Machine Learning Discoveries of RHNO1-X Synergy in ETC-1922159 Treated Colorectal Cancer Cells

1. Introduction

1.1. RHNO1

In response to DNA damage and replication stress, the DNA damage response (DDR) a protein kinase cascade is intiated. This initiation leads to cell cycle arrest. RHNO1, interacts with RAD9-HUS1-RAD1 (9-1-1) clamp and TOPBP1, which is then recruited to the sites of DNA damage to activate ATR signaling pathway (Cotta-Ramusino et al. [1]). RHNO1, previously known as chromosome 12 open reading frame 32 (C12orf32), was found to be overexpressed in breast cancer cells, as discovered by Kim et al. [2]. They showed that depletion of C12orf32 expression suppressed the growth of breast cancer cell lines. Lindsey-Boltz et al. [3] show that RHNO1 directly binds to TOPBP1 and forms a stable stoichiometric complex with 9-1-1, which further mediates ATR DNA damage checkpoint signaling in mammalian cells.

In hepatocellular carcinoma (HCC), Du et al. [4] found that RHNO1 protein levels were increased in most cells and knockdown of RHNO1 inhibited the proliferation and triggered cell mitochondrial apoptosis by inactivating the PI3K/AKT pathway. Recently, in their review, Jirapongwattana et al. [5] discuss the structure and role of RHNO1, in DNA replication stress, DNA repair, and cancer. In colorectal cancer (CRC) cells treated with ETC-1922159, RHNO1 was found to be down regulated along with other genes. Some combinations of RHNO1 have been confirmed in wet lab, however, many of the combinations have not been explored/tested or are known. To reveal these combinations, I use a modification of a recently published machine learning based search engine, details of which are given 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. 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 [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 2^nd order gene combinations.

2. Results & Discussion

2.1. RHNO1 Related Synergies

2.1.1. RHNO1 - TOP

From the references in introduction section, it is known that RHNO1 binds with TOPBP1, along with 9-1-1 clamp to activate ATR signaling pathway, in response to DNA damage and replication stress. A further advancement by Day et al. [9] has lead to the indentification of TOPBP1-BRCT-1/2 binding site in RHNO1. In colorectal cancer cells treated with ETC-1922159, TOP family members and RHNO1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2^nd order combination of these TOP members along with RHNO1.

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 TOP members w.r.t RHNO1. TOPBP1 - RHNO1 shows low ranking of 1201 (laplace), 136 (linear) and 357 (rbf). TOP1MT - RHNO1 shows low ranking of 325 (laplace), 1254 (linear) and 889 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment.

Further, TOPA2 and TOP2B showed high ranking with RHNO1, thus indicating that they might not be working synergistically with RHNO1, before the drug treatment.

One can also interpret the results of the Table 1 graphically, with the following influences - • TOP members w.r.t RHNO1 with RHNO1 $->$ TOP-BP1/1MT.

2.1.2. RHNO1 - NEIL

McDonald et al. [10] study the protein-protein interactions of NEIL1 glycosylase and the checkpoint protein 9-1-1 complex and discovered interactions between the two. Since RHNO1 also interacts with 9-1-1 complex, but it is not known that there is an interaction (direct/indirect) between RHNO1 and NEIL family, it would be great to see what the search engine points to regarding RHNO1 and NEIL family members. In colorectal cancer cells treated with ETC-1922159, NEIL family members and RHNO1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2^nd order combination of these NEIL members along with RHNO1.

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 NEIL members w.r.t RHNO1. NEIL1 - RHNO1 shows low ranking of 592 (laplace) and 851 (rbf). NEIL2 - RHNO1 shows low ranking of 1351 (laplace) and 68 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment.

Further, NEIL3 showed high ranking with RHNO1, thus indicating that they might not be working synergistically with RHNO1, before the drug treatment.

One can also interpret the results of the Table 3 graphically, with the following influences - • NEIL members w.r.t RHNO1 with RHNO1 $->$ NEIL-1/2.

2.1.3. RHNO1 - FEN1

Similar to the case of NEIL1 above, Shi et al. [11] observe in their previous study that in response to DNA damage, FEN1 interacts with the RAD9-HUS1-RAD1 complex instead of PCNA to engage in DNA repair activities, and undergoes SUMOylation by SUMO1. Further, FEN1 succinylation regulates DNA replication and repair, thus maintaining genome stability. Since FEN1 also interacts with 9-1-1 complex, but it is not known that there is an interaction (direct/indirect) between RHNO1 and FEN1, it would be great to see what the search engine points to regarding RHNO1 and FEN1. In colorectal cancer cells treated with ETC-1922159, FEN1 and RHNO1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2^nd order combination of FEN1 along with RHNO1.

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 Table 5 shows rankings of FEN1 members w.r.t RHNO1. FEN1 - RHNO1 shows low ranking of 212 (laplace) and 1354 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment.

One can also interpret the results of the Table 5 graphically, with the following influences - • FEN1 members w.r.t RHNO1 with RHNO1 $->$ FEN1.

2.1.4. RHNO1 - TP53

Bigot et al. [12] show that the DNA damage checkpoint regulating S-phase entry is controlled by a phosphorylation-dependent interaction of 53BP1 (or TP53BP1) and TOPBP1, through BRCT domains. Also, TOPBP1 interaction with 53BP1 is structurally complimentary to its interaction with RAD9-HUS1-RAD1. Since RHNO1 also interacts with 9-1-1 complex, but it is not known that there is an interaction (direct/indirect) between RHNO1 and TP53 family, it would be great to see what the search engine points to regarding RHNO1 and TP53 family members. In colorectal cancer cells treated with ETC-1922159, TP53 family members and RHNO1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2^nd order combination of these TP53 members along with RHNO1.

