.t CASP7, WNT-2B/4/9A show promise of up regulation. These are reflected with rankings of 2505 (laplace) and 1891 (linear) for WNT2B - CASP7; rankings of 2456 (linear) and 2455 (rbf) for WNT4 - CASP7;325 and rankings of 2183 (laplace) and 1941 (linear) for WNT9A - CASP7, respectively. W.r.t CASP9, WNT-9A shows promise of up regulation. This is reflected with rankings of 2378 (laplace), 2396 (linear) and 2058 (rbf) for WNT9A - CASP9. W.r.t CASP10, WNT-4/9A show promise of up regulation. These are reflected with rankings of 1830 (laplace), 2229 (linear) and 1847 (rbf) for WNT4 - CASP10; and rankings330 of 2185 (laplace) and 1977 (linear) for WNT9A - CASP10, respectively. Finally, w.r.t CASP16, WNT-2B/4/9A show promise of up regulation. These are reflected with rankings of 2197 (laplace), 2489 (linear) and 1775 (rbf) for WNT2B - CASP16; rankings of 2508 (laplace), 1820 (linear) and 1867 (rbf) for WNT7B - CASP16; and rankings of 1943 (laplace) and 1839 (linear) for WNT9A - CASP16, respectively.335 One can also interpret the results of the table 11 graphically, with the following influences - •CASP w.r.t WNT with CASP5 <−WNT2B; CASP9 <−WNT-4/7B/9A; CASP16 <−WNT4 and •WNT w.r.t CASP with. WNT-7B/9A <−CASP4; WNT7B <−CASP5; WNT-2B/4/9A <−CASP7; WNT9A <−CASP9; WNT-4/9A <− CASP10; WNT-2B/7B/9A <−CASP16.340 16 RANKING CASP FAMILY VS WNT FAMILY RANKING OF CASP4 W.R.TWNTS FAMILY RANKING OF CASP5 W.R.TWNTS FAMILY laplace linear rbf laplace linear rbf CASP4 - WNT2B 2265 320 1517 CASP5 - WNT2B 975 2171 2366 CASP4 - WNT4 1050 1081 558 CASP5 - WNT4 1788 1356 569 CASP4 - WNT7B 622 9 632 CASP5 - WNT7B 716 978 606 CASP4 - WNT9A 446 1413 583 CASP5 - WNT9A 383 808 147 RANKING OF CASP7 W.R.TWNTS FAMILY RANKING OF CASP9 W.R.TWNTS FAMILY laplace linear rbf laplace linear rbf CASP7 - WNT2B 1152 305 248 CASP9 - WNT2B 1345 1501 1328 CASP7 - WNT4 936 1260 1787 CASP9 - WNT4 1344 2472 2200 CASP7 - WNT7B 901 1403 1303 CASP9 - WNT7B 2196 1935 1713 CASP7 - WNT9A 1330 1527 2436 CASP9 - WNT9A 1863 428 2002 RANKING OF CASP10 W.R.TWNTS FAMILY RANKING OF CASP16 W.R.TWNTS FAMILY laplace linear rbf laplace linear rbf CASP10 - WNT2B 1607 1108 739 CASP16 - WNT2B 240 621 193 CASP10 - WNT4 432 689 132 CASP16 - WNT4 2070 1783 711 CASP10 - WNT7B 1906 1171 1165 CASP16 - WNT7B 411 713 103 CASP10 - WNT9A 1611 2152 1451 CASP16 - WNT9A 14 2512 181 RANKING OF WNTS FAMILY W.R.TCASP4 RANKING OF WNTS FAMILY W.R.TCASP5 laplace linear rbf laplace linear rbf CASP4 - WNT2B 609 1317 2372 CASP5 - WNT2B 1849 1192 1590 CASP4 - WNT4 105 711 1062 CASP5 - WNT4 890 682 714 CASP4 - WNT7B 1093 2479 1739 CASP5 - WNT7B 2112 1919 2440 CASP4 - WNT9A 456 2278 1939 CASP5 - WNT9A 315 1880 1437 RANKING OF WNTS FAMILY W.R.TCASP7 RANKING OF WNTS FAMILY W.R.TCASP9 laplace linear rbf laplace linear rbf CASP7 - WNT2B 2505 1891 1120 CASP9 - WNT2B 282 639 1414 CASP7 - WNT4 108 2456 2455 CASP9 - WNT4 572 1788 378 CASP7 - WNT7B 1380 1559 1681 