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

Machine learning discoveries of ABC transporter-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 ATPbinding cassette (ABC) transporters-X (X, a particular gene/protein) at 2nd order level after drug administration. These rankings reveal which ABC-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 ABC and X. In this research work, we cover combinations of ABC with ubiquitinconjugating enzymes (UBE2), Interleukin (IL), BCL and Caspase (CASP). Keywords: ATP-binding cassette (ABC) transporters, 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 Introduction In the unpublished preprint Sinha [1], a frame work of a search engine was developed which can rank combinations of factors (genes/proteins) in a signaling pathway.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 Interested readers are advised to go through unpublished preprints Sinha [1] and Sinha [2] for details regarding the search engine and the discoveries mentioned in there.10 IML dicoveries of ABC transporter synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at (1) the recently concluded first ever Wnt Gordon Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA.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 31, 2024 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. ABC transporter related synergies 3.1.1. ABC transporters - UBE2 cross family analysis Not much is known about the interaction or any possible direct/indirect synergy of40 ABC transporters and the Ubiquitin-conjugating enzyme E2 family. In CRC cells treated with ETC-1922159, family members of both were found to be up regulated. The search engine also assigned numerically high valued ranks to a few of 2nd order synergies between the the two. We document here these synergies and show the possible unexplored combinations between the two familes. Tables 1 and 2 show the45 rankings of ABC w.r.t UBE2 and vice versa, respectively. In table 1 we found ABC-C3 up regulated w.r.t UBE2-A. This is reflected in the rankings of 2137 (laplace) and 2491 (linear) for ABC-C3 - UBE2-A. ABC-C5 was up regulated w.r.t UBE2-B. This is reflected in the rankings of 2317 (laplace) and 2266 (rbf) for ABC-C5 - UBE2-B. ABC-A5/D1/G2 were up regulated w.r.t UBE2-F. These50 2 RANKING ABC FAMILY W.R.TUBE2 FAMILY RANKING OF ABC FAMILY W.R.TUBE2-A RANKING OF ABC FAMILY W.R.TUBE2-B laplace linear rbf laplace linear rbf ABC-A5 - UBE2-A 2101 185 382 ABC-A5 - UBE2-B 1223 1193 194 ABC-B11 - UBE2-A 129 2487 304 ABC-B11 - UBE2-B 125 103 571 ABC-C3 - UBE2-A 2137 2491 1023 ABC-C3 - UBE2-B 606 791 1411 ABC-C5 - UBE2-A 1630 490 2408 ABC-C5 - UBE2-B 1515 2317 2266 ABC-C13 - UBE2-A 742 1604 475 ABC-C13 - UBE2-B 2199 2254 2362 ABC-D1 - UBE2-A 316 620 596 ABC-D1 - UBE2-B 1082 374 1057 ABC-G1 - UBE2-A 46 819 533 ABC-G1 - UBE2-B 48 843 551 ABC-G2 - UBE2-A 398 259 261 ABC-G2 - UBE2-B 189 189 41 RANKING OF ABC FAMILY W.R.TUBE2-F RANKING OF ABC FAMILY W.R.TUBE2-H laplace linear rbf laplace linear rbf ABC-A5 - UBE2-F 997 2408 1784 ABC-A5 - UBE2-H 1247 2068 2438 ABC-B11 - UBE2-F 141 1122 578 ABC-B11 - UBE2-H 932 429 409 ABC-C3 - UBE2-F 931 2420 681 ABC-C3 - UBE2-H 540 1962 563 ABC-C5 - UBE2-F 628 1373 217 ABC-C5 - UBE2-H 1551 865 1450 ABC-C13 - UBE2-F 403 2464 1307 ABC-C13 - UBE2-H 1192 2492 2051 ABC-D1 - UBE2-F 2069 1959 1235 ABC-D1 - UBE2-H 1094 1016 1474 ABC-G1 - UBE2-F 209 1216 1450 ABC-G1 - UBE2-H 683 173 18 ABC-G2 - UBE2-F 690 1995 2120 ABC-G2 - UBE2-H 