A Facile Method to Append a Bio-ID Tag to Endogenous Mutant Kras Alleles.

KRAS mutations occur in approximately ~50% of colorectal cancers (CRCs) and are associated with poor prognosis and resistance to therapy. While these most common mutations found at amino acids G12, G13, Q61, and A146 have long been considered oncogenic drivers of CRC, emerging clinical data suggest that each mutation may possess different biological functions, resulting in varying consequences in oncogenesis. Currently, the mechanistic underpinnings associated with each allelic variation remain unclear. Elucidating the unique effectors of each KRAS mutant could both increase the understanding of KRAS biology and provide a basis for allele-specific therapeutic opportunities. Biotinylation identification (BioID) is a method to label and identify proteins located in proximity of a protein of interest. These proteins are captured through the strong interaction between the biotin label and streptavidin bead and subsequently identified by mass spectrometry. Here, we developed a protocol using CRISPR-mediated gene editing to generate endogenous BioID2-tagged KrasG12D and KrasG12V isogenic murine colon epithelial cell lines to identify unique protein proximity partners by BioID.

KRAS mutation-selective requirement for ACSS2 in colorectal adenoma formation.

Oncogenic KRAS mutations are prevalent in colorectal cancer (CRC) and are associated with poor prognosis and resistance to therapy. There is a substantial diversity of KRAS mutant alleles observed in CRC. Emerging clinical and experimental analysis of common KRAS mutations suggest that each mutation differently influences the clinical properties of a disease and response to therapy. Although there is some evidence to suggest biological differences between mutant KRAS alleles, these are yet to be fully elucidated. One approach to study allelic variation involves the use of isogenic cell lines that express different endogenous Kras mutants. Here, we generated Kras isogenic Apc-/- mouse colon epithelial cell lines using CRISPR-driven genome editing by altering the original G12D Kras allele to G12V, G12R, or G13D. We utilized these cell lines to perform transcriptomic and proteomic analysis to compare different signaling properties between these mutants. Both screens indicate significant differences in pathways relating to cholesterol and lipid regulation that we validated with targeted metabolomic measurements and isotope tracing. We found that these processes are upregulated in G12V lines through increased expression of nuclear SREBP1 and higher activation of mTORC1. G12V cells showed higher expression of ACSS2 and ACSS2 inhibition sensitized G12V cells to MEK inhibition. Finally, we found that ACSS2 plays a crucial role early in the development of G12V mutant tumors, in contrast to G12D mutant tumors. These observations highlight differences between KRAS mutant cell lines in their signaling properties. Further exploration of these pathways may prove to be valuable for understanding how specific KRAS mutants function, and identification of novel therapeutic opportunities in CRC.

Unique vulnerability of RAC1-mutant melanoma to combined inhibition of CDK9 and immune checkpoints.

RAC1P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S-harboring melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1P29S-mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.

Unique vulnerability of RAC1-mutant melanoma to combined inhibition of CDK9 and immune checkpoints.

RAC1P29S is the third most prevalent hotspot mutation in sun-exposed melanoma. RAC1 alterations in cancer are correlated with poor prognosis, resistance to standard chemotherapy, and insensitivity to targeted inhibitors. Although RAC1P29S mutations in melanoma and RAC1 alterations in several other cancers are increasingly evident, the RAC1-driven biological mechanisms contributing to tumorigenesis remain unclear. Lack of rigorous signaling analysis has prevented identification of alternative therapeutic targets for RAC1P29S-harboring melanomas. To investigate the RAC1P29S-driven effect on downstream molecular signaling pathways, we generated an inducible RAC1P29S expression melanocytic cell line and performed RNA-sequencing (RNA-seq) coupled with multiplexed kinase inhibitor beads and mass spectrometry (MIBs/MS) to establish enriched pathways from the genomic to proteomic level. Our proteogenomic analysis identified CDK9 as a potential new and specific target in RAC1P29S-mutant melanoma cells. In vitro, CDK9 inhibition impeded the proliferation of in RAC1P29S-mutant melanoma cells and increased surface expression of PD-L1 and MHC Class I proteins. In vivo, combining CDK9 inhibition with anti-PD-1 immune checkpoint blockade significantly inhibited tumor growth only in melanomas that expressed the RAC1P29S mutation. Collectively, these results establish CDK9 as a novel target in RAC1-driven melanoma that can further sensitize the tumor to anti-PD-1 immunotherapy.

RAC1 as a Therapeutic Target in Malignant Melanoma.

Small GTPases of the RAS and RHO families are related signaling proteins that, when activated by growth factors or by mutation, drive oncogenic processes. While activating mutations in KRAS, NRAS, and HRAS genes have long been recognized and occur in many types of cancer, similar mutations in RHO family genes, such as RAC1 and RHOA, have only recently been detected as the result of extensive cancer genome-sequencing efforts and are linked to a restricted set of malignancies. In this review, we focus on the role of RAC1 signaling in malignant melanoma, emphasizing recent advances that describe how this oncoprotein alters melanocyte proliferation and motility and how these findings might lead to new therapeutics in RAC1-mutant tumors.

Radiation injury to cardiac arteries and myocardium is reduced by soy isoflavones.

OBJECTIVE: The negative effects of incidental radiation on the heart and its vessels, particularly in the treatment of locally advanced non-small cell lung cancer, esophageal cancer, left-sided breast cancer, and lymphoma, are known. Late cardiac events induced by radiotherapy including coronary artery disease, ischemia, congestive heart failure, and myocardial infarction can manifest months to years after radiotherapy. We have previously demonstrated that soy isoflavones mitigate inflammatory responses induced in lungs by thoracic irradiation resulting in decreased vascular damage, inflammation, and fibrosis. In the current study, we investigate the use of soy isoflavones to protect cardiac vessels and myocardium from radiation injury. METHODS: Mice received a single dose of 10-Gy thoracic irradiation and daily oral treatment with soy isoflavones. At different time points, hearts were processed for histopathology studies to evaluate the effect of soy isoflavones on radiation-induced damage to cardiac vessels and myocardium. RESULTS: Radiation damage to arteries and myocardium was detected by 16 weeks after radiation. Soy isoflavones given in conjunction with thoracic irradiation were found to reduce damage to the artery walls and radiation-induced fibrosis in the myocardium. CONCLUSION: Our histopathological findings suggest a radioprotective role of soy isoflavones to prevent cardiac injury. This approach could translate to the use of soy isoflavones as a safe complement to thoracic radiotherapy with the goal of improving the overall survival in patients whose cancer has been successfully controlled by the radiotherapy but who otherwise succumb to heart toxicity.