CASP9 - WNT7B 979 901 676 CASP7 - WNT9A 2183 1941 1632 CASP9 - WNT9A 2378 2396 2058 RANKING OF WNTS FAMILY W.R.TCASP10 RANKING OF WNTS FAMILY W.R.TCASP16 laplace linear rbf laplace linear rbf CASP10 - WNT2B 625 1471 81 CASP16 - WNT2B 2197 2489 1775 CASP10 - WNT4 1830 2229 1847 CASP16 - WNT4 1382 954 1017 CASP10 - WNT7B 1965 937 147 CASP16 - WNT7B 2508 1820 1867 CASP10 - WNT9A 2185 1977 1350 CASP16 - WNT9A 1943 1154 1839 Table 10: 2nd order interaction ranking between WNT VS CASP family members. 17 UNEXPLORED COMBINATORIAL HYPOTHESES CASP w.r.t WNT CASP5 WNT2B CASP9 WNT4/WNT7B/WNT9A CASP16 WNT4 WNT w.r.t CASP WNT7B/WNT9A CASP4 WNT7B CASP5 WNT2B/WNT4/WNT9A CASP7 WNT9A CASP9 WNT4/WNT9A CASP10 WNT2B/WNT7B/WNT9A CASP16 Table 11: 2nd order combinatorial hypotheses between CASP and WNT family members. 18 3.1.7. TP53 - WNT cross family analysis Sadot et al. [43] have shown that down regulation of β-catenin is activated by TP53. Wnt/β-catenin signaling is known to regulate the proliferation and differentiation of mesenchymal progenitor cells through the TP53 Pathway, as shown by Peng et al. [44]. Zhukova et al. [45] show that WNT activation by lithium abrogates TP53 mutation345 associated radiation resistance in medulloblastoma. In mouse cochlea, Liu et al. [46] show that WNT signaling activates TP53-induced glycolysis and apoptosis regulator and protects against cisplatin-induced spiral ganglion neuron damage. These range of interactions of TP53 with WNT points towards definite synergy. Okayama et al. [47] show that TP53 protein regulates Hsp90 ATPase activity and thereby Wnt signaling by350 modulating Aha1 expression. Family members belonging to each of the factors like TP53, WNT etc, might be involved synergistically in pathological case or otherwise. TP53 and WNT members were found to be up regulated after the treatment of ETC-159 in colorectal cancer cells. Table 12 contains rankings of TP53 w.r.t WNTs and vice versa. Followed by this is355 the unexplored combinatorial hypotheses in table 13 generated from two-way analysis of the ranks in table 12. On the left half of table 12 are rankings of TP53 w.r.t WNTs and on the right half are the rankings of WNTs w.r.t TP53 family. Beginning with the left half, TP53I3 - WNT2B shows synergistic up regulation with rankings of 2056 (laplace) and 1712 (linear); TP53INP1 - WNT2B shows synergistic up regulation with360 rankings of 1805 (linear) and 2056 (rbf) and TP53BP2 - WNT9A shows synergistic up regulation with rankings of 2232 (linear) and 2143 (rbf). On the right half the table, TP53INP1 - WNT2B shows synergistic up regulation with rankings of 1853 (laplace) and 2089 (linear); TP53INP2 - WNT2B shows synergistic up regulation with rankings of 1723 (linear) and 2335 (rbf); TP53INP1 - WNT4 shows synergistic up regulation365 with rankings of 2414 (linear) and 2493 (rbf); TP53I3 - WNT7B shows synergistic up regulation with