Machine Learning Discoveries of BUB1-X Synergy in ETC-1922159 Treated Colorectal Cancer Cells

Machine learning discoveries of BUB1-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 The multidomain protein kinase, budding uninhibited by benzimidazoles 1 (BUB1) is a central component of the mitotic checkpoint for spindle assembly (SAC). It works with many different components. A recently developed search engine ranked combinations of mitotic checkpoint serine/threonine-protein kinase BUB1-X (X, a particular gene/protein) at 2nd order level after drug administration. Some of these rankings point to already confirmed wet lab results, while other point to possible BUB1-X synergistic combinations that might be working in CRC, but haven’t been tested/explored. In this research work, I cover combinations of BUB1 with spindle checkpoint protein mitotic arrest deficient 2 (MAD2), inner centromere protein (INCENP), zwilch kinetochore protein (ZWILCH), aurora kinase (AURK), ZW10 interacting kinetochore protein (ZWINT), BUB-1B/3, centromere protein (CENP), cell division cycle (CDC), transforming growth factor beta (TGFB), DNA topoisomerase II (TOP), shugoshin (SGO), kinesin family member (KIF) and cyclin dependent kinase (CDK) family. Keywords: BUB1, Porcupine inhibitor ETC-1922159, Sensitivity analysis, Colorectal cancer. 1. Introduction 1.1. Spindle assembly checkpoint Zirkle [1] first observed that, when just one chromosome is retarded to arrive at the metaphase plate, anaphase onset is postponed until some minutes after its arrival. Later, Hoyt et al. [2] identified mutant strains of S. cerevisiae that fail to properly arrest their5 cell cycles at mitosis in response to the loss of microtubule function. Their studies defined three genes (BUB) required for normal cell cycle arrest. The multidomain protein kinases BUB1 and BUBR1 are central components of the mitotic checkpoint for IML dicoveries of BUB1-X synergy in ETC-1922159 treated CRC cells Email address: [email protected] (shriprakash sinha) 1Aspects of unpublished work were presented in a poster session at (1) the recently concluded first ever Wnt Gordon Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA.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 December 27, 2024 spindle assembly (SAC). In most colorectal cancers, a chromosomal instability (CIN) leading to an abnormal chromosome number (aneuploidy) is observed Strand et al. [3].10 Cahill et al. [4] show that CIN is associated with the loss of function of a mitotic checkpoint. In some cancers displaying CIN, the loss of this checkpoint was associated with the mutational inactivation of a human homologue of the yeast BUB1 gene. The normal mitotic checkpoints of cells displaying microsatellite instability become defective upon transfer of mutant hBUB1 alleles from either of two CIN cancers.15 The missegregation of sister chromatids during mitosis results in the loss or gain of chromosomes in daughter cells (aneuploidy). To avoid this disastrous outcome, BUB works in synergy with multiple proteins (Bolanos-Garcia and Blundell [5]). Many of the different synergies represented as combinations of genes/proteins have been experimentally tested and published. However, there are combinations that have yet to be20 explored. I address the problem of identifying these unexplored combinations via use of a machine learning based search engine in the next section. 1.2. Combinatorial search problem and a possible solution In a recently published work Sinha [6], a frame work of a search engine was developed which can rank combinations of factors (genes/proteins) in a signaling pathway. Read-25 ers 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 [7]. The work uses SVM package by Joachims [8] in https://www.cs.cornell.edu/people/tj/svm_light/svm_ rank.html. I use the adaptation to rank 2nd order gene combinations.30 2. Results & Discussion 2.1. BUB1 related synergies 2.1.1. BUB1-MAD2/INCENP/ZWILCH/AURKB/ZWINT/BUB1B/BUB3 Johnson et al. [9] show that BUB1 plays a key role in the assembly of checkpoint proteins at the kinetochore. It is required for the subsequent localization of CENPF,35 BUBR1 (or BUB1B), CENPE and MAD2. Carvalhal et al. [10] state that proper kinetochore-microtubule attachments are supported by BUB1 dependent centromeric H2A-T120 phosphorylation, which