Revealing ETC-1922159 Affected Unknown 3 rd Order Combinations of DNA Repair & Genomic Stability Factor RAD 51 Family , in Silico †

DNA repair helps in maintaining the proper and healthy functioning for the cells in the human body. Failure in DNA repair process can lead to aberrations as well as tumorous stages. There are various types of damages that a DNA can go through, one of which is the DNA double strand breaks (DSB) that can be repaired via homologous recombination (HR). RAD51 plays a central role in HR and has been implicated as a negative/poor prognostic marker for colorectal adenocarcinoma, with high expression in colorectal cancer. Mechanistically, RAD51AP1 facilitates RAD51 during the repairing process by binding with RAD51 via two DNA binding sites, thus helping in the D-loop formation in the HR process. Often, in biology, we are faced with the problem of exploring relevant unknown biological hypotheses in the form of myriads of combination of factors that might be affecting the pathway under certain conditions. For example, RAD51AP1-XRCC2 is one such 2nd order combination whose relation needs to be tested under the influence recently developed Porcupine-WNT inhibitor ETC-1922159. The x-ray repair cross complementing XRCC family is known to work as a mediator or stabilizer for RAD51 during the HR process. The inhibitor is known to suppress Porcupine and thus inhibit a range of oncogenes known to be directly or indirectly affected by the Wnts. In a recent unpublished work in bioRxiv, we had the opportunity to rank these unknown biological hypotheses for down regulated genes at 2nd order level after the drug was administered. The in silico observations showed that the combination of RAD51AP1-XRCC2 was assigned a relatively lower rank, thus validating the pipeline’s efficacy with the confirmed wet lab experiment that indicate that both RAD51AP1 and XRCC2 were down regulated after treatment in cancer cells. Here, we take one step further by in silico analysis of the 3rd order combinations of RAD51-X-X & RAD51AP1-X-X (X can be known or unknown factor), from a range of 100 randomly picked down regulated genes after ETC-1922159 treatment. The pipeline uses the density based HSIC (Hibert Schmidth Information Criterion) sensitivity index with an rbf (radial basis function) kernel, which is known to be highly effective in sensitivity analysis. Various unknown/unexplored/untested RAD51/RAD51AP1 related 3rd order biological hypotheses emerge some of which are confirmed in wet lab, while others need to be tested.


DNA repair and genomic stability
DNA repair is an important aspect in maintaining the proper and healthy functioning for the cells in the human body.Failure in DNA repair process can lead to aberrations as well as tumorous stages.There are various types of damages that a DNA can go through, one of which is the DNA double strand breaks (DSB) that can be repaired via homologous recombination (HR).RAD51 plays a central role in HR and is known to function in the three phases of HR namely : presynapsis, synapsis and post-synapsis ? .Recently, RAD51 has been implicated as a negative/poor prognostic marker for colorectal adenocarcinoma and has been found to be highly expressed ? .A negative/poor prognostic marker indicates that it is harder to control the malignancy.Since RAD51 helps in the repair of the DNA damage via HR and is implicated as a poor prognostic marker in colorectal adenocarcinoma, this suggests its functionality in maintaining genomic stability and therapeutic resistance to cancer drugs ?& ? .Mechanistically, RAD51AP1 facilitates RAD51 during the repairing process by DNA binding via two DNA binding sites, thus helping in the D-loop formation in the HR process ?& ? .

RAD51
RAD51, an ortholog of Esherichia coli RECA ? is found to be operating in mammals and work in three steps during the homologous recombination process.During the presynaptic phase there is a formation of the RAD51-ssDNA filament as RAD51 is loaded to form a stretch of the filment with single stranded DNA (ss-DNA).This ssDNA is generated from degradation and deletion of unwound double strand of DSBs.During the synaptic phase there is a formation of the D-loop were RAD51 facilitates the intruding DNA to form a connection with the homologous duplex DNA.This leads to formation of RAD51-dsDNA (ds -double strand) from the intruding and the donar ssDNA.Finally, DNA synthesis takes place via DNA polymerases and an invading primer, during the postsynaptic phase when the RAD51 disassociates from the RAD51-dsDNA filament to leave the dsDNA homologous pair for further processing.A good survey of this process has been described in ? .The formation of the RAD51 nucleoprotein filaments has been described in detail in ?& ? .

