Machine Learning Discoveries of ATG3-X Synergy in ETC-1922159 Treated Colorectal Cancer Cells

Machine learning discoveries of ATG3-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 Autophagy related 3 (ATG3) is one of the genes that plays a major role in autophagy. It has been found to be highly expressed in colon caner. In colorectal cancer (CRC) cells treated with ETC-1922159, ATG3 was found to be down regulated along with other genes. A recently developed search engine ranked combinations of ATG3-X (X, a particular gene/protein) at 2nd order level after drug administration. Some of these combinations have been tested in wet lab, however, there are other that have yet to be explored or tested. These rankings reveal which ATG3-X combinations might be working synergistically in CRC. In this research work, I cover combinations of ATG3 with programmed cell death (PDCD), bromodomain containing (BRD), aspartyl-tRNA synthetase (DARS), methyltransferase N6-adenosine-methyltransferase non-catalytic subunit (METTL), eukaryotic translation initiation factor (EIF), forkhead box (FOX), growth arrest specific (GAS), RAB member RAS oncogene GTPases (RAB), FA complementation groups (RAD51), tripartite motif containing (TRIM) and TNF alpha induced protein (TNFAIP) family. Keywords: ATG3, Porcupine inhibitor ETC-1922159, Sensitivity analysis, Colorectal cancer. 1. Introduction 1.1. Autophagy Authophagy is a natural process in which cells works towards degradation of dysfunctional proteins and other cytoplasmic cargo via lysosome dependent mechanism. Hitherto, there are three ways by which autophagy happens - (1) macroautopahgy, (2) chaperone mediated autophagy and (3) microautophagy. Out of these the first one is the most widely researched topic. Autophagy was coined by Christian de Duve in 1963, IML dicoveries of ATG3-X synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at the first Wnt Gordon Research Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA. Preprint submitted to Preprint January 2, 2025 after his discovery of the exitence of lysosomes which were involved in the process (De Duve and Wattiaux [1] and Klionsky [2]). Later, Takeshige et al. [3] first observed the autophagy degradation in yeast cells. This was followed by isolation and characterization of autophagy causing genes in Tsukada and Ohsumi [4]. Introductory reviews on autophagy can be found in Levine and Kroemer [5] and Levine and Kroemer [6]. The reviews also cover the roles of autophagy genes in cancer, briefly. A recent study by Li et al. [7], discusses the role of autophagy and its related genes (i.e ATGs) in both cancer suppression as well as cancer promotion. 1.2. ATG3 ATG3 is a protein that lacks rigid structure and more specifically it is a ubiquitin carrier protein E2-like enzyme. The crystal structure of ATG3 has been elucidated in Yamada et al. [8]. ATG3 engages with many binding partners and binding sites. Fang et al. [9] indicate that ATG3 interacts with ATG7 via formation of an E1-E2 complex, LC3/ATG8 via a thioester bond and ATG12 via leucine of the LC3-interacting region motif, while providing necessary references for the same. They also cite references which show that ATG3 is implicated in various types of cancers. In a recent finding, Huang et al. [10] show that ATG3 promotes colon cancer. ATG3 was found to be down regulated in colorectal cancer cell lines after the treatment of ETC-1922159 drug as observed in Madan et al. [11]. Most studies till now, have dealt with how the ATG3 works and there is very less informantion reagarding which gene/protein combinations might be working synergistically along with it. It would be nice to observe if there is any connection between the independently observed factors in the form of unknown biological hypotheses. To solve the issue, the next section discusses a solution to the problem. 