1328 1374 78 RANKING OF ABC FAMILY W.R.TUBE2-J1 RANKING OF ABC FAMILY W.R.TUBE2-Z laplace linear rbf laplace linear rbf ABC-A5 - UBE2-J1 634 222 711 ABC-A5 - UBE2-Z 454 1059 1287 ABC-B11 - UBE2-J1 1182 1075 403 ABC-B11 - UBE2-Z 134 503 436 ABC-C3 - UBE2-J1 1232 719 1285 ABC-C3 - UBE2-Z 975 1722 2095 ABC-C5 - UBE2-J1 964 1342 2373 ABC-C5 - UBE2-Z 2348 845 1859 ABC-C13 - UBE2-J1 2095 2412 2360 ABC-C13 - UBE2-Z 1157 651 1335 ABC-D1 - UBE2-J1 542 1198 704 ABC-D1 - UBE2-Z 392 1660 943 ABC-G1 - UBE2-J1 306 97 122 ABC-G1 - UBE2-Z 545 142 354 ABC-G2 - UBE2-J1 335 668 591 ABC-G2 - UBE2-Z 747 285 530 Table 1: 2nd order interaction ranking between ABC w.r.t UBE2 family members. are reflected in the rankings of 2408 (linear) and 1784 (rbf) for ABC-A5 - UBE2-F, 2069 (linear) and 1959 (rbf) for ABC-D1 - UBE2-F and 1995 (linear) and 2120 (rbf) for ABC-G2 - UBE2-F. ABC-A5/C13 were up regulated w.r.t UBE2-H. These are reflected in 2068 (linear) and 2438 (rbf) for ABC-A5 - UBE2-H and 2492 (linear) and 2051 (rbf) for ABC-C13 - UBE2-H. ABC-C13 was up regulated w.r.t UBE2-J1. This55 is reflected in the rankings of 2095 (laplace), 2412 (linear) and 2360 (rbf). ABC-C5 was up regulated w.r.t UBE2-Z. This is reflected in rankings of 2348 (laplace) and 1859 (rbf) for ABC-C5 - UBE2-Z. In table 2 we found UBE2-A up regulated w.r.t ABC-C5/G2. This is reflected in the rankings of 2122 (linear) and 2297 (rbf) for ABC-C5 - UBE2-A; and 204860 (laplace) and 1829 (linear) for ABC-G2 - UBE2-A. UBE2-B up regulated w.r.t ABCA5/C3/C13/D1/G2. This is reflected in the rankings of 1846 (laplace) and 2038 (linear) for ABC-A5 - UBE2-B; 1999 (laplace) and 2050 (rbf) for ABC-C3 - UBE2-B; 1863 (linear) and 2496 (rbf) for ABC-C13 - UBE2-B; 2322 (laplace), 1917 (linear) and 2426 (rbf) for ABC-D1 - UBE2-B and 1833 (laplace), 2445 (linear) and 2506 (rbf)65 3 RANKING UBE2 FAMILY W.R.TABC FAMILY RANKING OF UBE2-A W.R.TABC RANKING OF UBE2-B W.R.TABC FAMILY laplace linear rbf laplace linear rbf ABC-A5 - UBE2-A 1037 253 2091 ABC-A5 - UBE2-B 1846 2038 936 ABC-B11 - UBE2-A 1491 1269 2179 ABC-B11 - UBE2-B 1623 1304 1995 ABC-C3 - UBE2-A 1726 1906 1390 ABC-C3 - UBE2-B 1999 832 2050 ABC-C5 - UBE2-A 880 2122 2297 ABC-C5 - UBE2-B 612 2276 1681 ABC-C13 - UBE2-A 412 234 670 ABC-C13 - UBE2-B 467 1863 2496 ABC-D1 - UBE2-A 2507 237 1319 ABC-D1 - UBE2-B 2322 1917 2426 ABC-G1 - UBE2-A 907 2291 1573 ABC-G1 - UBE2-B 1194 1592 1239 ABC-G2 - UBE2-A 2048 1829 1376 ABC-G2 - UBE2-B 1833 2445 2506 RANKING OF UNE2-F W.R.TABC FAMILY RANKING OF UBE2-H W.R.TABC FAMILY laplace linear rbf laplace linear rbf ABC-A5 - UBE2-F 2485 406 66 ABC-A5 - UBE2-H 508 2339 1110 ABC-B11 - UBE2-F 2003 1203 2422 ABC-B11 - UBE2-H 1950 1770 2461 ABC-C3 - UBE2-F 2132 2163 861 ABC-C3 - UBE2-H 2439 1972 2305 ABC-C5 - UBE2-F 406 1651 1838 ABC-C5 - UBE2-H 398 2473 2355 ABC-C13 - UBE2-F 821 959 1196 ABC-C13 - UBE2-H 2004 2317 1847 ABC-D1 - UBE2-F 2421 686 2176 ABC-D1 - UBE2-H 164 1641 648 ABC-G1 - UBE2-F 115 2202 1953 ABC-G1 - UBE2-H 201 1921 2288 ABC-G2 - UBE2-F 983 883 1012 ABC-G2 - UBE2-H 2063 1631 1354 RANKING OF UBE2-J1 W.R.TABC FAMILY RANKING OF UBE2-Z W.R.TABC FAMILY laplace linear rbf laplace linear rbf ABC-A5 - UBE2-J1 1740 1467 1244 ABC-A5 - UBE2-Z 2336 1710 35 ABC-B11 - UBE2-J1 1806 991 1935 ABC-B11 - UBE2-Z 521 645 2168 ABC-C3 - UBE2-J1 2073 2291 631 ABC-C3 - UBE2-Z 1978 1823 1859 ABC-C5 - UBE2-J1 126 525 1409 ABC-C5 - UBE2-Z 1237 148 1928 ABC-C13 - UBE2-J1 2329 2153 1951 ABC-C13 - UBE2-Z 1185 137 