Machine learning discoveries of ANTXR2-X synergy in ETC-1922159 treated colorectal cancer cells

Machine learning discoveries of ANTXR2-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 ANTXR cell adhesion molecule 2 (ANTXR2)-X (X, a particular gene/protein) at 2nd order level after drug administration. These rankings reveal which ANTXR2-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 ANTXR2 and X. In this research work, we cover combinations of ANTXR2 with Collagen (COL), Integrin (ITG), Matrix metalloproteinases (MMP), WNT, TNF and Interleukin (IL). Keywords: ANTXR2, 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 Introduction In the unpublished preprint Sinha [1], a frame work of a search engine was developed which can rank combinations of factors (genes/proteins) in a signaling pathway.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 Interested readers are advised to go through unpublished preprints Sinha [1] and Sinha [2] for details regarding the search engine and the discoveries IML dicoveries of ANTXR2-X synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at (1) the recently concluded first ever Wnt Gordon Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA.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 September 1, 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. Anthrax toxin receptor related synergies 3.1.1. ANTXR2 - Collagen cross family analysis Anthrax toxin receptor ANTRX is known to capture the Bacillus anthracis toxin and40 form the cause of the anthrax disease. Regulatory mechanism of the ANTXR1 has been demostrated essential component in the fibrosis processes in fibroproliferative diseases. Loss of ANTXR1 (a.k.a TEM8) in fibroblasts leads to increased rates of synthesis of fiber-forming collagens, resulting in progressive fibrosis in skin and other organs Besschetnova et al. [5]. TEM8 interacts with the cleaved C5 domain of collagen 3(VI)45 Nanda et al. [6]. Hotchkiss et al. [7] also indicate the interaction of TEM8 and collagens. Bell et al. [8] indicate that a recombinant portion of ANTXR2 (a.k.a CMG2) was 2 RANKING ANTRX2 VS COL FAMILY RANKING OF ANTRX2 W.R.TCOL FAMILY RANKING OF COL FAMILY W.R.TANTXR2 laplace linear rbf laplace linear rbf COL5A3-ANTXR2 2006 1473 2217 COL5A3-ANTXR2 984 1782 933 COL6A1-ANTXR2 1935 1061 1366 COL6A1-ANTXR2 324 2211 398 COL7A1-ANTXR2 1002 2119 1690 COL7A1-ANTXR2 1722 956 2121 COL9A2-ANTXR2 1498 552 1361 COL9A2-ANTXR2 2391 80 135 COL17A1-ANTXR2 1906 1086 780 COL17A1-ANTXR2 576 2504 229 COL28A1-ANTXR2 1409 2259 2296 COL28A1-ANTXR2 478 1731 2362 Table 1: 2nd order combinatorial hypotheses between ANTRX2 and COL family found to bind collagen type IV and laminin, suggesting a potential role in basement membrane matrix synthesis and assembly. B¨ urgi et al. [9] show that CMG2/ANTXR2 regulates extracellular collagen VI which accumulates in hyaline fibromatosis syn-50 drome. A distinctive early childhood-onset disorder, systemic hyalinosis, is characterized by mutations in the anthrax toxin receptor 2 gene (ANTRX2) as shown by Shieh et al. [10]. Not much is known about the