1.3. Combinatorial search problem and a possible solution In a recently published work Sinha [12], a frame work of a search engine was developed which can rank combinations of factors (genes/proteins) in a signaling pathway. Readers are requested to go through the adaptation of the above mentioned work for gaining deeper insight into the working of the pipeline and its use of published data set generated after administration of ETC-1922159, Sinha [13]. The work uses SVM package by Joachims [14] in https://www.cs.cornell.edu/people/tj/svm_light/ svm_rank.html. I use the adaptation to rank 2nd order gene combinations. 2. Results & Discussion 2.1. ATG3 related synergies 2.1.1. ATG3-PDCD Murrow et al. [15] identify an interaction between ATG12-ATG3 and PDCD6IP and demonstrate that the interaction controls multiple PDCD6IP dependent processes like 2 exosome biogenesis, late endosome distribution,, and viral budding. In colorectal cancer cells treated with ETC-1922159, PDCD family members and ATG3, were found to be down regulated and recorded independently. I was able to rank 2nd order combinations of PDCD family members and ATG3, that were down regulated. Table 1 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 2 generated from analysis of the ranks in table 1. The table 1 shows rankings of PDCD family w.r.t ATG3. PDCD2 - ATG3 shows low ranking of 338 (laplace) and 272 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, PDCD2L, PDCD11 and PDCD7 showed high ranking with ATG3, thus indicating that they might not be working synergistically with ATG3, before the drug treatment. RANKING PDCD FAMILY VS ATG3 RANKING OF PDCD FAMILY W.R.TATG3 laplace linear rbf PDCD2 - ATG3 338 272 1974 PDCD2L - ATG3 643 1890 1069 PDCD11 - ATG3 1683 2573 1307 PDCD7 - ATG3 2706 1722 1648 Table 1: 2nd order interaction ranking between ATG3 VS PDCD family members. One can also interpret the results of the table 1 graphically, with the following influences - •PDCD family w.r.t ATG3 with ATG3 −>PDCD2. UNEXPLORED COMBINATORIAL HYPOTHESES PDCD family w.r.t ATG3 PDCD2 ATG3 Table 2: 2nd order combinatorial hypotheses between ATG3 and PDCD family members. 2.1.2. ATG3-BRD In acute myelogenous leukemia cells, Huang et al. [16] found that BRD4 binds to the promoters of ATG3, and expression of this gene is reduced by inhibitors of BRD4. 3 Thus BRD4 plays a direct role in autophagy by regulating the transcription of ATG3. In colorectal cancer cells treated with ETC-1922159, BRD family members and ATG3, were found to be down regulated and recorded independently. I was able to rank 2nd order combinations of BRD family members and ATG3, that were down regulated. Table 3 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 4 generated from analysis of the ranks in table 3. The table 3 shows rankings of BRD family w.r.t ATG3. BRD8 - ATG3 shows low ranking of 453 (laplace), 574 (linear) and 1550 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. RANKING BRD FAMILY VS ATG3 RANKING OF BRD FAMILY W.R.TATG3 laplace linear rbf BRD8 - ATG3 453 574 1550 Table 3: 2nd order interaction ranking between ATG3 VS BRD family members. One can also interpret the results of the table 3 graphically, with the following influences - •BRD family w.r.t ATG3 with ATG3 −>BRD8. UNEXPLORED COMBINATORIAL HYPOTHESES BRD family w.r.t ATG3 BRD8 ATG3 Table 4: 2nd order combinatorial hypotheses between ATG3 and BRD family members. 2.1.3. ATG3-DARS/METTL In cervical cancer (CC), Shen et al. [17] experimentally confirmed that DARS-AS1 regulated the expression of ATG3 to affect CC cell autophagy by modulating DARS expression. Further, they show that DARS-AS1 recruits METTL3 and METTL14 mediated m6A methylation to translate DARS translation. In colorectal cancer cells treated with ETC-1922159, DARS (and METTL) family members and ATG3, were found to be down regulated and recorded independently. I was able to rank 2nd order combinations of DARS (and METTL) family members and ATG3, that were down regulated. Table 5 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 