2475 ABC-D1 - UBE2-J1 2263 1886 2249 ABC-D1 - UBE2-Z 2292 21 2381 ABC-G1 - UBE2-J1 1262 2418 2277 ABC-G1 - UBE2-Z 426 2515 1858 ABC-G2 - UBE2-J1 1558 2408 1304 ABC-G2 - UBE2-Z 2270 2080 2448 Table 2: 2nd order interaction ranking between UBE2 w.r.t ABC family members. for ABC-G2 - UBE2-B. UBE2-F was found up regulated w.r.t ABC-B11/C3/D1/G1. These were reflected in 2003 (laplace) and 2422 (rbf) for ABC-B11 - UBE2-F; 2132 (laplace) and 2163 (linear) for ABC-C3 - UBE2-F; 2421 (laplace) and 2176 (rbf) for ABC-D1 - UBE2-F; and 2202 (laplace) and 1953 (rbf) for ABC-G1 - UBE2-F. UBE2Hwas found to be up regulated w.r.t ABC-B11/C3/C5/C13/G1. These are reflected in70 rankings of 1950 (laplace), 1770 (linear) and 2461 (rbf) for ABC-B11 - UBE2-H; 2439 (laplace), 1972 (linear) and 2305 (rbf) for ABC-C3 - UBE2-H; 2473 (linear) and 2355 (rbf) for ABC-C5 - UBE2-H; 2004 (laplace), 2317 (linear) and 1847 (rbf) for ABCC13 - UBE2-H; and 1921 (linear) and 2288 (rbf) for ABC-G1 - UBE2-H; UBE2-J1 was found to be up regulated w.r.t ABC-B11/C3/C13/D1/G1/G2; 1806 (laplace) and75 1935 (rbf) for ABC-B11 - UBE2-J1; 2073 (laplace) and 2291 (linear) for ABC-C3 - UBE2-J1; 2329 (laplace), 2153 (linear) and 1951 (rbf) ABC-C13 - UBE2-J1; 2263 (laplace), 1886 (linear) and 2249 (rbf) for ABC-D1 - UBE2-J1; and 2418 (linear) and 2277 (rbf) for ABC-G1 - UBE2-J1; Finally, UBE2-Z was found up regulated w.r.t ABC-C3/D1/G1/G2. These are reflected in rankings of 1978 (laplace), 1823 (linear)80 4 UNEXPLORED COMBINATORIAL HYPOTHESES ABC w.r.t UBE2 ABC-C3 UBE2-A ABC-C5 UBE2-B ABC-A5/D1/G2 UBE2-F ABC-A5/C13 UBE2-H ABC-C13 UBE2-J1 ABC-C5 UBE2-Z UBE2 w.r.t ABC UBEA-2 ABC-C5/G2 UBE2-B ABC-A5/C3/C13/D1/G2 UBE2-F ABC-B11/C3/D1/G1 UBE2-H ABC-B11/C3/C5/C13/G1 UBE2-J1 ABC-B11/C3/C13/D1/G1/G2 UBE2-Z ABC-C3/D1/G1/G2 Table 3: 2nd order combinatorial hypotheses between ABC and UBE2. and 1859 (rbf) for ABC-C3 - UBE2-Z; 2292 (laplace) and 2381 (linear) for ABC-D1 - UBE2-Z; 2515 (linear) and 1858 (rbf) for ABC-G1 - UBE2-Z; 2270 (laplace), 2080 (linear) and 2448 (rbf) for ABC-G2 - UBE2-Z. Table 3 shows the derived influences which can be represented graphically, with the following influences - •ABC w.r.t UBE2 with ABC-C3 <−UBE2-A; ABC-C5 <−85 UBE2-B; ABC-A5/D1/G2 <−UBE2-F; ABC-A5/C13 <−UBE2-H; ABC-C13 <− UBE2-J1; ABC-C5 <−UBE2-Z; and •UBE2 w.r.t ABC with UBEA-2 <−ABCC5/G2; UBE2-B <−ABC-A5/C3/C13/D1/G2; UBE2-F <−ABC-B11/C3/D1/G1; UBE2-H <−ABC-B11/C3/C5/C13/G1; UBE2-J1 <−ABC-B11/C3/C13/D1/G1/G2; UBE2-Z <−ABC-C3/D1/G1/G2.90 5 3.1.2. ABC transporters intra cross family analysis A range of ABC transporters were found to be up regulated in CRC cells after ETC1922159 treatment. We checked the rankings of the ABC transporters within the ABC family and found multiple synergistic upregulation at 2nd order level that were ranked appropriately. Table 4 shows intra family rankings of ABC members among them-95 selves. We found ABC-C13 upregulated w.r.t ABC-A5. These were reflected in rankings of 1943 (linear) and 2151 (rbf); ABC-C5/C13/G1 were up regulated w.r.t ABCB11. These are reflected in rankings of 2226 (laplace) and 2241 (rbf) for ABC-C5 - ABC-B11; 1971 (laplace) and 2150 (rbf) for ABC-C13 - ABC-B11 and 1957 (laplace) and 1920 (linear) for ABC-G1 - ABC-B11; ABC-C3/C13 were found to be up reg-100 ulated w.r.t ABC-C5. These are reflected in 2084 (laplace), 2274 (linear) and 1758 (rbf) for ABC-C3 - ABC-C5 and 2476 (linear) and 2446 (rbf) for ABC-C13 - ABCC5. ABC-C5/C13 were found to be up regulated w.r.t ABC-D1. 