behaviour of ANTRX2 with collagens in colorectal cancer. In CRC cells treated with ETC-1922159 these were up regulated. Our search engine was able to rank the 2nd order combinations between these two to55 see if there is a possible existing synergy based on the already explored pathological functionality in the above works. At in silico level we found possible high numerical valued ranks pointed to some of the combinations of ANTXR2 and collagen family. On the left side of table 1 we found ANTXR2 to be up regulated w.r.t COL5A3/28A1 (probably COL-7A1 also). These are reflected in rankings of 2006 (laplace)60 and 2217 (rbf) for COL5A3-ANTXR2; 2259 (linear) and 2296 (rbf) for COL28A1ANTXR2 and probably 2119 (linear) and 1690 (rbf) for COL7A1-ANTXR2. On the right side, we found, COL-7A1/28A1 up regulated w.r.t ANTXR2. These are reflected in rankings of 1722 (laplace) and 2121 (rbf) for COL7A1-ANTXR2 and 1731 (linear) and 2362 (rbf) for COL28A1-ANTXR2. Table 2 shows the derived influences which65 can be represented graphically, with the following influences - •ANTXR2 w.r.t COL with COL5A3 −>ANTXR2 and COL28A1 −>ANTXR2; and •COL w.r.t ANTXR2 with COL7A1 <−ANTXR2 and COL28A1 <−ANTXR2. 3.1.2. ANTXR2 - Integrin cross family analysis To define whether there is a possible functional cooperation between TEM8/ANTXR170 and integrins to accomplish cell spreading, Werner et al. [11] sought to disrupt the function of collagen binding integrins, which mostly belong to β1 family of integrins, with β1 integrin-blocking antibodies. Their experiments suggest that the participation of β1 integrins can be excluded in TEM8-mediated cell spreading on collagen in primary fibroblasts. However, collectively, they indicate that the participation of these integrins75 in TEM8 spreading on collagen vary with cell type. The cytoplasmic domain of ANTXR1 affects binding of the protective antigen which is similar to integrin I domains that convert between open and closed conformations that bind ligand with high and low affinities, respectively. These findings are observed by Go et al. [12]. Scobie et al. [13] 3 UNEXPLORED COMBINATORIAL HYPOTHESES ANTXR2 w.r.t COL COL5A3 ANTXR2 COL28A1 ANTXR2 COL w.r.t ANTXR2 COL7A1 ANTXR2 COL28A1 ANTXR2 Table 2: 2nd order combinatorial hypotheses between ANTRX2 and COL family. observe similar behaviour. Abnormal clustering of TEM8/ANTXR1 with integrin β180 and vascular endothelial growth factor receptor 2 (VEGFR2) occurs in endothelial cells within cutaneous infantile hemangiomas, the most common vascular anomaly in childhood Besschetnova et al. [5]. These findings suggest the possibility to synergy between ANTXR2 and Integrin family. In CRC cells treated with ETC-1922159, ANTXR2 and integrin families were up regulated. The search engine alloted high numerical valued85 ranks to some of the 2nd order combinations of ANTXR2 and integrin family members thus pointing to possible synergy in CRC cells. Table 3 shows the rankings of ANTRX2 along with integrin family members. On the left side, we found ANTRX2 to be up regulated w.r.t ITG-A2/A3/B1BP1/B5/B8. These are