Machine learning discoveries of MYC-HOXB8-X gene combinations in ETC-1922159 treated colorectal can- cer cells

Machine learning discoveries of MYC-HOXB8-X gene combinations in ETC-1922159 treated colorectal cancer cells † Shriprakash Sinha ☯,? The three families of (c,L,N)-MYC are known to encode proteins that play significant role as transcription factors in cancer and target various kinds of genes, thus contributing to regrowth and proliferation. Homeobox protein Hox-B8 (HOXB8), is a protein that is implicated in development and known to be highly expressed in colorectal cancer. Treatment of colorectal cancer cells with Porcupine-Wnt inhibitor ETC-1922159 showed down regulation of HOXB8 and MYC, independently. Recently it has been confirmed experimentally that there exists a synergy between MYC-HOXB8. 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. Using a machine learning based search engine, I had the opportunity to rank these unknown biological hypotheses for down regulated genes at 3rd order level after the drug was administered. Here, an in silico analysis of the 3rd order combinations of MYC-HOXB8-X (X can be known or unknown factor), from a range of 100 randomly picked down regulated genes after ETC-1922159 treatment, have been presented. Various unknown/unexplored/untested MYC-HOXB8 related 3rd order biological hypotheses emerge, some of which have been confirmed in wet lab, while others need to be tested. 1 Significance c-MYC and HOXB8 are known to be expressed in colorectal cancer case. Separately, in acute leukaemia, the mechanism by which the combinatorial regulation of c-MYC and HOXB8 happens is not determined. Often, we are faced with the problem of exploring relevant unknown biological hypotheses in the form of myriads of combination of factors that might be involved in the pathway. The current work reveals at in silico level, the 3rd order c-MYCHOXB8 associated combinations that might be affected by the administration of Porcupine-Wnt inhibitor ETC-1922159 in colorectal cancer. In silico, the current work advances our knowledge of c-MYC-HOXB8-X combinations to facilitate the understanding of Wnt pathway and entail recommended wet lab tests. †Aspects of unpublished work were presented in a poster session at the first Wnt Gordon Research Conference, from 6-11 August 2017, held in Stowe, VT 05672, USA. ?Corresponding Authors : [email protected] ☯Worked as independent researcher ‡104 Madhurisha Heights Phase 1, Risali, Bhilai - 490006, India 2 Introduction 2.1 Combinatorial search problem and a possible solution A search engine design has recently been developed in Sinha1, which showed promise by revealing existing confirmatory published wet lab results. Additionally and of import, adaptation of the engine mined up a range of unexplored/untested/unknown combinations of genetic factors in the Wnt pathway that were affected by ETC-1922159 enantiomer, a PORCN-WNT inhibitor, after the colorectal cancer cells were treated with the inhibitor drug. It was possible to generate the rankings for 3rd order combinations. 100 genes were randomly selected from the list of down regulated genes, by the pipeline and a 3rd order combination was generated from those 100 genes. The total number of gene combination with c100 3=161700. Out of these the MYC-HOXB8 associated 3rd order combinations were selected, which account to a total of 4851 combinations. The goal of this manuscript is to analyse these 3rd order ranked MYC-HOXB8 associations. 1 2.2 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 Chen et al.2&Wang et al.3, C59 Proffitt et al.4, LGK974 Liu et al.5 and ETC-1922159 Duraiswamy et al.6. 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 Duraiswamy et al.6. 