2423 (laplace) and 2388 (rbf) for ABC-C5 - ABC-D1 and 2383 (laplace) and 2029 (linear) for ABC-C13 - ABC-D1. ABC-A5 was found to be up regulated w.r.t ABC-G1. This is reflected in105 rankings of 2488 (laplace) and 1776 (linear) for ABC-A5 - ABC-G1. ABC-A5 was found to be up regulated w.r.t ABC-G2 also. This is reflected in rankings of 2284 (laplace), 1904 (linear) and 1829 (rbf) for ABC-A5 - ABC-G2. Table 5 shows the derived influences which can be represented graphically, with the following influences - •ABC intra family with ABC-C13 <−ABC-A5; ABC-110 C5/C13/G1 <−ABC-B11; ABC-C3/C13 <−ABC-C5; ABC-C5/C13 <−ABC-D1; ABC-A5 <−ABC-G1; ABC-C5 <−ABC-G2. 6 RANKING ABC FAMILY W.R.TABC FAMILY RANKING OF ABC FAMILY W.R.TABC-A5 RANKING OF ABC FAMILY W.R.TABC-B11 laplace linear rbf laplace linear rbf ABC-B11 - ABC-A5 733 471 26 ABC-A5 - ABC-B11 1148 1443 1782 ABC-C3 - ABC-A5 111 493 2264 ABC-C3 - ABC-B11 845 527 1257 ABC-C5 - ABC-A5 1717 519 1921 ABC-C5 - ABC-B11 2226 1644 2241 ABC-C13 - ABC-A5 1243 1943 2151 ABC-C13 - ABC-B11 1971 609 2150 ABC-D1 - ABC-A5 1262 2387 1573 ABC-D1 - ABC-B11 891 217 854 ABC-G1 - ABC-A5 657 991 533 ABC-G1 - ABC-B11 1957 1920 669 ABC-G2 - ABC-A5 587 397 104 ABC-G2 - ABC-B11 685 1978 226 RANKING OF ABC FAMILY W.R.TABC-C3 RANKING OF ABC FAMILY W.R.TABC-C5 laplace linear rbf laplace linear rbf ABC-A5 - ABC-C3 163 861 1672 ABC-A5 - ABC-C5 2086 411 1243 ABC-B11 - ABC-C3 410 613 1501 ABC-B11 - ABC-C5 2398 272 464 ABC-C5 - ABC-C3 1591 2435 927 ABC-C3 - ABC-C5 2084 2274 1758 ABC-C13 - ABC-C3 405 880 1282 ABC-C13 - ABC-C5 226 2476 2446 ABC-D1 - ABC-C3 18 1145 2187 ABC-D1 - ABC-C5 2010 891 1257 ABC-G1 - ABC-C3 1858 173 842 ABC-G1 - ABC-C5 2402 894 741 ABC-G2 - ABC-C3 1462 275 1373 ABC-G2 - ABC-C5 2463 736 661 RANKING OF ABC FAMILY W.R.TABC-C13 RANKING OF ABC FAMILY W.R.TABC-D1 laplace linear rbf laplace linear rbf ABC-A5 - ABC-C13 2251 1219 1614 ABC-A5 - ABC-D1 163 1068 291 ABC-B11 - ABC-C13 1106 56 1171 ABC-B11 - ABC-D1 1273 130 1655 ABC-C3 - ABC-C13 2279 1431 365 ABC-C3 - ABC-D1 568 251 149 ABC-C5 - ABC-C13 1537 2178 690 ABC-C5 - ABC-D1 2423 538 2388 ABC-D1 - ABC-C13 2370 171 362 ABC-C13 - ABC-D1 2383 2029 425 ABC-G1 - ABC-C13 833 1544 1343 ABC-G1 - ABC-D1 1462 1175 827 ABC-G2 - ABC-C13 329 1323 1755 ABC-G2 - ABC-D1 467 670 2491 RANKING OF ABC FAMILY W.R.TABC-G1 RANKING OF ABC FAMILY W.R.TABC-G2 laplace linear rbf laplace linear rbf ABC-A5 - ABC-G1 2488 1776 1078 ABC-A5 - ABC-G2 1011 1640 1705 ABC-B11 - ABC-G1 2312 253 52 ABC-B11 - ABC-G2 988 481 1849 ABC-C3 - ABC-G1 273 1415 1139 ABC-C3 - ABC-G2 1102 1082 1563 ABC-C5 - ABC-G1 220 1988 437 ABC-C5 - ABC-G2 2284 1904 1829 ABC-C13 - ABC-G1 2389 427 1125 ABC-C13 - ABC-G2 929 1238 222 ABC-D1 - ABC-G1 1836 485 597 ABC-D1 - ABC-G2 814 995 1152 ABC-G2 - ABC-G1 2506 692 1143 ABC-G1 - ABC-G2 596 460 848 Table 4: 2nd order interaction ranking between ABC family members. 7 UNEXPLORED COMBINATORIAL HYPOTHESES ABC intra family ABC-C13 ABC-A5 ABC-C5/C13/G1 ABC-B11 ABC-C3/C13 ABC-C5 ABC-C5/C13 ABC-D1 ABC-A5 ABC-G1 ABC-C5 ABC-G2 Table 5: 2nd order combinatorial hypotheses between ABC family members. 