reflected in the rankings of 2261 (laplace) and 2444 (rbf) for ITGA2-ANTXR2;90 2027 (laplace) and 2134 (linear) for ITGA3-ANTXR2; 2444 (laplace) and 2128 (rbf) for ITGB1BP1-ANTXR2; 1860 (linear)and 2315 (rbf) for ITGB5-ANTXR2; and 2354 (linear)and 2136 (rbf) for ITGB8-ANTXR2. On the right side, we found ITGB6 to be up regulated w.r.t ANTRX2. These are reflected in the rankings of 2000 (linear) and 1896 (rbf) for ITGB6-ANTXR2; Table 4 shows the derived influences which can95 be represented graphically, with the following influences - •ANTXR2 w.r.t ITG with ITG-A2/A3 −>ANTXR2 and ITG-B1BP1/B5/B8 −>ANTXR2; and •ITG w.r.t ANTXR2 with ITGB6 <−ANTXR2. 3.1.3. ANTXR2 - MMP cross family analysis Compromised interactions between TEM8/ANTXR1-deficient endothelial and fibrob-100 lastic cells cause dramatic reduction in the activity of the matrix-degrading enzyme MMP2 Besschetnova et al. [5]. They observe experimentally that loss of MMP2 activity requires loss of TEM8/ANTXR1 function in both endothelial and fibroblastic cells. Matrix metalloproteinases (MMP) are members of the metzincin group of proteases which share the conserved zinc-binding motif in their catalytic active site L¨ offek et al.105 [14]. these enzymes are capable of degrading all kinds of extracellular matrix proteins, but also can process a number of bioactive molecules as well as play a major role in 4 RANKING ANTRX2 VS ITG FAMILY RANKING OF ANTRX2 W.R.TITG FAMILY RANKING OF ITG FAMILY W.R.TANTXR2 laplace linear rbf laplace linear rbf ITGA2-ANTXR2 2261 1129 2444 ITGA2-ANTXR2 657 1662 215 ITGA3-ANTXR2 2027 2134 179 ITGA3-ANTXR2 305 1402 278 ITGA6-ANTXR2 1065 1850 1660 ITGA6-ANTXR2 352 2029 583 ITGB1-ANTXR2 2192 1273 1431 ITGB1-ANTXR2 1538 987 1593 ITGB1BP1-ANTXR2 2444 498 2128 ITGB1BP1-ANTXR2 1743 152 534 ITGB4-ANTXR2 1484 699 249 ITGB4-ANTXR2 123 1420 2116 ITGB5-ANTXR2 1318 1860 2315 ITGB5-ANTXR2 2216 718 1182 ITGB6-ANTXR2 1205 1262 1244 ITGB6-ANTXR2 1200 2000 1896 ITGB8-ANTXR2 1710 2354 2136 ITGB8-ANTXR2 296 1724 1485 Table 3: 2nd order combinatorial hypotheses between ANTRX2 and ITG family UNEXPLORED COMBINATORIAL HYPOTHESES ANTXR2 w.r.t ITG ANTXR2 ITG-A2/A3 ANTXR2 ITG-B1BP1/B5/B8 ITG w.r.t ANTXR2 ITGB6 ANTXR2 Table 4: 2nd order combinatorial hypotheses between ANTRX2 and ITG family. cell behaviors such as proliferation, migration, differention, apoptosis and host defense, Wikipedia contributors [15]. In CRC cells treated with ETC-1922159, ANTXR2 and integrin families were up regulated. The search engine alloted high numerical valued110 ranks to some of the 2nd order combinations of ANTXR2 and integrin family members thus pointing to possible synergy in CRC cells. Table 5 shows the rankings of ANTRX2 along with MMP family members. On the left side, we found ANTXR2 to be upregulated w.r.t MMP28. This is reflected in rankings of 2468 (laplace) and 1765 (linear) for MMP28-ANTXR2. On the115 right side we found MMP-1/15/28 up regulated w.r.t ANTXR2. These are reflected in rankings of 2009 (linear) and 2142 (rbf) for MMP1-ANTXR2; 2219 (linear) and 1926 (rbf) for MMP15-ANTXR2; and 1857 (linear) and 2092 (rbf) for MMP28-ANTXR2. Table 6 shows the derived influences which can be represented graphically, with the following influences - •ANTXR2 w.r.t MMP with ANTXR2 <−MMP28 and •MMP120 w.r.t ANTXR2 with MMP-1/15/28 <−ANTXR2. 