2.3 MYC c-MYC (MYC) is a gene that encodes for transcription factor. It belongs to the family of MYC genes which includes B-MYC, N-MYC, L-MYC and S-MYC. Mutations in c-MYC have been found to be involved in various kinds of cancers. MYC contains a helix-loophelix (HLH) and leucine zipper (LZ) domain that helps it to bind to interact with DNA and form a complex with MAX Nair and Burley7, respectively. A good introductory review on c-MYC can be found in Dang8which covers topics on transcription factors and binding sites, transcriptional properties, MYC affected genes and finally role of MYC in cell cycle, apoptosis Hoffman and Liebermann9and metabolism Miller et al.10. A lot of work has been done in MYC at various levels as it is implicated in various types of cancer. Additionally, MYC has been found to have influence on the chromatin structure also Knoepfler et al.11. In colon cancer, c-MYC has been found to be highly expressed Sikora et al.12 & Smith et al.13. 2.4 Homeobox protein B8 or HOXB8 Homeobox protein B8 or HOXB8 belongs to the HOX family and is implicated in development Fujimura et al.14. Homeobox proteins are known to play major role in embryogenesis, however, their transition and role in oncogenesis is not clearly established, as there are cases where there is loss of function, while in others, there is gain of function Abate-Shen15. The HOX family Acampora et al.16 is known to play multiple roles in various tumor cases and varied affects have been found in colorectal tumor and normal cases Kanai et al.17. HOXB8 and HOXB9 are highly expressed in colorectal cancer cases Vider et al.18 & Shen et al.19. 2.5 MYC-HOXB8 interaction It has been observed that in presence of expressed HOXB8, there is enhancement in the expresson of MYC in acute myeloid leukaemia Salmanidis et al.20. However, the mechanism by which the combinatorial regulation of HOXB8 and MYC happens in leukaemia, is yet to be determined. In human embryonic stem cells, MYC is found to bind with MIZ1 via recruitment of DNMT3A (DNA (cytosine-5)-methyltransferase 3A) and HDAC (histone deacetylases) to silence HOXB8 Varlakhanova et al.21. Reversibly, decrease in levels of MYC have lead to increase in HOXB8 expression. Separately, MYC-HOXB8 interaction in colorectal cancer has yet to be investigated and is a subject of on going research. As observed previously, MYC and HOXB8 have been found to be up regulated in colorectal cancer cases and after the administration of ETC-1922159 in specific subtype of colorectal cancer Madan et al.22, both were found to be down regulated. Recently, in 2020, Ying et al.23 showed that oncogenic HOXB8 is driven by MYC-regulated super-enhancer and potentiates colorectal cancer invasiveness via BACH1. This wet lab experiment confirms our search engine’s indication of the synergy of combination of MYC-HOXB8 in colorectal cancer. Using the in silico analysis, we generated and prioritized 3rd order combinations of MYCHOXB8-X in these treated colorectal cancer cells Madan et al.22, to reveal unknown combinatorial hypotheses regarding probable synergistic down regulation of factors in the Wnt pathway. These emerging unknown hypotheses facilitate oncologists/biologists to navigate a vast combinatorial forest of search space. 3 Results and Discussion We present here the 3rd order combinations associated with MYC and represent them as MYC-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, MYC was one of them. Here we analyse some of the ranked combinations out of 4851 3rd order interactions for MYC-HOXB8-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. 