8 3.1.3. Interleukin - ABC transporters cross family analysis Zhou et al. [5] have observed that the ABCA1 contributes to the secretion of interleukin 1βfrom macrophages. Hask´ o et al. [6] show that inhibitors of ABC trans-115 porters suppress interleukin-12 p40 production and major histocompatibility complex II up-regulation in macrophages. Park et al. [7] conclude that anti-cancer drug-induced IL-8 secretion increased the expression of ABC transporters and SP cells, promoting the growth of HCC in vitro. Marty et al. [8] show that ABC1 is required for the release of interleukin-1βby P2X7-stimulated and lipopolysaccharide-primed mouse120 Schwann cells. Lottaz et al. [9] observe that inhibition of ABC transporter downregulates interleukin-1β-mediated autocrine activation of human dermal fibroblasts. These findings and many more indicate the synergy between IL family and ABC transporters. In colorectal cancer cells treated with ETC-1922159, some of the members of both families were up regulated. Given the studied synergies, our search engine found multiple125 combinations which were ranked with high numerical values, thus indicating possible dual combinatorial role. Tables 6 and 7, each show rankings of ABC transporters w.r.t IL family on the left half and vice versa on the right half. On the left half we found IL-17REL up regulated w.r.t ABCA5. This is reflected in the rankings of 2405 (linear) and 2202 (rbf) for IL17REL - ABCA5. IL-2RG/6ST/15/15RA130 up regulated w.r.t ABCB11. This is reflected in the rankings of 2182 (laplace), 2102 (linear) and 550 (rbf) for IL2RG - ABCB11; 1793 (laplace), 2140 (linear) and 1938 (rbf) for IL6ST - ABCB11; 2438 (laplace) and 2512 (linear) for IL15 - ABCB11; and 2271 (laplace) and 1784 (rbf) for IL15RA - ABCB11. IL-8/15RA up regulated w.r.t ABCC3. This is reflected in the rankings of 1767 (laplace) and 2419 (rbf) for IL8 -135 ABCC3 and 2403 (linear) and 1795 (rbf) for IL15RA - ABCC3. IL-15RA/17REL up regulated w.r.t ABCC5. These are reflected in rankings of 2255 (linear) and 1861 (rbf) for IL15RA - ABCC5 and 2462 (linear) and 2509 (rbf) for IL17REL - ABCC5. IL15RA/17REL were up regulated w.r.t ABCC13. These are reflected in 2248 (laplace), 1955 (linear) and 2456 (rbf) for IL15RA - ABCC13 and 2339 (laplace) and 2137 (lin-140 ear) for IL17REL - ABCC13. IL-1A/1RAP/8/15RA were up regulated w.r.t ABCD1. These are reflected in rankings of 1932 (laplace) and 2203 (rbf) for IL1A - ABCD1; 2508 (laplace), 2006 (linear) and 1907 (rbf) for IL1RAP - ABCD1; 2010 (laplace), 2315 (linear) and 1814 (rbf) for IL8 - ABCD1; and 2097 (laplace) and 1765 (linear) for IL15RA - ABCD1. IL-1RAP was up regulated w.r.t ABCG1. This was reflected145 in rankings of 2205 (linear) and 2339 (rbf) for IL1RAP - ABCG1. IL-1RAP/15RA were up regulated w.r.t ABCG2. These were reflected in rankings of 2184 (laplace) and 2167 (linear) for IL1RAP - ABCG2 and 1910 (laplace), 2428 (linear) and 1921 (rbf) for IL15RA - ABCG2. On the right half we found ABCA5 up regulated w.r.t IL-1B/1RAP/10RB. These150 are reflected in the rankings of 2069 (laplace) and 2301 (rbf) for IL1B - ABCA5; 1763 (laplace) and 2345 (rbf) for IL1RAP - ABCA5; and 2230 (laplace), 2184 (linear) and 2240 (rbf) for IL10RB - ABCA5; ABCB11 was up regulated w.r.t IL-10RB. This is reflected in the rankings of 2101 (laplace) and 2419 (linear) for IL10RB - ABCB11. ABCC3 was up regulated w.r.t IL-1A/17REL. This is reflected in the rankings of 1798155 (linear) and 2459 (rbf) for IL1A - ABCC3 and 2089 (laplace) and 2388 (linear) for IL17REL - ABCC3. ABCC5 was up regulated w.r.t IL-1A/1RAP/15/17C. This are 9 RANKING BCL FAMILY VS ABC FAMILY RANKING OF BCL6 W.R.TABC FAMILY RANKING OF ABC FAMILY W.R.TBCL6 laplace linear rbf laplace linear rbf ABCA5 - BCL6 2045 557 1384 ABCA5 - BCL6 211 283 1615 