rankings of 1988 (laplace) and 2393 (rbf) and finally, TP53INP1 - WNT9A shows synergistic up regulation with rankings of 2045 (linear) and 2437 (rbf). One can also interpret the results of the table 11 graphically, with the following influences - •TP53 family w.r.t WNTs with TP53I3 <−WNT2B; TP53INP1 <−370 WNT2B and TP53BP2 <−WNT9A; and •WNT family VS TP53 with TP53INP1 −>WNT2B; TP53INP2 −>WNT2B; TP53INP1 −>WNT4; TP53I3 −>WNT7B and TP53INP1 −>WNT9A. 19 RANKING TP53 FAMILY VS WNT RANKING OF TP53 FAMILY W.R.TWNT2B RANKING OF WNT2B W.R.TTP53 FAMILY laplace linear rbf laplace linear rbf TP53BP2 - WNT2B 2286 234 1550 TP53BP2 - WNT2B 313 908 2457 TP53I3 - WNT2B 2056 1712 1461 TP53I3 - WNT2B 713 1223 1720 TP53INP1 - WNT2B 945 1805 2056 TP53INP1 - WNT2B 1853 2089 762 TP53INP2 - WNT2B 369 1277 453 TP53INP2 - WNT2B 754 1723 2335 RANKING OF TP53 FAMILY W.R.TWNT4 RANKING OF WNT4 W.R.TTP53 FAMILY laplace linear rbf laplace linear rbf TP53BP2 - WNT4 1034 315 1734 TP53BP2 - WNT4 678 1464 2500 TP53I3 - WNT4 1738 1631 232 TP53I3 - WNT4 297 319 493 TP53INP1 - WNT4 645 498 450 TP53INP1 - WNT4 131 2414 2493 TP53INP2 - WNT4 671 1440 405 TP53INP2 - WNT4 529 467 154 RANKING OF TP53 FAMILY W.R.TWNT7B RANKING OF WNT7B W.R.TTP53 FAMILY laplace linear rbf laplace linear rbf TP53BP2 - WNT7B 2333 1282 1673 TP53BP2 - WNT7B 1442 2217 1068 TP53I3 - WNT7B 324 712 284 TP53I3 - WNT7B 1712 1988 2393 TP53INP1 - WNT7B 1227 1585 1019 TP53INP1 - WNT7B 1226 1685 1497 TP53INP2 - WNT7B 845 1004 470 TP53INP2 - WNT7B 1017 1746 1925 RANKING OF TP53 FAMILY W.R.TWNT9A RANKING OF WNT9A W.R.TTP53 FAMILY laplace linear rbf laplace linear rbf TP53BP2 - WNT9A 908 2232 2143 TP53BP2 - WNT9A 1035 371 1218 TP53I3 - WNT9A 1707 2297 1018 TP53I3 - WNT9A 1351 1281 1695 TP53INP1 - WNT9A 447 243 1245 TP53INP1 - WNT9A 295 2045 2437 TP53INP2 - WNT9A 22 2497 1138 TP53INP2 - WNT9A 421 1765 1121 Table 12: 2nd order interaction ranking between WNT VS TP53 family members. UNEXPLORED COMBINATORIAL HYPOTHESES TP53 family w.r.t WNT TP53I3 WNT2B TP53INP1 WNT2B TP53BP2 WNT9A WNT family w.r.t TP53 TP53INP1 WNT2B TP53INP2 WNT2B TP53INP1 WNT4 TP53I3 WNT7B TP53INP1 WNT9A Table 13: 2nd order combinatorial hypotheses between TP53 and WNT family members. 20 3.1.8. BCL - WNT cross family analysis Wang et al. [48] observed that silencing Wnt2B by siRNA interference inhibits metasta-375 sis and enhances chemotherapy sensitivity in ovarian cancer. More specifically, Wang et al. [48] show that in the presence of Wnt2B siRNA treatment, the caspase-9/Bcell lymphoma 2 (BCL2)/B-cell lymphoma-xL (BCL-xL) pathway and the epithelialmesenchymal transition/phosphorylated protein kinase B pathway were inhibited. Takada et al. [49] show that targeted disruption of the BCL9/β-catenin complex inhibits onco-380 genic WNT signaling. CDK1-mediated BCL9 phosphorylation inhibits clathrin to promote mitotic Wnt signaling as shown by Chen et al. [50]. These findings