6 generated from analysis of the ranks in table 5. The 4 table 5 shows rankings of DARS family w.r.t ATG3. DARS2 - ATG3 shows low ranking of 1242 (laplace), 1451 (linear) and 512 (rbf). METTL1 - ATG3 shows low ranking of 472 (laplace), 337 (linear) and 391 (rbf). METTL8 - ATG3 shows low ranking of 1287 (laplace) and 1469 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, METTL13, METTL3, METTL21A, METTL17, METTL16, METTL5, METTL12, METTL21B and METTL2B showed high ranking with ATG3, thus indicating that they might not be working synergistically with ATG3, before the drug treatment. RANKING DARS (AND METTL) FAMILY VS ATG3 RANKING OF DARS (AND METTL) FAMILY W.R.TATG3 laplace linear rbf DARS2 - ATG3 1242 1451 512 METTL1 - ATG3 472 337 391 METTL8 - ATG3 1287 1971 1469 METTL13 - ATG3 1515 2540 1811 METTL3 - ATG3 1681 2358 2070 METTL21A - ATG3 1817 2034 1709 METTL17 - ATG3 1990 2695 2157 METTL16 - ATG3 2125 2260 1061 METTL5 - ATG3 2166 2530 2039 METTL12 - ATG3 2222 1756 1772 METTL21B - ATG3 2403 2003 2366 METTL2B - ATG3 2436 2322 1941 Table 5: 2nd order interaction ranking between ATG3 VS DARS family members. One can also interpret the results of the table 5 graphically, with the following influences - •DARS family w.r.t ATG3 with ATG3 −>DARS2 and ATG3 −>METTL1/8. 2.1.4. ATG3-EIF EIF5A function is well-described in yeast and bacteria, but little is known about its translational targets in human cells. Frankel [18] using liquid chromatography-mass spectrometry (LC-MS) analysis, revealed that EIF5A affects the translation of ATG3. It was confirmed that ATG3 protein levels were reduced upon knockdown of EIF5A. In colorectal cancer cells treated with ETC-1922159, EIF family members and ATG3, were found to be down regulated and recorded independently. I was able to rank 2nd order combinations of EIF family members and ATG3, that were down regulated. 5 UNEXPLORED COMBINATORIAL HYPOTHESES DARS (and METTL) family w.r.t ATG3 DARS2 ATG3 METTL-1/8 ATG3 Table 6: 2nd order combinatorial hypotheses between ATG3 and DARS family members. Table 7 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 8 generated from analysis of the ranks in table 7. The table 7 shows rankings of EIF family w.r.t ATG3. EIF2B1 - ATG3 shows low ranking of 784 (laplace) and 485 (linear). EIF2D - ATG3 shows low ranking of 1300 (laplace), 1108 (linear) and 1497 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, EIF2B3, EIF3F, EIF3L, EIF3E, EIF2AK4, EIF4B, EIF2B5 and EIF4EBP1 showed high ranking with ATG3, thus indicating that they might not be working synergistically with ATG3, before the drug treatment. RANKING EIF FAMILY VS ATG3 RANKING OF EIF FAMILY W.R.TATG3 laplace linear rbf laplace linear rbf EIF2B1 - ATG3 784 485 2014 EIF2D - ATG3 1300 1108 1497 EIF2B3 - ATG3 1601 1947 1165 EIF3F - ATG3 1826 2612 1861 EIF3L - ATG3 1883 1801 2185 EIF3E - ATG3 1936 2142 1131 EIF2AK4 - ATG3 2099 2654 2052 EIF4B - ATG3 2270 2577 1524 EIF2B5 - ATG3 2614 2678 2387 EIF4EBP1 - ATG3 2729 579 2622 Table 7: 2nd order interaction ranking between ATG3 VS EIF family members. One can also interpret the results of the table 7 graphically, with the following influences - •EIF family w.r.t ATG3 with ATG3 −>EIF-2B1/2D. UNEXPLORED COMBINATORIAL HYPOTHESES EIF family w.r.t ATG3 EIF-2B1/2D ATG3 Table 8: 2nd order combinatorial hypotheses between ATG3 and EIF family members. 6 2.1.5. ATG3-FOX In hypoxic granulosa cells, Li et al. [19] show that members of forkhead box proteins FOXO, stimulate the upregulation of ATG3. In colorectal cancer cells treated with ETC-1922159, FOX family members and ATG3, were found to be down regulated and recorded independently. I was able to rank 2nd order combinations of FOX family members and ATG3, that were down regulated. Table 9 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 10 generated from analysis of the ranks in table 9. The table 9 shows rankings of FOX family w.r.t ATG3. FOXM1 - ATG3 shows low ranking of 14 (laplace), 