3.1.4. ANTXR2 - WNT cross family analysis Abrami et al. [16] show that LRP6 can indeed form a complex with ATRs (anthrax toxin 5 RANKING ANTRX2 VS MMP FAMILY RANKING OF ANTRX2 W.R.TMMP FAMILY RANKING OF MMP FAMILY W.R.TANTXR2 laplace linear rbf laplace linear rbf MMP1-ANTXR2 1428 1407 1620 MMP1-ANTXR2 244 2009 2142 MMP14-ANTXR2 1067 1141 900 MMP14-ANTXR2 866 971 443 MMP15-ANTXR2 1457 740 1881 MMP15-ANTXR2 121 2219 1926 MMP28-ANTXR2 2468 1765 1202 MMP28-ANTXR2 11 1857 2092 Table 5: 2nd order combinatorial hypotheses between ANTRX2 and MMP family UNEXPLORED COMBINATORIAL HYPOTHESES ANTXR2 w.r.t MMP ANTXR2 MMP28 MMP w.r.t ANTXR2 MMP-1/15/28 ANTXR2 Table 6: 2nd order combinatorial hypotheses between ANTRX2 and MMP family. receptors), and that this interaction plays a role both in Wnt signalling and in anthrax toxin endocytosis. Through the ATR-LRP6 interaction, adhesion to the extracellular125 matrix could locally control Wnt signalling. The authors demonstrated that physical and functional interaction between CMG2/ANTXR2 and LRP6 also raised the possibility that the complex clinical manifestation of Systemic Hyalinosis might be due in part to defects in Wnt signalling. Fluorescence microscopy and biochemical analyses showed that LRP6 enables toxin internalization by interacting at the cell surface with130 PA receptors TEM8/ATR and/or CMG2/ANTXR2 to form a multi-component complex that enters cells upon PA binding (Wei et al. [17]). Verma et al. [18] postulate that the developmentally controlled expression of TEM8 modulates endothelial cell response to canonical Wnt signaling to regulate vessel patterning and density. These findings definitely indicate the synergy of ANTRX with Wnts. In CRC cells treated with ETC-135 1922159, ANTXR2 and WNT families were up regulated. The search engine alloted high numerical valued ranks to some of the 2nd order combinations of ANTXR2 and WNT family members thus pointing to possible synergy in CRC cells. Table 7 shows the rankings of ANTRX2 along with WNT family members. On the left side, we found ANTXR2 to be upregulated w.r.t WNT4. This is re-140 flected in rankings of 1833 (linear) and 2341 (rbf) for WNT4-ANTXR2. Table 8 shows the derived influences which can be represented graphically, with the following influences - •ANTXR2 w.r.t WNT with ANTXR2 <−WNT4. This synergistic upregulation of the WNT4 with ANTXR2 might indicate possible control over the signalling in CRC cells treated with ETC-1922159.145 6 RANKING ANTRX2 VS WNT FAMILY RANKING OF ANTRX2 W.R.TWNT FAMILY RANKING OF WNT FAMILY W.R.TANTXR2 laplace linear rbf laplace linear rbf WNT2B-ANTXR2 1160 1013 2286 WNT2B-ANTXR2 1577 1367 944 WNT4-ANTXR2 1735 1833 2341 WNT4-ANTXR2 175 1643 97 WNT7B-ANTXR2 2453 304 1196 WNT7B-ANTXR2 2106 242 1144 WNT9A-ANTXR2 1618 487 1766 WNT9A-ANTXR2 2317 162 845 Table 7: 2nd order combinatorial hypotheses between ANTRX2 and WNT family UNEXPLORED COMBINATORIAL HYPOTHESES ANTXR2 w.r.t WNT ANTXR2 WNT4 Table 8: 2nd order combinatorial hypotheses between ANTRX2 and WNT family. 