3.1 MYC-HOXB8-X, X - known/unknown/untested factor DHX9 is a RNA helicase that belongs to the family of DExD/H-box family and is found to be involved in the transcriptional regulation in cancer Lin et al.24 & Mi et al.25. DHX9 has been found to be up regulated in colorectal cancer cases and after the treatment of the ETC-1922159, it was down regulated. The combination of HOXB8-DHX9-MYC acquires lower priority of 4, indicating a possible potential synergistic affect (within the selected 100 genes). METTL12 (methyltransferase-like protein 12)Malecki et al.26 was found to be down regulated and the pipeline assigned a low rank of 19 along with MYC-HOXB8. This is in accordance with the down regulation by ETC-1922159 Madan et al.22. However, how METTL12 plays a role in colorectal cancer needs to be investigated and research is ongoing. METTL8 (methyltransferase-like protein 8) was also found to be down regulated after the treatment of ETC-1922159. It has been found that the frameshift mutations have occurred in METTL8 in CRC and might be a cause of tumorigenesis through their inactivation in CRC Yeon et al.27. It is not known if this is the case with the samples used in experiments of Madan et al.22. However, this does point to the fact that if the frameshift mutations played a role in CRC samples of Madan et al.22, then ETC-1922159 treatment would have suppressed frameshift mutated METTL8. The pipeline assigns this down regulation with a low rank of 19. PABPC1L (as recorded by Madan et al.22) or the cytoplasmic 1-like poly(A)-binding protein Gray et al.28, belong to a family of multifunctional proteins PABPs which regulate and stabilise the mRNA translation. They are observed to be helpful in the transportation of the mRNA also from the nucleus and there exists a nucleic version of PABP also Kini et al.29. PABPC1 contains four non identical RRMs that are joined with the main PABC domain and separated by a linker. Though their mechanism of import and export of mRNA has not been understood deeply, a few models/observations have been made. Association with translation complexes or mRNA at cytoplasmic level inhibits the transportation of PABPC1 to the nucleus. Contrary to this, its release leads to binding with Importin-α/β complex that facilitates in nuclear import. There are various mechanisms by which PABPC1L might be exported out of the nucleus. Modes of export involve association with (1) cytoplasmic eEF1α(2) mRNA as well as TAP that mediates the mRNA export and (3) Paxillin via CRM1 pathway. In gastric cancer cases, PABPC1 has been found to be an oncoprotein Zhu et al.30 and observed to exert carcinogenesis. In colon cancer mutations in PABPC1 have been found in minor tumor clones Yu et al.31. Genomic correlates of immune-cell infiltrates in CRC have found the existence of significantly mutated CRC genes Giannakis et al.32. PABPC1L was found to be down regulated after the treatment of ETC-1922159 and a low ranking (of 1167) is associated with MYC-HOXB8. In CRC, PABPC1L might be mutated and work as onco-protein and facilitate the transmission of other oncogenic factors. The administration of the drug down regulated the gene in drug treated CRC cells and the low ranking confirms at in silico level the suppression at cytoplasmic level. Hydroxyacyl-Coenzyme A dehydrogenase is encoded by gene HADH and mutations in the same have been found to cause hyperinsulinemic hypoglycemia Molven et al.33. Deficiency in HADH can lead to a rare condition where body stops converting fat into energy. The authors are not aware of HADH in colorectal cancer and the pipeline indicated a low rank of 1555 along with MYC-HOXB8. After the administration of the drug, HADH was found to be down regulated in the treated colorectal cancer cells with ETC-1922159. Reduced HUGL1, a homologue of LGL tumor suppressor, is found to contribute to progression of colorectal cancer Schimanski et al.34. LGL has been found to arrest G1 cell cycle via formation of a complex involving LGL-VPRBP-DDB1 Yamashita et al.35 & Huang and Chen36. Yamashita et al.35 show that depletion of depletion of VPRBP leads to rescue of over proliferation of LGLdepleted cells. Mutations in VPRBP might lead to cell proliferation in colorectal cancer cases and ETC-1922159 administration show down regulation of VPRBP. Reversibly, down regulation of wild type VPRBP leads to phase progression. Our pipeline shows a down regulation with a high