ABCB11 - BCL6 1611 2010 2350 ABCB11 - BCL6 841 427 2320 ABCC3 - BCL6 1895 983 958 ABCC3 - BCL6 1084 570 594 ABCC5 - BCL6 615 597 567 ABCC5 - BCL6 1370 1841 2389 ABCC13 - BCL6 1097 2431 1731 ABCC13 - BCL6 2172 2456 1063 ABCD1 - BCL6 1446 1139 1953 ABCD1 - BCL6 1097 1297 827 ABCG1 - BCL6 1462 1688 1918 ABCG1 - BCL6 192 27 1111 ABCG2 - BCL6 947 1503 978 ABCG2 - BCL6 129 745 719 RANKING OF BCL9L W.R.TABC FAMILY RANKING OF ABC FAMILY W.R.TBCL9L laplace linear rbf laplace linear rbf ABCA5 - BCL9L 67 1008 94 ABCA5 - BCL9L 1753 1167 2312 ABCB11 - BCL9L 1989 158 1705 ABCB11 - BCL9L 1033 494 48 ABCC3 - BCL9L 1307 2249 1357 ABCC3 - BCL9L 457 2296 971 ABCC5 - BCL9L 1694 432 477 ABCC5 - BCL9L 1775 1551 2073 ABCC13 - BCL9L 1724 1410 862 ABCC13 - BCL9L 110 2475 2325 ABCD1 - BCL9L 1366 2344 1666 ABCD1 - BCL9L 1016 2440 2411 ABCG1 - BCL9L 1248 1680 536 ABCG1 - BCL9L 1146 676 16 ABCG2 - BCL9L 2451 1119 224 ABCG2 - BCL9L 1263 1421 218 RANKING OF BCL10 W.R.TABC FAMILY RANKING OF ABC FAMILY W.R.TBCL10 laplace linear rbf laplace linear rbf ABCA5 - BCL10 687 176 808 ABCA5 - BCL10 1753 1167 2312 ABCB11 - BCL10 2234 2382 322 ABCB11 - BCL10 1033 494 48 ABCC3 - BCL10 589 379 492 ABCC3 - BCL10 457 2296 971 ABCC5 - BCL10 1489 397 1643 ABCC5 - BCL10 1775 1551 2073 ABCC13 - BCL10 956 538 1491 ABCC13 - BCL10 110 2475 2325 ABCD1 - BCL10 1009 470 1597 ABCD1 - BCL10 1016 2440 2411 ABCG1 - BCL10 1613 310 1115 ABCG1 - BCL10 1146 676 16 ABCG2 - BCL10 361 676 2020 ABCG2 - BCL10 1263 1421 218 Table 10: 2nd order interaction ranking between ABC and BCL family members. 16 UNEXPLORED COMBINATORIAL HYPOTHESES BCL w.r.t ABC ABC-C5 BCL2L1 ABC-B11/C5/C13/D1 BCL2L2 ABC-B11/C5/C13/D1/G1 BCL2L13 ABC-D1/G1 BCL3 ABC-B11 BCL6 ABC-B11 BCL10 ABC w.r.t BCL ABC-C3 BCL2L1 ABC-C5/C13 BCL2L13 ABC-C3 BCL3 ABC-C5/C13 BCL6 ABC-C5/C13/D1 BCL9L ABC-A5/C5/C13/D1 BCL10 Table 11: 2nd order combinatorial hypotheses between BCL and ABC family members. 17 3.1.5. CASPASE - ABC transporters cross family analysis225 Hu et al. [13] observe that the loss of ABCB4 attenuates the caspase-dependent apoptosis regulating resistance to 5-Fu in colorectal cancer. Ihlefeld et al. [14] analyze whether the observed upregulation of the multidrug transporters contributed to the resistance of Sgpl1/-MEFs against chemotherapy-induced apoptosis by measuring the influence of ABC transporter inhibitors on cell viability and caspase-3 cleavage. Though230 recent developements, they point to the synergy between the transporters and the CASP family. In CRC cells, treated with ETC-1922159, these were found to be UP regulated. The engine alotted high numerical valued ranks to some of the 2nd order combinations between the members of the two families. Tables 12 and 13 show the rankings of ABC transporters w.r.t CASP and vice versa.235 In table 12, we found ABC-C5 to be up regulated w.r.t CASP4. These are reflected in rankings of 2495 (laplace) and 2257 (rbf) for CASP4 - ABC-C5. ABC-C5 was up regulated w.r.t CASP5. These are reflected in rankings of 2475 (laplace) and 2234 (rbf) for CASP5 - ABC-C5. ABC-A5/C13/D1 were up regulated w.r.t CASP7. These are reflected in rankings of 2515 (laplace) and 1742 (linear) for CASP7 - ABC-C5;240 2489 (laplace) and 2418 (linear) for CASP7 - ABC-C13; and 2323 (laplace) and 2004 (linear) for CASP7 - ABC-D1. ABC-B11/C5/D1/G1 were up regulated w.r.t CASP9. These are reflected in rankings of 2001 (linear) and 2051 (rbf) for CASP9 - ABC-B11; 2180 (laplace) and 2343 (linear) for