66 (linear) and 147 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, FOXD2-AS1, FOXA2 and FOXJ1 showed high ranking with ATG3, thus indicating that they might not be working synergistically with ATG3, before the drug treatment. RANKING FOX FAMILY VS ATG3 RANKING OF FOX FAMILY W.R.TATG3 laplace linear rbf FOXM1 - ATG3 14 66 147 FOXD2-AS1 - ATG3 1667 1571 2633 FOXA2 - ATG3 1946 1898 1198 FOXJ1 - ATG3 2711 2644 2740 Table 9: 2nd order interaction ranking between ATG3 VS FOX family members. One can also interpret the results of the table 9 graphically, with the following influences - •FOX family w.r.t ATG3 with ATG3 −>FOXM1. UNEXPLORED COMBINATORIAL HYPOTHESES FOX family w.r.t ATG3 FOXM1 ATG3 Table 10: 2nd order combinatorial hypotheses between ATG3 and FOX family members. 7 2.1.6. ATG3-GAS Li et al. [20] showed that knockdown of GAS5 suppressed the expression of LC3II, ATG3 and ATG5-ATG12 complex formation, thus suggesting that GAS5/miR-23a/ATG3 axis might be a regulatory network contributing to autophagy and cell viability. In colorectal cancer cells treated with ETC-1922159, GAS family members and ATG3, were found to be down regulated and recorded independently. I was able to rank 2nd order combinations of GAS family members and ATG3, that were down regulated. The table 11 shows rankings of GAS family w.r.t ATG3. Interestingly, all recorded family members, i.e GAS5, GAS5-AS1, GAS2L3, GAS6 and GAS6-AS1 showed high ranking with ATG3, thus indicating that they might not be working synergistically with ATG3, before the drug treatment. RANKING GAS FAMILY VS ATG3 RANKING OF GAS FAMILY W.R.TATG3 laplace linear rbf GAS5 - ATG3 954 2092 1870 GAS5-AS1 - ATG3 1607 2016 1841 GAS2L3 - ATG3 2354 1931 2264 GAS6 - ATG3 2491 1407 2328 GAS6-AS1 - ATG3 2604 1368 2705 Table 11: 2nd order interaction ranking between ATG3 VS GAS family members. 2.1.7. ATG3-RAB ATG16L facilitates LC3/ATG8-conjugation to phos-phatidylethanolamine by forming a complex with ATG12-conjugated ATG5 and recruiting an LC3-ATG3 intermediate to elongating isolation membranes. Fukuda and Itoh [21] report that ATG16L interacts with the Golgi-resident small GTPase RAB33B and RAB33A. Thus there exists a synergy or connection between RAB33-A/B and ATG3. In colorectal cancer cells treated with ETC-1922159, RAB family members and ATG3, were found to be down regulated and recorded independently. I was able to rank 2nd order combinations of RAB family members and ATG3, that were down regulated. Table 12 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 13 generated from analysis of the ranks in table 12. The table 12 shows rankings of RAB family w.r.t ATG3. RABEPK - ATG3 shows low ranking of 634 (laplace) and 1261 (rbf). RAB26 - ATG3 shows low ranking of 8 851 (laplace), 1180 (linear) and 157 (rbf). RAB23 - ATG3 shows low ranking of 1121 (laplace) and 1073 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. Further, RAB40B, RAB11FIP3, RABL5, RAB36 and RAB40A showed high ranking with ATG3, thus indicating that they might not be working synergistically with ATG3, before the drug treatment. RANKING RAB FAMILY VS ATG3 RANKING OF RAB FAMILY W.R.TATG3 laplace linear rbf RABEPK - ATG3 634 1626 1261 RAB26 - ATG3 851 1180 157 RAB23 - ATG3 1121 2057 1073 RAB40B - ATG3 2007 2584 1810 RAB11FIP3 - ATG3 2117 2672 2106 RABL5 - ATG3 2353 1197 2241 RAB36 - ATG3 2521 1510 2598 RAB40A - ATG3 2664 2242 2629 Table 12: 2nd order interaction ranking between ATG3 VS RAB family members. One can also interpret the results of the table 12 graphically, with the following influences - •RAB family w.r.t ATG3 with ATG3 −>RAB-EPK/26/23. UNEXPLORED COMBINATORIAL HYPOTHESES RAB family w.r.t ATG3 RAB-EPK/26/23 ATG3 Table 13: 2nd order combinatorial hypotheses between ATG3 and RAB family members. 2.1.8. ATG3-RAD51 RAD51 plays a major role in homologous recombination but it is unclear whether RAD51 can be involved in gene regulation as a co-factor. Kang et al. [22] show results 9