promotes the recruitment of the chromosomal passenger complex, consisting of Aurora B (AURKB), inner centromere protein (INCENP), survivin, and borealin. BUB1 contributes to the recruitment of ROD-ZWILCH-40 ZW10 (RZZ) in human cells (Cheeseman and Desai [11] , Foley and Kapoor [12]). Wang et al. [13] show that AURKB phosphorylates HASPIN to promote generation of H3T3ph and that AURKB activity is required for normal chromosomal localization of the chromosomal passenger complex. This indicates an intimate linkage between AURKB and HASPIN functions in mitosis. They also propose that AURKB activity45 triggers a CPC-HASPIN-H3T3ph feedback loop that promotes generation of H3T3ph on chromatin and provide evidence that the BUB1-SGO-CPC pathway supplies a signal that boosts the CPC-HASPIN-H3T3ph feedback loop specifically at centromeres 2 to produce the well-known accumulation of the CPC in these regions. Kiyomitsu et al. [14] show that the kinetochore target of BUBs in human cells is a kinetochore protein50 (blinkin), which interacts with ZWINT-1 (or ZWINT) and the two subunits hMis13 and hMis14 of the hMis12 complex. Bolanos-Garcia and Blundell [5] also state that BUBR1 (or BUB1B) forms part of the mitotic checkpoint complex (MCC) that contains BUB3, MAD2 and cell division cycle 20 (CDC20). All the above experimental validations have been done and each of them were55 found to be down regulated in ETC-1922159 treated CRC. Individual recordings of these down regulations have been documented. Using the adaptation of the above mentioned search engine, I was able to rank 2nd order combination of MAD2 / INCENP / ZWILCH / AURKB / ZWINT / BUB1B / BUB3 with BUB1, that were down regulated. Table 1 shows rankings of these combinations. Followed by this is the unexplored60 combinatorial hypotheses in table 2 generated from analysis of the ranks in table 1. The table 1 shows rankings of individual members w.r.t BUB1. MAD2L1 - BUB1 shows low ranking of 227 (laplace) and 17 (rbf). MAD2L2 - BUB1 shows low ranking of 977 (laplace) and 219 (linear). INCENP - BUB1 shows low ranking of 959 (laplace), 842 (linear) and 1188 (rbf). ZWILCH - BUB1 shows low ranking of 345 (laplace) and65 1561 (linear). AURKB - BUB1 shows low ranking of 97 (laplace) and 491 (linear). ZWINT - BUB1 shows low ranking of 219 (laplace), 187 (linear) and 27 (rbf). BUB3 - BUB1 shows low ranking of 1657 (laplace) and 1042 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. AURKA and BUB1B did not show synergistic down regulation70 with BUB1. RANKING INDIVIDUAL MEMBERS VS BUB1 RANKING OF INDIVIDUAL MEMBERS W.R.TBUB1 laplace linear rbf MAD2L1 - BUB1 227 2601 17 MAD2L2 - BUB1 977 219 2067 INCENP - BUB1 959 842 1188 ZWILCH - BUB1 345 1561 2722 AURKA - BUB1 1008 1835 2613 AURKB - BUB1 97 491 2561 ZWINT - BUB1 219 187 27 BUB1B - BUB1 41 2188 2031 BUB3 - BUB1 1657 2711 1042 Table 1: 2nd order interaction ranking between BUB1 VS Individual members. 3 One can also interpret the results of the table 1 graphically, with the following influences - •Individual members w.r.t BUB1 with BUB1 >MAD2-L1/L2; BUB1 >INCENP; BUB1 >ZWILCH; BUB1 >AURKB; BUB1 >ZWINT; and BUB1 >BUB3.75 UNEXPLORED COMBINATORIAL HYPOTHESES Individual members w.r.t BUB1 MAD2-L1/L2 BUB1 INCENP BUB1 ZWILCH BUB1 AURKB BUB1 ZWINT BUB1 BUB3 BUB1 Table 2: 2nd order combinatorial hypotheses between BUB1 and Individual members. 2.1.2. BUB1-CENP family Johnson et al. [9] show that BUB1 plays a key role in the assembly of checkpoint proteins at the kinetochore. It is required for the subsequent localization of CENPF, BUBR1 (or BUB1B), CENPE and MAD2. Johnson et al. [9] also mention a number of proteins, including CENPA, CENPC and CENPI, that localize to the centromere re-80 gion throughout the cell cycle and many other proteins only localize to kinetochores transiently during mitosis, while pointing to relevant literature. All the above experimental validations have been done and each of them were found to be down regulated in ETC-1922159 treated CRC. Individual recordings of these down regulations have been documented. I was able to rank 2nd order combination of CENP family mem-85 bers with BUB1, that were down regulated. Rankings confirm some of the existing combinations while giving insight about unexplored combinations also. 