3.1.5. ANTXR2 - TNF cross family analysis The author could not find much about TNF-ANTXR2 combinations in pathological cases in existing literature, however, Lee et al. [19] report the ”both LeTx and EdTx markedly inhibited LPS-induced transcription of tumour necrosis factor alpha (TNFα), interleukin (IL)-1β, and IL-6 in J774A.1 cells. In contrast, EdTx synergised with150 LPS to increase the transcription of IL-6 and IL-8 in HAECs. We showed that HAECs are suitable for anthrax toxin research and express higher levels of the two anthrax toxin receptors - tumour endothelial marker 8 (TEM8/ANTXR1) and capillary morphogenesis protein 2 (CMG2/ANTXR2) - than do J774A.1 cells”. The high expression of the ANTXR-1/2 is shown, however, the possible synergy between ANTXR and TNFs in155 not shown. Our search engine pointed to some of the combinations in CRC cells treated with ETC-1922159 treatment. In table 9, on the left we found ANTXR2 to be up regulated w.r.t TNF-AIP1/RSF10B. These are reflected in rankings of 1769 (linear) and 1946 (rbf) for TNFAIP1-ANTXR2 and 2278 (linear) and 2218 (rbf) for TNFRSF10BANTXR2. On the right we found, TNF-RSF10A/RSF10D/RSF12A/RSF14 was up160 regulated w.r.t ANTXR2. These are reflected in rankings of 2260 (laplace) and 2377 (rbf) for TNFRSF10A-ANTXR2, 2258 (laplace) and 2363 (rbf) for TNFRSF10DANTXR2, 2190 (laplace) and 2061 (rbf) for TNFRSF12A-ANTXR2 and 2370(laplace) and 1777 (linear) for TNFRSF14-ANTXR2. Table 10 shows the derived influences which can be represented graphically, with165 the following influences - •ANTXR2 w.r.t TNF with ANTXR2 <−TNFAIP1 and ANTXR2 <−TNFRSF10B and •TNF w.r.t ANTXR2 with TNFRSF10A <−ANTXR2; TNFRSF10D <−ANTXR2; TNFRSF12A <−ANTXR2 and TNFRSF14 <−ANTXR2. This synergistic upregulation of the TNF with ANTXR2 might indicate possible control over the signalling in CRC cells treated with ETC-1922159.170 7 RANKING ANTRX2 VS TNF FAMILY RANKING OF ANTRX2 W.R.TTNF FAMILY RANKING OF TNF FAMILY W.R.TANTXR2 laplace linear rbf laplace linear rbf TNF-ANTXR2 1439 1568 1285 TNF-ANTXR2 709 1758 1479 TNFAIP1-ANTXR2 1552 1769 1946 TNFAIP1-ANTXR2 1252 2177 218 TNFAIP2-ANTXR2 125 962 2134 TNFAIP2-ANTXR2 659 1156 2109 TNFAIP3-ANTXR2 1184 1253 1558 TNFAIP3-ANTXR2 1429 2485 1731 TNFRSF1A-ANTXR2 1063 310 2145 TNFRSF1A-ANTXR2 1557 2471 935 TNFRSF10A-ANTXR2 351 1358 1280 TNFRSF10A-ANTXR2 2260 32 2377 TNFRSF10B-ANTXR2 2278 2218 982 TNFRSF10B-ANTXR2 852 715 216 TNFRSF10D-ANTXR2 1352 891 1685 TNFRSF10D-ANTXR2 2258 454 2363 TNFRSF12A-ANTXR2 551 1283 1794 TNFRSF12A-ANTXR2 2190 1150 2061 TNFRSF14-ANTXR2 999 442 498 TNFRSF14-ANTXR2 2370 1777 1014 TNFRSF21-ANTXR2 897 997 298 TNFRSF21-ANTXR2 1474 343 510 TNFRSF10-ANTXR2 2151 966 324 TNFRSF10-ANTXR2 2065 112 339 TNFRSF15-ANTXR2 868 967 1590 TNFRSF15-ANTXR2 664 1211 1669 Table 9: 2nd order combinatorial hypotheses between ANTRX2 and TNF family UNEXPLORED COMBINATORIAL HYPOTHESES ANTXR2 w.r.t TNF ANTXR2 TNFAIP1 ANTXR2 TNFRSF10B TNF w.r.t ANTXR2 TNFRSF10A ANTXR2 TNFRSF10D ANTXR2 TNFRSF12A ANTXR2 TNFRSF14 ANTXR2 Table 10: 2nd order combinatorial hypotheses between ANTRX2 and TNF family. 