CASP9 - ABC-C5; 2267 (laplace) and 2382 (rbf) for CASP9 - ABC-C13; 1890 (linear) and 2286 (rbf) for CASP9 - ABC-G1; ABC-245 A5/C13 were up regulated w.r.t CASP10. These are reflected in rankings of 2292 (laplace), 2311 (linear) and 1108 (rbf) for CASP10 - ABC-A5; 2139 (laplace) and 2203 (linear) for CASP10 - ABC-C13; In table 13, we found ABC-C5 to be up regulated w.r.t CASP4. These are reflected in rankings of 2495 (laplace) and 2257 (rbf) for CASP4 - ABC-C5. ABC-C5 was250 up regulated w.r.t CASP5. These are reflected in rankings of 2475 (laplace) and 2234 (rbf) for CASP5 - ABC-C5. ABC-A5/C13/D1 were up regulated w.r.t CASP7. These are reflected in rankings of 2515 (laplace) and 1742 (linear) for CASP7 - ABC-C5; 2489 (laplace) and 2418 (linear) for CASP7 - ABC-C13; and 2323 (laplace) and 2004 (linear) for CASP7 - ABC-D1. ABC-B11/C5/D1/G1 were up regulated w.r.t CASP9.255 These are reflected in rankings of 2001 (linear) and 2051 (rbf) for CASP9 - ABC-B11; 2180 (laplace) and 2343 (linear) for CASP9 - ABC-C5; 2267 (laplace) and 2382 (rbf) for CASP9 - ABC-C13; 1890 (linear) and 2286 (rbf) for CASP9 - ABC-G1; ABCA5/C13 were up regulated w.r.t CASP10. These are reflected in rankings of 2292 (laplace), 2311 (linear) and 1108 (rbf) for CASP10 - ABC-A5; 2139 (laplace) and260 2203 (linear) for CASP10 - ABC-C13; In table 13, we found CASP4 to be up regulated w.r.t ABC-D1. These are reflected in rankings of 1791 (laplace) and 1954 (rbf) for CASP4 - ABC-D1. CASP5 was up regulated w.r.t ABC-C13. These are reflected in rankings of 2286 (laplace) and 1905 (rbf) for CASP5 - ABC-C13. CASP7 was up regulated w.r.t ABC-C5. This is reflected265 in rankings of 2168 (laplace), 1881 (linear) and 2016 (rbf) for CASP7 - ABC-C5. CASP9 were up regulated w.r.t ABC-C5/C13/D1/G1. These are reflected in rankings of 2404 (laplace) and 2374 (linear) for CASP9 - ABC-A5; 2449 (laplace) and 2506 (rbf) for CASP9 - ABC-C13; 1858 (laplace) and 2430 (rbf) for CASP9 - ABC-D1; and 18 RANKING ABC FAMILY W.R.TCASP FAMILY RANKING OF ABC FAMILY W.R.TCASP4 RANKING OF ABC FAMILY W.R.TCASP5 laplace linear rbf laplace linear rbf CASP4 - ABC-A5 957 682 991 CASP5 - ABC-A5 733 1986 421 CASP4 - ABC-B11 19 727 158 CASP5 - ABC-B11 513 406 355 CASP4 - ABC-C3 1242 857 1848 CASP5 - ABC-C3 685 1694 1558 CASP4 - ABC-C5 2495 1316 2257 CASP5 - ABC-C5 2475 1038 2234 CASP4 - ABC-C13 154 1537 1206 CASP5 - ABC-C13 1660 1581 853 CASP4 - ABC-D1 1494 964 999 CASP5 - ABC-D1 354 725 1304 CASP4 - ABC-G1 1405 70 326 CASP5 - ABC-G1 298 485 382 CASP4 - ABC-G2 157 176 523 CASP5 - ABC-G2 706 846 1598 RANKING OF ABC FAMILY W.R.TCASP7 RANKING OF ABC FAMILY W.R.TCASP9 laplace linear rbf laplace linear rbf CASP7 - ABC-A5 2515 1742 25 CASP9 - ABC-A5 1125 1863 694 CASP7 - ABC-B11 1299 207 348 CASP9 - ABC-B11 729 2001 2051 CASP7 - ABC-C3 992 511 2222 CASP9 - ABC-C3 1108 1470 1465 CASP7 - ABC-C5 1232 1449 2154 CASP9 - ABC-C5 2180 2343 1732 CASP7 - ABC-C13 2489 2418 1623 CASP9 - ABC-C13 2267 1472 2382 CASP7 - ABC-D1 1544 2323 2004 CASP9 - ABC-D1 1011 1086 174 CASP7 - ABC-G1 665 382 670 CASP9 - ABC-G1 580 1890 2286 CASP7 - ABC-G2 1930 23 963 CASP9 - ABC-G2 647 2374 310 RANKING OF ABC FAMILY W.R.TCASP10 RANKING OF ABC FAMILY W.R.TCASP16 laplace linear rbf laplace linear rbf CASP10 - ABC-A5 2292 2311 1108 CASP16 - ABC-A5 165 408 113 CASP10 - ABC-B11 2245 1467 1182 CASP16 - ABC-B11 495 949 1417 CASP10 - ABC-C3 760 2479 923 CASP16 - ABC-C3 50 4 556 CASP10 - ABC-C5 326 485 1429 CASP16 - ABC-C5 1635 2487 1309 CASP10 - ABC-C13 2139 2203 1524 CASP16 - ABC-C13 1517 936 1236 CASP10 - ABC-D1 2210 475 1655 CASP16 - ABC-D1 1029 1210 1285 CASP10 - ABC-G1 2337 128 71 CASP16 - ABC-G1 350 756 109 CASP10 - ABC-G2 2075 1693 1306 CASP16 - ABC-G2 318 476 515 Table 12: 2nd order interaction ranking between ABC and CASP family members. 