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 CENP family w.r.t BUB1. CENPH - BUB1 shows low rank-90 ing of 43 (laplace), 261 (linear) and 482 (rbf). CENPA - BUB1 shows low ranking of 67 (laplace), 41 (linear) and 31 (rbf). CENPM - BUB1 shows low ranking of 90 (laplace), 1492 (linear) and 1105 (rbf). CENPJ - BUB1 shows low ranking of 142 (laplace), 69 (linear) and 1484 (rbf). CENPU - BUB1 shows low ranking of 342 (laplace), 632 (linear) and 852 (rbf). CENPE - BUB1 shows low ranking of 385 (laplace) and 10595 (linear). CENPF - BUB1 shows low ranking of 606 (laplace), 430 (linear) and 1419 (rbf). CENPK - BUB1 shows low ranking of 607 (laplace), 3 (linear) and 1371 (rbf). 4 CENPI - BUB1 shows low ranking of 672 (laplace), 170 (linear). CENPW - BUB1 shows low ranking of 730 (laplace), 64 (linear) and 304 (rbf). CENPN - BUB1 shows low ranking of 917 (laplace), 482 (linear) and 447 (rbf). These rankings point to the100 synergy existing between the two components, which have been down regulated after the drug treatment. CENPO, CENPL and CENPV did not show synergistic down regulation with BUB1. RANKING CENP FAMILY VS BUB1 RANKING OF CENP FAMILY W.R.TBUB1 laplace linear rbf CENPH - BUB1 43 261 482 CENPA - BUB1 67 41 31 CENPM - BUB1 90 1492 1105 CENPJ - BUB1 142 69 1484 CENPU - BUB1 342 632 852 CENPE - BUB1 385 105 2360 CENPF - BUB1 606 430 1419 CENPK - BUB1 607 3 1371 CENPO - BUB1 650 2578 1694 CENPI - BUB1 672 170 2384 CENPW - BUB1 730 64 304 CENPL - BUB1 819 1994 2181 CENPN - BUB1 917 482 447 CENPV - BUB1 1955 1423 2226 Table 3: 2nd order interaction ranking between BUB1 VS CENP family members. One can also interpret the results of the table 3 graphically, with the following influences - •CENP family w.r.t BUB1 with BUB1 >CENP-H/A/M/J/U/E/F/K/I/W/N.105 5 UNEXPLORED COMBINATORIAL HYPOTHESES CENP family w.r.t BUB1 CENP-H/A/M/J/U/E/F/K/I/W/N BUB1 Table 4: 2nd order combinatorial hypotheses between BUB1 and CENP family members. 2.1.3. BUB1-CDC family Goto et al. [15] suggest that CDC28 mediates phosphorylation of BUB1 at T566. This plays an important role for the degradation of BUB1 in anaphase, and the phosphorylation is required for adaptation of the spindle checkpoint to prolonged mitotic arrest. Bolanos-Garcia and Blundell [5] depict a model where BUB1 KEN boxes me-110 diate BUB1 degradation by the APC/CCDH1 in the G1 phase of the cell cycle and BUB1CDC20 binding. Phosphorylation of CDC20 S153 by BUB1 is required for efficient SAC activity. All the above experimental validations have been done and each of them were found to be down regulated in ETC-1922159 treated CRC. Individual recordings of these down regulations have been documented. I was able to rank 2nd 115 order combination of CDC family members with BUB1, that were down regulated. Rankings confirm some of the existing combinations while giving insight about unexplored combinations also. 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. The120 table 5 shows rankings of CDC family w.r.t BUB1. CDC45 - BUB1 shows low ranking of 12 (laplace) and 228 (rbf). CDC25C - BUB1 shows low ranking of 53 (laplace), 303 (linear) and 188 (rbf). CDC20 - BUB1 shows low ranking of 253 (laplace) and 362 (rbf). CDC7 - BUB1 shows low ranking of 305 (laplace), 34 (linear) and 853 (rbf). CDC6 - BUB1 shows low ranking of 647 (laplace), 776 (linear) and 444 (rbf).125 CDC25A - BUB1 shows low ranking of 853 (laplace) and 447 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. CDC23 and CDC123 did not show synergistic down regulation with BUB1. One can also interpret the results of the table 5 graphically, with the following130 influences - •CDC family w.r.t BUB1 with BUB1 >CDC-45/25C/20/7/6/25A. 2.1.4. BUB1-TGFbfamily Nyati et al. [16] identified BUB1 as a mediator of TGFbsignaling. BUB1 interacted with TGFBRI in response to stimulation with TGFband promoted the heterodimerization of TGFBRI and TGFBRII. Nyati et al. [17] demonstrate that TGFBR2 phosphory-135 lates BUB1 at Serine-318. All the above experimental validations have been done and each of them were found to be down regulated in ETC-1922159 treated CRC. Individual recordings of these down regulations have been documented. I was able to rank 2nd order combination of TGFbfamily members with BUB1, that were down regulated. 