EWS is a member of the FET family of RNA/DNA binding proteins that regulate crucial phases of nucleic acid metabolism. EWS comprises an N-terminal low-complexity domain (LCD) and a C-terminal RNA-binding domain (RBD). The RBD is further divided into three RG-rich regions, which flank an RNA-recognition motif (RRM) and a zinc finger (ZnF) domain. Recently, EWS was shown to regulate R-loops in Ewing sarcoma, a pediatric bone and soft-tissue cancer in which a chromosomal translocation fuses the N-terminal LCD of EWS to the C-terminal DNA binding domain of the transcription factor FLI1. Though EWS was shown to directly bind R-loops, the binding mechanism was not elucidated. In the current study, the RBD of EWS was divided into several constructs, which were subsequently assayed for binding to various nucleic acid structures expected to form at R-loops, including RNA stem-loops, DNA G-quadruplexes, and RNA:DNA hybrids. EWS interacted with all three nucleic acid structures with varying affinities and multiple domains contributed to binding each substrate. The RRM and RG2 region appear to bind nucleic acids promiscuously while the ZnF displayed more selectivity for single-stranded structures. With these results, the structural underpinnings of EWS recognition and binding of R-loops and other nucleic acid structures is better understood.

Mediator complex subunit 12 (MED12) is a subunit of Mediator, a large multi-subunit protein complex that acts an important regulator of transcription. Specifically, MED12 is an integral part of the kinase module of Mediator along with MED13, CyclinC (CycC) and CDK8. Structural studies have indicated that MED12 makes a direct connection to CycC through a specific interface and thereby functions to create a link between MED13 and CycC-CDK8. Disruption of the MED12-CycC interface often leads to dysregulated CDK8 kinase activity, which has important physiological implications. For example, a number of studies have indicated that mutations within MED12 can lead to the formation of benign or malignant tumors, either as a result of MED12-CycC disruption or through distinct independent mechanisms. Furthermore, recent studies have indicated that the N-terminal portion of MED12 forms a direct connection to CDK8. Mutations within MED12 do not appear to disrupt the physical connection to CDK8, but rather abrogate CDK8 kinase activity. Thus, mutations in MED12 can cause disruption of CDK8 kinase activity through two separate mechanisms. The aim of the present review article was to discuss the MED12 mutational landscape in a variety of benign and malignant tumors, as well as the mechanistic basis behind tumorigenesis. Furthermore, the link between MED12 and drug resistance has also been discussed, as well as potential cancer therapeutics related to MED12-altered tumors. Contents 1. Introduction 2. MED12 mutations and tumorigenesis 3. Therapeutics 4. Conclusions

Ewing sarcoma (EwS) is an aggressive pediatric cancer of bone and soft tissue. A chromosomal translocation that joins the low-complexity domain of EWS (EWSLCD) with the DNA-binding domain of FLI1 (FLI1DBD) creates EWS-FLI1, a fusion oncoprotein essential for EwS development and accounts for 85% of all EwS cases. EWS-FLI1 acts as an aberrant transcription factor and interferes with the normal functions of nucleic acid-binding proteins via multivalent interactions and biomolecular condensation. The FLI1DBD was found to directly interact with the EWSLCD causing enhanced phase separation and induced hardening of EWSLCD condensates. Three related ETS DBDs (ERG, ETV1 and PU.1) also induced EWSLCD condensate hardening. DNA binding blocked the interaction with the EWSLCD, and NMR spectroscopy confirmed that ETS DBDs interact with EWSLCD via the DNA-binding interface. Our results provide a physical basis for the dominant-negative effect EWS-FLI1 exerts on EWS and highlight the need for further investigations of the FLI1DBD-EWSLCD interaction in vivo.