2342 (linear) and 2468 (rbf) for CASP9 - ABC-G1; CASP16 were up regulated w.r.t270 ABC-A5. This is reflected in rankings of 2477 (linear) and 2315 (rbf) for CASP16 - ABC-A5. Table 14 shows the derived influences which can be represented graphically, with the following influences - •ABC w.r.t CASP with CASP-4 −>ABC-C5; CASP-5 −>ABC-C5; CASP-7 −>ABC-A5/C13/D1; CASP-9 −>ABC-B11/C5/D1/G1;275 CASP-10 −>ABC-A5/C13; and •CASP w.r.t ABC with CASP-4 <−ABC-D1; CASP-5 <−ABC-C13; CASP-7 <−ABC-C5; CASP-9 <−ABC-C5/C13/D1/G1; CASP-16 <−ABC-A5; 19 RANKING CASP FAMILY W.R.TABC FAMILY RANKING OF CASP4 W.R.TABC FAMILY RANKING OF CASP5 W.R.TABC FAMILY laplace linear rbf laplace linear rbf CASP4 - ABC-A5 791 586 753 CASP5 - ABC-A5 696 427 48 CASP4 - ABC-B11 462 263 427 CASP5 - ABC-B11 1470 1300 242 CASP4 - ABC-C3 1013 54 1140 CASP5 - ABC-C3 821 286 459 CASP4 - ABC-C5 2396 26 209 CASP5 - ABC-C5 2368 665 171 CASP4 - ABC-C13 1305 775 2193 CASP5 - ABC-C13 2286 739 1905 CASP4 - ABC-D1 1791 591 1954 CASP5 - ABC-D1 653 440 972 CASP4 - ABC-G1 593 99 173 CASP5 - ABC-G1 2176 446 317 CASP4 - ABC-G2 423 109 1364 CASP5 - ABC-G2 332 122 533 RANKING OF CASP7 W.R.TABC FAMILY RANKING OF CASP9 W.R.TABC FAMILY laplace linear rbf laplace linear rbf CASP7 - ABC-A5 1726 697 1874 CASP9 - ABC-A5 2404 2374 1265 CASP7 - ABC-B11 1549 189 1692 CASP9 - ABC-B11 998 1258 2046 CASP7 - ABC-C3 2331 1572 69 CASP9 - ABC-C3 1398 2358 1445 CASP7 - ABC-C5 2168 1881 2016 CASP9 - ABC-C5 1023 965 1080 CASP7 - ABC-C13 1822 865 1239 CASP9 - ABC-C13 2449 1545 2506 CASP7 - ABC-D1 111 813 2230 CASP9 - ABC-D1 1858 2430 412 CASP7 - ABC-G1 1609 983 1994 CASP9 - ABC-G1 305 2342 2468 CASP7 - ABC-G2 1094 952 102 CASP9 - ABC-G2 1868 1621 1154 RANKING OF CASP10 W.R.TABC FAMILY RANKING OF CASP16 W.R.TABC FAMILY laplace linear rbf laplace linear rbf CASP10 - ABC-A5 683 1453 1437 CASP16 - ABC-A5 960 2477 2315 CASP10 - ABC-B11 1301 774 558 CASP16 - ABC-B11 402 1860 794 CASP10 - ABC-C3 369 683 1453 CASP16 - ABC-C3 171 825 23 CASP10 - ABC-C5 1823 346 761 CASP16 - ABC-C5 2467 585 258 CASP10 - ABC-C13 1320 832 868 CASP16 - ABC-C13 428 177 64 CASP10 - ABC-D1 249 1440 387 CASP16 - ABC-D1 651 153 2010 CASP10 - ABC-G1 1687 1232 156 CASP16 - ABC-G1 2398 421 1120 CASP10 - ABC-G2 1151 651 464 CASP16 - ABC-G2 1193 734 479 Table 13: 2nd order interaction ranking between CASP and ABC family members. 20 UNEXPLORED COMBINATORIAL HYPOTHESES ABC w.r.t CASP CASP-4 ABC-C5 CASP-5 ABC-C5 CASP-7 ABC-A5/C13/D1 CASP-9 ABC-B11/C5/D1/G1 CASP-10 ABC-A5/C13 CASP w.r.t ABC CASP-4 ABC-D1 CASP-5 ABC-C13 CASP-7 ABC-C5 CASP-9 ABC-C5/C13/D1/G1 CASP-16 ABC-A5 Table 14: 2nd order combinatorial hypotheses between BCL and ABC family members. 21 Conclusion Presented here are a range of multiple synergistic ABC transporter 2nd order combina-280 tions that 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 com-285 ponents 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.290 Author’s contributions Concept, design, in silico implementation - SS. Analysis and interpretation of results - SS. Manuscript writing - SS. Manuscript revision - SS. 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