point to the existing synergy of BCL family with WNTs. Family members belonging to each of the factors like BCL, WNT etc, might be involved synergistically in pathological case or otherwise. BCL and WNT members were found to be up regulated after the treatment385 of ETC-159 in colorectal cancer cells. Table 14 contains rankings of BCL w.r.t WNTs and vice versa. Followed by this is the unexplored combinatorial hypotheses in table 15 generated from two-way analysis of the ranks in table 14. On the left half of table 14 are rankings of BCL w.r.t WNTs. WNT4 - BCL2L2 shows high ranking with 2364 (laplace) and 2042 (linear); WNT7B -390 BCL2L2 shows high ranking with 1877 (laplace) and 2456 (linear); WNT9A - BCL2L2 shows high ranking with 1877 (laplace) and 2447 (linear); WNT4 - BCL2L13 shows high ranking with 1938 (laplace), 2425 (linear) and 1900 (rbf); WNT7B - BCL2L13 shows high ranking with 1993 (linear) and 2284 (rbf) and WNT2B - BCL10 shows high ranking with 2321 (laplace) and 2023 (linear).395 On the right side are rankings of WNTs w.r.t BCL. WNT7B - BCL2L1 shows high ranking with 2213 (laplace) and 2266 (linear); WNT7B - BCL2L2 shows high ranking with 2456 (laplace), 2512 (linear) and 2286 (rbf); WNT9A - BCL2L2 shows high ranking with 1868 (laplace) and 2333 (rbf); WNT9A - BCL2L13 shows high ranking with 1858 (laplace), 2422 (linear) and 1934 (rbf); WNT2B - BCL3 shows high ranking400 with 1846 (laplace), 2056 (linear) and 1896 (rbf); WNT4 - BCL6 shows high ranking with 2483 (laplace) and 2488 (linear); WNT7B - BCL6 shows high ranking with 1893 (laplace) and 2284 (linear); WNT9A - BCL6 shows high ranking with 2098 (linear) and 1905 (rbf); WNT2B - BCL9L shows high ranking with 1918 (laplace) and 1882 (rbf) and WNT4 - BCL9L shows high ranking with 2498 (linear) and 2509 (rbf); One405 can also interpret the results of the table 15 graphically, with the following influences - •BCL family w.r.t WNTs with WNT4 −>BCL2L2; WNT7B −>BCL2L2; WNT9A −>BCL2L2; WNT4 −>BCL2L13; WNT7B −>BCL2L13; WNT2B −>BCL10 and •WNT family w.r.t BCL with WNT7B <−BCL2L1; WNT7B <−BCL2L2; WNT9A <−BCL2L2; WNT9A <−BCL2L13; WNT2B <−BCL3; WNT4 <−410 BCL6; WNT7B <−BCL6; WNT9A <−BCL6; WNT2B <−BCL9L; WNT4 <− BCL9L. Conclusion Presented here are a range of multiple synergistic WNT 2nd order combinations that were ranked via a search engine. Later, two way cross family analysis between compo-415 21 RANKING BCL FAMILY VS WNT RANKING OF BCL2L1 W.R.TWNT FAMILY RANKING OF WNT FAMILY W.R.TBCL2L1 laplace linear rbf laplace linear rbf WNT2B - BCL2L1 1884 101 966 WNT2B - BCL2L1 1854 1666 1699 WNT4 - BCL2L1 98 1162 719 WNT4 - BCL2L1 21 107 16 WNT7B - BCL2L1 1434 1891 620 WNT7B - BCL2L1 2213 2266 1511 WNT9A - BCL2L1 1088 1020 1318 WNT9A - BCL2L1 1019 1462 1345 RANKING OF BCL2L2 W.R.TWNT FAMILY RANKING OF WNT FAMILY W.R.TBCL2L2 laplace linear rbf laplace linear rbf WNT2B - BCL2L2 625 2204 1677 WNT2B - BCL2L2 1574 