There is a critical need for superior breast cancer treatment since this disease remains the second leading cause of cancer deaths among American women. Though radiation and chemotherapy have been the frontline choice of treatment for cancer over the past few decades, due to the detrimental side effects, personalized treatment is rapidly rising as a superior treatment method. This is largely due to recent advances in genome‐wide DNA sequencing that have allowed for the identification of cancer‐related mutational landscapes. Through these technologies, MED12, a subunit of Mediator protein, has been found to be commonly mutated in a significant number of breast cancer patients. Therefore, uncovering the mechanistic basis behind MED12 mutant breast cancer is critical in order to find a personalized treatment regimen for this subclass of breast cancer. Previous work has shown that MED12 restricts GLI3‐dependent Sonic Hedgehog (SHH) signaling, a pathway that is often hyper activated in advanced breast cancer. We therefore hypothesize that mutated MED12 promotes GLI3‐dependent SHH signaling in breast cancer cells thereby promoting tumorigenesis and that this signaling pathway could be an effective therapeutic target in MED12 mutant breast cancer. To study the effect of mutant MED12, a lentivirus carrying an shRNA against MED12 was generated and infected into MCF‐7 cells. Since the vast majority of breast cancer‐associated MED12 mutations lead to loss of protein function, the knockdown strategy through lentiviral shRNA is assumed to mimic the MED12 mutant setting. Proliferation assays were utilized to confirm that downregulated MED12 plays a role in the increased proliferation of breast cancer cells. Next, quantitative PCR was performed to determine the effect of downregulated MED12 on the expression of genes that are known to be regulated by GLI3. Finally, a screening strategy was employed by using a natural compound library to find potential novel treatment strategies for MED12 mutant breast cancer. Our findings confirmed that downregulation of MED12 increases proliferation of breast cancer cells in vitro thereby providing a strong argument that mutant MED12 would have the same effect. Through quantitative PCR it was confirmed that GLI3 target genes are upregulated in cancer cells when MED12 expression is low. Importantly, our natural compound screen identified several novel therapeutic compounds that specifically target MED12 downregulated breast cancer cells through a mechanism that involves SHH signaling. We therefore uncovered that MED12 mutations promote GLI3‐dependent SHH signaling in breast cancer and we identified potential novel therapeutic strategies for MED12 mutant breast cancer patients.

Current statistics show that breast cancer is the second leading cause of cancer deaths among American women. Therefore, there is a strong need to find improved treatment strategies for women suffering from this lethal disease. Though radiation and chemotherapy have been the frontline choice of treatment for cancer over the past few decades, due to the detrimental side effects, personalized treatment is rapidly rising as a superior treatment method. This is largely due to recent advances in genome‐wide DNA sequencing that have allowed for the identification of cancer‐related mutational landscapes. Through these studies, it has been found that up to 67% of breast cancer tumors carry a mutation in the Mediator subunit MED12 thus indicating that MED12 likely has a critical tumor suppressive role in breast cancer. Previous results from our lab, and others, have indicated that MED12 plays a critical role in restricting GLI3‐dependent SHH signaling. This finding is of particular interest to this study since hyper‐activated SHH signaling is known to play a major role in promoting breast cancer oncogenesis. Due to these findings, we hypothesize that mutations in MED12 cause hyper‐activated SHH signaling in breast cancer to promote oncogenesis and, furthermore, that natural compounds could provide useful as a personalized treatment for MED12 mutant breast cancer. To study the effect of mutant MED12, a lentivirus carrying an shRNA against MED12 was generated and infected into MCF‐7 cells. Since the vast majority of breast cancer‐associated MED12 mutations lead to loss of protein function, the knockdown strategy through lentiviral shRNA is assumed to mimic the MED12 mutant setting. Proliferation assays were utilized to confirm that downregulated MED12 plays a role in the increased proliferation of breast cancer cells. Next, quantitative PCR was performed to determine the effect of downregulated MED12 on the expression of genes that are known to be regulated by GLI3. Finally, a screening strategy was employed by using a natural compound library to find potential novel treatment strategies for MED12 mutant breast cancer. Our findings confirmed that downregulation of MED12 increases proliferation of breast cancer cells in vitro thereby providing a strong argument that mutant MED12 would have the same effect. Through quantitative PCR it was confirmed that GLI3 target genes are upregulated in cancer cells when MED12 expression is low. Importantly, our natural compound screen identified several novel therapeutic compounds that specifically target MED12 downregulated breast cancer cells through a mechanism that involves SHH signaling. We therefore uncovered that MED12 mutations promote GLI3‐dependent SHH signaling in breast cancer and, importantly, we identified potential novel therapeutic strategies for MED12 mutant breast cancer patients.