rank of 2997 for the mutated version, however, across different tables, the majority voting points to higher rank. This high rank indicates the wild type VPRBP to be working reversibly and thus point to inhibition of progression of cell proliferation. Alternatively, higher ranks also mean that these combinations might not be important in one specific condition while it might be in another. Further tests are needed for VPRBP. FZD7 has been found to be highly expressed in colorectal cancer cells Ueno et al.37 & Vincan et al.38. After the ETC-1922159 treatment, FZD7 was found to be highly suppressed. However, the combination of FZD7-MYC-HOXB8 showed and extremely high rank of 3571, indicating that either the combination of not much importance or FZD7 increases MYC levels Ueno et al.39 which then decreases HOXB8 levels (similar to Varlakhanova et al.21)(if this model works). Reversibly, suppression of FZD7 by ETC-1922159 might lead to suppression of MYC, however, this should increase the levels of HOXB8 (according to the model in Varlakhanova et al.21), which is not the case after ETC-1922159 treatment. Thus this combination might not be of interest or there is an intricate mechanism between MYC-HOXB8 that is yet to be explored. Unknown factors XX133 (rank 143), XX229 (rank 362), XX170 (rank 481), XX210 (rank 923), XX115 (rank 1018), XX216 (rank 1160), XX157 (rank 2094), XX92 (rank 2147) were found to be assigned low ranks by the pipeline and this is in accordance with the down regulation of the same due to ETC-1922159 Madan et al.22. Probable insight might be that these unknown factors have similar priority fo up regulation in colorectal cancer and vice versa after the drug treatment. Biologists might want to investigate these unknown factors and explore their influence apropos MYC-HOXB8. On the other hand, XX13 (rank 2458), XX63 (rank 2459), XX46 (rank 2774), XX198 (rank 3818) and XX144 (rank 4015) were found to be assigned a low rank along with MYC-HOXB8 by the pipeline. These rankings indicate possible non significance after the drug treatment or, there is a reverse mechanism between the components (MYC-HOXB8-XX) that is not known. These unknown factors require investigation in wet lab. Zinc finger is motif that helps in the stabilisation of the folds via the zinc ions. There are numerous variants of the zinc finger motifs. ZNF215 was recorded to be down regulated after the colorectal cancer cells were treated with ETC-1922159 and not much has been studied regarding ZNF215 in colorectal cancer. The pipeline assigns a low rank of 17 along with MYC-HOXB8 out of the 4851 3rd order interactions with MYC-HOXB8, using 99 randomly chosen down regulated genes. Similar interpretations for ZNF780B (with low rank of 562) and ZNF614 (with low rank of 635) can be derived as the pipeline assigned low ranks for down regulation. Wet lab study regarding these zinc finger variants needs to done as they emerge as untested 3rd order combination hypotheses. EZH2 encodes enhancer of zeste homolog 2 and is involved in transcriptional repression via epigenetic modifications. It has been found to be either mutated or over-expressed in many forms of cancer. Over expression of EZH2 leads to silencing of various tumor suppressor genes and thus implicating it for potential roles in tumorigenesis Simon and Lange40. EZH2 is a subunit of the highly conserved Polycomb repressive complex 2 (PRC2) which executes the methylation of the histone H3 at lysine-27 O⢠A´ ZMeara and Simon41. Thus targeting EZH2 has become a major research domain for cancer therapeutics Kim and Roberts42. In colon cancer, it has been shown that depletion of EZH2 has led to blocking of proliferation of the cancer Fussbroich et al.43. This indicates the fact that tumor suppressor genes get activated and lead to subsequent blocking of the cancer. Also, EZH2 is recruited by PAF to bind with β-catenin transcriptional complex