2206 955 WNT4 - BCL2L2 2364 2042 1610 WNT4 - BCL2L2 160 590 316 WNT7B - BCL2L2 843 1877 2456 WNT7B - BCL2L2 2456 2512 2286 WNT9A - BCL2L2 1877 538 2447 WNT9A - BCL2L2 1868 2333 990 RANKING OF BCL2L13 W.R.TWNT FAMILY RANKING OF WNT FAMILY W.R.TBCL2L13 laplace linear rbf laplace linear rbf WNT2B - BCL2L13 201 1862 1353 WNT2B - BCL2L13 1256 1254 1490 WNT4 - BCL2L13 1938 2425 1900 WNT4 - BCL2L13 922 270 187 WNT7B - BCL2L13 1105 1993 2284 WNT7B - BCL2L13 1610 1319 954 WNT9A - BCL2L13 1855 268 2387 WNT9A - BCL2L13 1858 2422 1934 RANKING OF BCL3 W.R.TWNT FAMILY RANKING OF WNT FAMILY W.R.TBCL3 laplace linear rbf laplace linear rbf WNT2B - BCL3 950 1328 2482 WNT2B - BCL3 1846 2056 1896 WNT4 - BCL3 1228 1562 1353 WNT4 - BCL3 591 359 1932 WNT7B - BCL3 591 615 553 WNT7B - BCL3 1687 2160 1428 WNT9A - BCL3 1037 1410 1102 WNT9A - BCL3 1539 1424 398 RANKING OF BCL6 W.R.TWNT FAMILY RANKING OF WNT FAMILY W.R.TBCL6 laplace linear rbf laplace linear rbf WNT2B - BCL6 455 2426 1529 WNT2B - BCL6 52 107 170 WNT4 - BCL6 256 486 787 WNT4 - BCL6 2483 2488 1273 WNT7B - BCL6 2147 1466 1105 WNT7B - BCL6 975 1893 2284 WNT9A - BCL6 1547 734 2012 WNT9A - BCL6 1558 2098 1905 RANKING OF BCL9L W.R.TWNT FAMILY RANKING OF WNT FAMILY W.R.TBCL9L laplace linear rbf laplace linear rbf WNT2B - BCL9L 2348 804 1558 WNT2B - BCL9L 1918 700 1882 WNT4 - BCL9L 1446 657 309 WNT4 - BCL9L 303 2498 2509 WNT7B - BCL9L 1539 253 1279 WNT7B - BCL9L 1608 811 2168 WNT9A - BCL9L 1923 677 688 WNT9A - BCL9L 941 1843 1238 RANKING OF BCL10 W.R.TWNT FAMILY RANKING OF WNT FAMILY W.R.TBCL10 laplace linear rbf laplace linear rbf WNT2B - BCL10 2321 69 2023 WNT2B - BCL10 1951 1101 1599 WNT4 - BCL10 285 1170 465 WNT4 - BCL10 2032 34 406 WNT7B - BCL10 1847 606 1252 WNT7B - BCL10 1297 74 2009 WNT9A - BCL10 217 798 1649 WNT9A - BCL10 1771 335 861 Table 14: 2nd order interaction ranking between WNT VS BCL family members. nents 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 components which serve as hypotheses for further tests. If found true, it paves way for biolo-420 22 UNEXPLORED COMBINATORIAL HYPOTHESES BCL w.r.t WNT family WNT-4/7B/9A BCL2L2 WNT-4/7B BCL2L13 WNT-2B BCL10 WNT family w.r.t BCL WNT-7B BCL2L1 WNT-7B/9A BCL2L2 WNT-9A BCL2L13 WNT-2B BCL3 WNT-4/7B/9A BCL6 WNT-2B/4 BCL9L Table 15: 2nd order combinatorial hypotheses between TP53 and WNT family members. gists/oncologists to further investigate and understand the mechanism behind the synergy through wet experiments. Conflict of interest There are no conflicts to declare. Author’s contributions425 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.430 23 Source of Data Data used in this research work was released in a publication in Madan et al. [51]. 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 of Singapore Graduate Medical School (Duke-NUS).435 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. 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