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 TP53 members w.r.t RHNO1. TP53BP1 - RHNO1 shows low ranking of 776 (laplace), 1507 (linear) and 229 (rbf). TP53 - RHNO1 shows low ranking of 1338 (laplace) and 1540 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment.

One can also interpret the results of the Table 7 graphically, with the following influences - • TP53 members w.r.t RHNO1 with RHNO1 $->$ TP-53/53BP1.

2.1.5. RHNO1 - ATR

Du et al. [4] indicate through references that RHNO1 was recognized as a DNA damage response (DDR) regulator through interaction with 9-1-1 clamp and TOPBP1, thus activating ATR and its downstream CHK1 pathway, to respond to replication stress. In colorectal cancer cells treated with ETC-1922159, ATR family members and RHNO1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2^nd order combination of these ATR members along with RHNO1.

The Table 9 shows rankings of ATR members w.r.t RHNO1. Interestingly, both ATRIP and ATR showed high ranking with RHNO1, thus indicating that they might not be working synergistically with RHNO1, before the drug treatment.

2.1.6. RHNO1 - CDC

Sorensen et al. [13] show that during the S phase, the regulation of the CHK1-CDC25A pathway depends on ATR, Claspin, RAD9, and HUS1. We also know from previously mentioned discussion that ATR is regulated by RHNO1. Thus it might be suspected that there exists a synergy between RHNO1 and CDC family members. In colorectal cancer cells treated with ETC-1922159, CDC family members and RHNO1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2^nd order combination of these CDC members along with RHNO1.

Table 10 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in Table 11 generated from analysis of the ranks in Table 10. The Table 10 shows rankings of CDC members w.r.t RHNO1. CDCA5 - RHNO1 shows low ranking of 100 (laplace) and 1106 (rbf). CDCA7 - RHNO1 shows low ranking of 119 (laplace) and 1431 (rbf). CDC45 - RHNO1 shows low ranking of 141 (laplace) and 47 (rbf). CDC25C - RHNO1 shows low ranking of 167 (laplace) and 1134 (rbf). CDCA7L - RHNO1 shows low ranking of 221 (laplace) and 275 (rbf). CDC25A - RHNO1 shows low ranking of 396 (laplace), 995 (linear) and 841 (rbf). CDC7 - RHNO1 shows low ranking of 426 (laplace), 1385 (linear) and 1152 (rbf). CDC6 - RHNO1 shows low ranking of 620 (laplace) and 1115 (linear). CDCA3 - RHNO1 shows low ranking of 679 (laplace) and 264 (rbf). CDCA2 - RHNO1 shows low ranking of 796 (laplace) and 671 (rbf). CDC123 - RHNO1 shows low ranking of 1097 (laplace) and 712 (rbf). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment.

Further, CDC20, CDC23, CDCA4 and CDCA8 showed high ranking with RHNO1, thus indicating that they might not be working synergistically with RHNO1, before the drug treatment.

One can also interpret the results of the Table 10 graphically, with the following influences - • CDC members w.r.t RHNO1 with RHNO1 $->$ CDC-A5/A7/45/25C/A7L/25A/7/6/A3/A2/123.

2.1.7. RHNO1 - FOX

In high-grade serous carcinoma, Barger et al. [14] demonstrate that FOXM1 and RHNO1 are head-to-head (i.e., bidirectional) genes regulated by a bidirectional promoter named F/R-BDP. Thus, there is a synergy between FOXM1 and RHNO1. It might be suspected that there exists a synergy between RHNO1 and other FOX family members. In colorectal cancer cells treated with ETC-1922159, FOX family members and RHNO1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2^nd order combination of these FOX members along with RHNO1.

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 FOX members w.r.t RHNO1. FOXM1 - RHNO1 shows low ranking of 377 (laplace) and 1521 (rbf). FOXD2-AS1 - RHNO1 shows low ranking of 1090 (laplace) and 156 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment.

Further, FOXA2 and FOXJ1 showed high ranking with RHNO1, thus indicating that they might not be working synergistically with RHNO1, before the drug treatment.

One can also interpret the results of the Table 12 graphically, with the following influences - • FOX members w.r.t RHNO1 with RHNO1 $->$ FOX-M1/D2-AS1.

2.1.8. RHNO1 - BMP

Xiong et al. [15] showed that lncRNA RHNO1/miR-6979-5p/BMP2 axis is a regulatory mechanism controlling osteoblast differentiation. In colorectal cancer cells treated with ETC-1922159, BMP family members and RHNO1, were found to be down regulated and their regulation was recorded independently. I was able to rank 2^nd order combination of these BMP members along with RHNO1.

Table 14 shows rankings of these combinations. Followed by this is the unexplored combinatorial hypotheses in Table 15 generated from analysis of the ranks in Table 14. The Table 14 shows rankings of BMP members w.r.t RHNO1. BMP7 - RHNO1 shows low ranking of 1179 (laplace) and 179 (linear). These rankings point to the synergy existing between the two components, which have been down regulated after the drug treatment.

One can also interpret the results of the Table 14 graphically, with the following influences - • BMP members w.r.t RHNO1 with RHNO1 $->$ BMP-7.

3. Conclusion

Presented here are a range of multiple synergistic RHNO1 2^nd order combinations that were ranked via a machine learning based search engine. Via majority voting across the ranking methods, it was possible to find plausible unexplored synergistic combinations of RHNO1-X that might be prevalent in CRC cells after treatment with ETC-1922159 drug.