3.1.6. ANTXR2 - IL cross family analysis The author could not find much about TNF-ANTXR2 combinations in pathological cases in existing literature, however, Lee et al. [19] report the ”both LeTx and EdTx markedly inhibited LPS-induced transcription of tumour necrosis factor alpha (TNFα), interleukin (IL)-1β, and IL-6 in J774A.1 cells. In contrast, EdTx synergised with175 LPS to increase the transcription of IL-6 and IL-8 in HAECs. We showed that HAECs are suitable for anthrax toxin research and express higher levels of the two anthrax toxin receptors - tumour endothelial marker 8 (TEM8/ANTXR1) and capillary morphogenesis protein 2 (CMG2/ANTXR2) than do J774A.1 cells”. The high expression 8 RANKING ANTRX2 VS IL FAMILY RANKING OF ANTRX2 W.R.TIL FAMILY RANKING OF IL FAMILY W.R.TANTXR2 laplace linear rbf laplace linear rbf IL1A-ANTXR2 1733 454 2253 IL1A-ANTXR2 275 2356 1859 IL1B-ANTXR2 1222 1302 714 IL1B-ANTXR2 330 2011 1762 IL1RAP-ANTXR2 1288 367 80 IL1RAP-ANTXR2 2339 442 747 IL1RN-ANTXR2 1389 1914 1894 IL1RN-ANTXR2 349 1031 1919 IL2RG-ANTXR2 1897 25 432 IL2RG-ANTXR2 368 1867 450 IL6ST-ANTXR2 1944 2219 1914 IL6ST-ANTXR2 46 1780 1865 IL8-ANTXR2 1169 1281 1398 IL8-ANTXR2 1343 2002 434 IL10RB-ANTXR2 1737 496 1545 IL10RB-ANTXR2 1403 800 754 IL15-ANTXR2 787 1812 927 IL15-ANTXR2 1002 1340 481 IL15RA-ANTXR2 840 800 1695 IL15RA-ANTXR2 1924 636 1901 IL17C-ANTXR2 1832 2334 1191 IL17C-ANTXR2 339 2121 2437 IL17REL-ANTXR2 29 1889 2303 IL17REL-ANTXR2 2406 111 960 Table 11: 2nd order combinatorial hypotheses between ANTRX2 and IL family of the ANTXR-1/2 is shown, however, the possible synergy between ANTXR and IL180 in not shown. Our search engine pointed to some of the combinations in CRC cells treated with ETC-1922159 treatment. In table 11, on the left we found ANTXR2 to be up regulated w.r.t IL-1RN/6ST/17C/17REL. These are reflected in rankings of 1914 (linear) and 1894 (rbf) for IL1RN-ANTXR2; 1944 (laplace), 2219 (linear) and 1914 (rbf) for IL6ST-ANTXR2; 1832 (laplace) and 2334 (linear) for IL17C-ANTXR2185 and 1889 (linear) and 2303 (rbf) for IL17REL-ANTXR2. On the right we found, IL-1A/1B/6ST/17C was up regulated w.r.t ANTXR2. These are reflected in rankings of 2356 (linear) and 1859 (rbf) for IL1A-ANTXR2; 1780 (linear) and 1865 (rbf) for IL6ST-ANTXR2; 1924 (laplace) and 1901 (rbf) for IL15RA-ANTXR2; and 2121 (linear) and 2437 (rbf) for IL17C-ANTXR2.190 Table 12 shows the derived influences which can be represented graphically, with the following influences - •ANTXR2 w.r.t IL with ANTXR2 <−IL1RN; ANTXR2 < −IL6ST; ANTXR2 <−IL17C and ANTXR2 <−IL17REL; and •IL w.r.t ANTXR2 with IL1A <−ANTXR2; IL1B <−ANTXR2; IL6ST <−ANTXR2; and IL17C <−ANTXR2.195 Conclusion Presented here are a range of multiple synergistic ANTXR2 2nd order combinations 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 combina-200 tions 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 biologists/oncologists to further investigate and understand the mechanism behind the 9