RAD51AP1
RAD51AP1 or RAD51 associated protein 1, enhances the activity of RAD51, by two DNA binding sites during the D-loop formation ? .Mutant studies ?show that both C-terminal and Nterminal binding sites are indispensable for RAD51AP1 to function along with RAD51.More specifically, RAD51AP1 associates with DMC1 mediated D-loop formation and ?provide evidence that RAD51AP1 cooperates with the DMC1 presynaptic filament to capture duplex DNA and to assemble the synaptic complex, in which the recombining DNA molecules are joined paranemically.Thus, RAD51AP1 maintain genomic stability via RAD51 recombinase enhancement ? .

PORCN-WNT inhibitors
The regulation of the Wnt pathway is dependent on the production and secretion of the WNT proteins.Thus, the inhibition of a causal factor like PORCN which contributes to the WNT secretion has been proposed to be a way to interfere with the Wnt cascade, which might result in the growth of tumor.Several groups have been engaged in such studies and known PORCN-WNT inhibitors that have been made available till now are IWP-L6 ?& ?, C59 ?, LGK974 ? and ETC-1922159 ? .In this study, the focus of the attention is on the implications of the ETC-1922159, after the drug has been administered.The drug is a enantiomer with a nanomolar activity and excellent bioavailability as claimed in ? .

Combinatorial search problem and a possible solution
We have already addressed the issue of combinatorial search problem and a possible solution in ?and ? .The details of the methodology of this manuscript have been explained in great detail in ?& its application in ?and readers are requested to go through the same for gaining deeper insight into the working of the pipeline and its use for published data set generated from ETC-1922159.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.Using the same code with minor modifications in ?and ?, it was possible to generate the rankings for 3 rd order combinations.100 genes were randomly selected from the list of down regulated genes, by the pipeline and a 3 rd order combination was generated from those 100 genes.The total number of gene combination with C 100 3 = 161700.Out of these the RAD51 and RAD51AP1 associated 3 rd order combinations were selected, which account to a total of 4851 combinations.The goal of this manuscript is to analyse some of these 3 rd order ranked RAD51 and RAD51AP1 associations.

Results and Discussion
We present here the 3rd order combinations associated with RAD51/RAD51AP1 and represent them as RAD51/RAD51AP1-X-X, were X can be known or unknown factor from a list of genes that were affected after the administration of the ETC-1922159 drug.There are a total of 4851 combinations of randomly selected 100 genes from a list of 2500± genes.Out of these 100, RAD51/RAD51AP1 was one of them.Here we analyse some of the ranked combinations out of 4851 3 rd order interactions for RAD51-X-X and RAD51AP1-X-X.Note that the rankings were generated using only the HSIC density index using the radial basis function kernel.Also, the rankings for a particular gene might change over different combinations and the biologists/oncologists are advised to cross check across the different tables presented.However, where possible, we report confirmatory results by the pipeline that fall in line with the published and known mechanism of a particular gene under consideration.Also, many of the combinations are yet to be tested and we make opennings for the deeper analysis and exploration of the combinations as future work.These combinations with their rankings have been recored in table 1 and 2.

RAD51-X-X, X -known/unknown/untested factor
In table 1, we present the rankings of 3 rd order combination at particular intervals, for RAD51.

RAD51AP1-X-X, X -known/unknown/untested factor
In table 2, we present the rankings of 3 rd order combination at particular intervals, for RAD51AP1.

Conclusion
RAD51 and the complementing paralogue RAD51AP1, work in tandem to maintain genomic stability and play a greater role during homologous recombination process when double strand breaks are encountered.Here we present a range of ranking for both RAD51 and RAD51AP1 associated 3rd order combinations after the treatment of ETC-1922159 in colorectal cancer cells.Rankings guide oncologists/biologists to study the influence of these genomic stability and DNA repair factors in context of other factors in colorectal cancer case and the in silico pointers of prioritization facilitate in the emergence of some unknown/untested/unexplored biological hypotheses.