for further Wnt target gene activation, independent of the EZH2 epigenetic modification activities Jung et al.44. Consistent with these, ETC-15922159 treatment lead to down regulation of EZH2 in colorectal cancer samples Madan et al.22. This would have activated a lot of tumor suppressor genes that led to subsequent suppression of regrowth in treated cancer samples. More importantly, MYC directly upregulates core components of PRC2, EZH2 being one of them, in embryonic stem cells Neri et al.45. Neri et al.45 show that silencing of c-MYC and N-MYC Dardenne et al.46 lead to reduction in the expression of PRC2 and thus EZH2. Furthermore, in colorectal cancer cases, Satoh et al.47 show that knockdown of MYC led to decrease in EZH2 levels. Similar findings have been observed in Yamaguchi and Hung48, Chen et al.49 & Fluge et al.50. Our in silico findings show consistent results with respect to this down regulation after assigning a low rank of 54 along with MYC-HOXB8. More specifically, our in silico pipeline is able to approximate the value of the 3rd order combination of MYC-HOXB8-EZH2 by assigning a rank that is consistent with wet lab findings of dual combinatorial behaviour of MYC-EZH2 and MYC-HOXB8. However, the since the mechanism of combination of MYC-HOXB8 is not known hitherto, it would be interesting to confirm the behaviour of MYC-HOXB8-EZH2 at 3rd order to reveal a portion of the Wnt pathway’s modus operandi in colorectal cancer. Further wet lab tests on these in silico findings will confirm the efficacy of the search engine in Sinha51, Sinha52. Not much is known about OSGEPL1 or the O-sialoglycoprotein endopeptidase-like 1 in colorectal cancer case. The experiments of ETC-1922159 Madan et al.22 show a down regulation of OSGEPL1 and the in silico pipeline assigns this down regulation with a rank of 75. Similarly, LYRM7 was found to be down regulated after the drug treatment and not much is known about the role of LYRM7 in colorectal cancer. The pipeline assigned a ranking of 131 along with MYC-HOXB8 combination. Chromosome 9 open reading frame 152 (C9orf152) has not been studied much, however, it has been found to be up regulated in intrinsic subtypes of gastric cancer Tan et al.53. Experiments using ETC-1922159 showed down regulation of C9orf152. The pipeline shows an indication of down regulation with a low rank of 128. Similar interpretations for other open reading frames like C4orf27 (with rank of 148), C1orf53 (with rank of 247) and C3orf33 (with rank of 1643) can be made regarding their down regulation after ETC-1922159 treatment and the rank assignment by the pipeline. Mitochondrial 39S ribosomal protein L18, is a ribosomal mitochondrial protein that is encoded by MRPL18, that works within the mitochondria for protein synthesis Koc et al.54. Sasso et al.55 show that MRPL18 is differentially expressed in Ileum tumors from APCmn/+mice crossed with iVP16LXRαand iVP16 transgenic mice. Not much has been studied in colorectal cancer case regarding MRPL18. Experiments in Madan et al.22 show MRPL18 is down regulated after ETC-15922159 and the in silico predictions point to this with a low rank 249. Similar interpretations can be made for ranks of other factors with MYC-HOXB8-X (X is variable factor) axis till 1167 in the table 1. 4 Conclusion Homeobox proteins are known to play major role in embryogenesis, however, their transition and role in oncogenesis is not clearly established, as there are cases where there is loss of function, while in others, there is gain of function. The three families of (c,L,N)-MYC are known to encode proteins that play significant role as transcription factors in cancer and target various kinds of genes, thus contributing to regrowth and proliferation. In colorectal cancer, MYC and HOXB8 are known to be up regulated, however, the dual combinatorial regulation mechanism of RANKING USING HSIC - RBF KERNEL W.R.TMYC-HOXB8-X 3nd odr