6 RANKING CDC FAMILY VS BUB1 RANKING OF CDC FAMILY W.R.TBUB1 laplace linear rbf CDC45 - BUB1 12 2527 228 CDC25C - BUB1 53 303 188 CDC20 - BUB1 253 2321 362 CDC7 - BUB1 305 34 853 CDC6 - BUB1 647 776 444 CDC25A - BUB1 853 447 2216 CDC23 - BUB1 1672 1018 1775 CDC123 - BUB1 2292 2633 1366 Table 5: 2nd order interaction ranking between BUB1 VS CDC family members. UNEXPLORED COMBINATORIAL HYPOTHESES CDC family w.r.t BUB1 CDC-45/25C/20/7/6/25A BUB1 Table 6: 2nd order combinatorial hypotheses between BUB1 and CDC family members. Table 7 shows rankings of these combinations. Followed by this is the unexplored140 combinatorial hypotheses in table 8 generated from analysis of the ranks in table 7. The table 7 shows rankings of TGFbfamily w.r.t BUB1. TGFBR3 - BUB1 shows low ranking of 1066 (laplace), 890 (linear) and 1525 (rbf). TGFB1 - BUB1 shows low ranking of 1350 (linear) and 1000 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment.145 TGFBRAP1 did not show synergistic down regulation with BUB1. One can also interpret the results of the table 7 graphically, with the following influences - •TGFbfamily w.r.t BUB1 with BUB1 >TGFb-R3/1. 7 RANKING TGFbFAMILY VS BUB1 RANKING OF TGFbFAMILY W.R.TBUB1 laplace linear rbf TGFBR3 - BUB1 1066 890 1525 TGFBRAP1 - BUB1 2332 1239 1867 TGFB1 - BUB1 2549 1350 1000 Table 7: 2nd order interaction ranking between BUB1 VS TGFbfamily members. UNEXPLORED COMBINATORIAL HYPOTHESES TGFbfamily w.r.t BUB1 TGFB-R3/1 BUB1 Table 8: 2nd order combinatorial hypotheses between BUB1 and TGFbfamily members. 2.1.5. BUB1-TOP family Chromosome segregation in mitosis requires the removal of catenation between sis-150 ter chromatids. Timely decatenation of sister DNAs at mitotic centromeres by topoisomerase IIa (TOP2A) is crucial to maintain genomic stability. Zhang et al. [18] show that BUB1 generated modification, that is histone H2A Thr-120 phosphorylation (H2ApT120), is necessary and sufficient for the centromeric localization of TOP2A. Individual recordings of these down regulations have been documented. All the above155 experimental validations have been done and each of them were found to be down regulated in ETC-1922159 treated CRC. I was able to rank 2nd order combination of TOP family members with BUB1, 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 9.160 The table 9 shows rankings of TOP family w.r.t BUB1. TOP2A - BUB1 shows low ranking of 392 (laplace), 423 (linear) and 984 (rbf). TOP1MT - BUB1 shows low ranking of 884 (laplace) and 476 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. TOP2B and TOPBP1 did not show synergistic down regulation with BUB1.165 One can also interpret the results of the table 9 graphically, with the following influences - •TOP family w.r.t BUB1 with BUB1 >TOP-2A/1MT. 8 RANKING TOP FAMILY VS BUB1 RANKING OF TOP FAMILY W.R.TBUB1 laplace linear rbf TOP2A - BUB1 392 423 984 TOP1MT - BUB1 884 476 2088 TOP2B - BUB1 2261 2606 347 TOPBP1 - BUB1 2300 2288 346 Table 9: 2nd order interaction ranking between BUB1 VS TOP family members. UNEXPLORED COMBINATORIAL HYPOTHESES TOP family w.r.t BUB1 TOP-2A/1MT BUB1 Table 10: 2nd order combinatorial hypotheses between BUB1 and TOP family members. 2.1.6. BUB1-SGO family Salic et al. [19] demonstrate that the vertebrate SGO localizes to kinetochores and is required to prevent premature sister centromere separation in mitosis. Further, Tang et al.170 [20] show that human BUB1 protects centromeric sister-chromatid cohesion through SGO during mitosis. All the above experimental validations have been done and each of them were found to be down regulated in ETC-1922159 treated CRC. Individual recordings of these down regulations have been documented. I was able to rank 2nd order combination of SGO family members with BUB1, that were down regulated.175 Table 11 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in table 12 generated from analysis of the ranks in table 11. The table 11 shows rankings of SGO family w.r.t BUB1. SGOL1 - BUB1 shows low ranking of 336 (laplace) and 123 (linear). SGOL2 - BUB1 shows low ranking of 537 (laplace) and 115 (linear). SGOL1.AS1 - BUB1 shows low ranking of 637 (laplace), 4180 (linear) and 1494 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment. SGO2B and SGOBP1 did not show synergistic down regulation with BUB1. One can also interpret the results of the table 11 graphically, with the following influences - •SGO family w.r.t BUB1 with BUB1 >SGO-L1/L2/L1.AS1.185 9