comb. rbf rank 3nd odr comb. rbf rank 3ndodrcomb. rbf rank HOXB8-DHX9-MYC 4 HOXB8-ZNF215-MYC 17 HOXB8-METTL8-MYC 19 HOXB8-EZH2-MYC 54 HOXB8-OSGEPL1-MYC 75 HOXB8-C9orf152-MYC 128 HOXB8-LYRM7-MYC 131 HOXB8-XX133-MYC 143 HOXB8-MYC-C4orf27 148 HOXB8-C1orf53-MYC 247 HOXB8-MRPL18-MYC 249 HOXB8-MYC-PAAF1 305 HOXB8-XX229-MYC 362 HOXB8-METTL12-MYC 408 HOXB8-PHF14-MYC 463 HOXB8-MYC-CENPF 472 HOXB8-XX170-MYC 481 HOXB8-SIRT3-MYC 491 HOXB8-TAF9-MYC 506 HOXB8-KBTBD7-MYC 556 HOXB8-ZNF780B-MYC 562 HOXB8-TESC-MYC 575 HOXB8-TNNC2-MYC 587 BANF1-HOXB8-MYC 599 HOXB8-PARS2-MYC 611 HOXB8-ZNF614-MYC 635 ALG8-HOXB8-MYC 651 HOXB8-SHF-MYC 661 HOXB8-E2F2-MYC 675 HOXB8-RPL18A-MYC 742 HOXB8-LRRC2-MYC 768 HOXB8-SLC43A3-MYC 798 HOXB8-AGMAT-MYC 850 HOXB8-ENOX2-MYC 859 HOXB8-FAM81A-MYC 916 HOXB8-XX210-MYC 923 HOXB8-VMA21-MYC 999 HOXB8-XX115-MYC 1018 HOXB8-PDHA1-MYC 1023 HOXB8-HSD17B8-MYC 1058 HOXB8-XX216-MYC 1160 HOXB8-PABPC1L-MYC 1167 HOXB8-GCAT-MYC 1267 HOXB8-TANGO6-MYC 1278 HOXB8-ZBTB2-MYC 1283 HOXB8-ESF1-MYC 1289 HOXB8-GTPBP3-MYC 1295 HOXB8-ANKS6-MYC 1304 HOXB8-CENPM-MYC 1343 HOXB8-ALDH7A1-MYC 1345 HOXB8-THAP11-MYC 1408 HOXB8-ATOH1-MYC 1425 HOXB8-SLC6A6-MYC 1456 HOXB8-PGAP2-MYC 1490 HOXB8-HADH-MYC 1555 HOXB8-ANKRD32-MYC 1615 HOXB8-DNMT3A-MYC 1625 HOXB8-C3orf33-MYC 1643 HOXB8-ACTL8-MYC 1803 HOXB8-KIAA1586-MYC 1809 HOXB8-CHAF1B-MYC 1833 HOXB8-MYC-NDUFS3 1875 HOXB8-UQCRH-MYC 1909 HOXB8-GDF11-MYC 1922 HOXB8-XX157-MYC 2094 HOXB8-TUFM-MYC 2117 HOXB8-XX92-MYC 2147 HOXB8-GPX1P1-MYC 2157 HOXB8-SNRPA1-MYC 2194 HOXB8-PRPF19-MYC 2221 HOXB8-STMN1-MYC 2274 HOXB8-BIRC5-MYC 2309 HOXB8-ATP6V0E2-MYC 2434 HOXB8-SLC25A26-MYC 2437 HOXB8-XX13-MYC 2458 HOXB8-XX63-MYC 2459 CCDC84-HOXB8-MYC 2630 HOXB8-NDUFAF4-MYC 2679 HOXB8-PAXBP1-MYC 2748 HOXB8-XX46-MYC 2774 HOXB8-PBK-MYC 2904 HOXB8-TFAM-MYC 2922 HOXB8-PRIM1-MYC 2981 HOXB8-ACN9-MYC 2992 HOXB8-MYC-VPRBP 2997 HOXB8-CCDC138-MYC 3090 KIF18A-HOXB8-MYC 3189 HOXB8-SCAMP5-MYC 3208 HOXB8-PTTG1-MYC 3437 HOXB8-FZD7-MYC 3571 HOXB8-ADCY3-MYC 3640 HOXB8-HPRT1-MYC 3807 HOXB8-XX198-MYC 3818 HOXB8-GLA-MYC 3835 HOXB8-XX144-MYC 4015 HOXB8-ATP5I-MYC 4127 HOXB8-MYC-UMPS 4252 HOXB8-CBY1-MYC 4564 Table 1 3rd order interaction ranking using HSIC for radial basis function kernel. Total number of 3rd order interactions in a set of 100 genes - 161700.4851 3rd order combinations for associated work. Rankings for MYC-HOXB8-X have been tabulated. MYC and HOXB8, has only recently been discovered experimentaly. More specifically, our in silico pipeline is able to approximate the value of the 3rd order combinations like that of MYC-HOXB8EZH2 by assigning a rank that is consistent with wet lab findings down regulation of dual combinatorial behaviour of MYC-EZH2 and MYC-HOXB8 after the ETC-1922159 treatment. It would be interesting to confirm the functional behaviour of MYC-HOXB8EZH2 at 3rd order to reveal a portion of the Wnt pathway’s modus operandi in colorectal cancer. Further wet lab tests on these in silico findings will confirm the efficacy of the search engine. 5 Conflict of interest There are no conflicts to declare. 6 Acknowledgements Special thanks to Mrs. Rita Sinha and Mr. Prabhat Sinha for supporting the author financially, without which this work could not have been made possible. 7 Source of Data Data used in this research work was released in a publication in Madan et al.22. References 1 S. Sinha, Integrative Biology, 2024. 2 B. Chen, M. E. Dodge, W. Tang, J. Lu, Z. Ma, C.-W. Fan, S. Wei, W. Hao, J. Kilgore, N. S. Williams et al.,Nature chemical biology, 2009, 5, 100–107. 3 X. Wang, J. Moon, M. E. Dodge, X. Pan, L. Zhang, J. M. Hanson, R. Tuladhar, Z. Ma, H. Shi, N. S. Williams et al.,Journal of medicinal chemistry, 2013, 56, 2700–2704. 4 K. D. Proffitt, B. Madan, Z. Ke, V. Pendharkar, L. Ding, M. A. Lee, R. N. Hannoush and D. M. Virshup, Cancer research, 2013, 73, 502–507. 5 J. Liu, S. Pan, M. H. Hsieh, N. Ng, F. Sun, T. Wang, S. 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