Recent Developments in Targeting RAS Downstream Effectors for RAS-Driven Cancer Therapy

Aberrant activity of oncogenic rat sarcoma virus (RAS) protein promotes tumor growth and progression. RAS-driven cancers comprise more than 30% of all human cancers and are refractory to frontline treatment strategies. Since direct targeting of RAS has proven challenging, efforts have been centered on the exploration of inhibitors for RAS downstream effector kinases. Two major RAS downstream signaling pathways, including the Raf/MEK/Erk cascade and the phosphatidylinositol-3-kinase (PI3K) pathway, have become compelling targets for RAS-driven cancer therapy. However, the main drawback in the blockade of a single RAS effector is the multiple levels of crosstalk and compensatory mechanisms between these two pathways that contribute to drug resistance against monotherapies. A growing body of evidence reveals that the sequential or synergistic inhibition of multiple RAS effectors is a more convenient route for the efficacy of cancer therapy. Herein, we revisit the recent developments and discuss the most promising modalities targeting canonical RAS downstream effectors for the treatment of RAS-driven cancers.

Clinical Translation of Combined MAPK and Autophagy Inhibition in RAS Mutant Cancer

RAS (rat sarcoma virus) mutant cancers remain difficult to treat despite the advances in targeted therapy and immunotherapy. Targeted therapies against the components of mitogen-activated protein kinase (MAPK) pathways, including RAS, RAF, MEK, and ERK, have demonstrated activity in BRAF mutant and, in limited cases, RAS mutant cancer. RAS mutant cancers have been found to activate adaptive resistance mechanisms such as autophagy during MAPK inhibition. Here, we review the recent clinically relevant advances in the development of the MAPK pathway and autophagy inhibitors and focus on their application to RAS mutant cancers. We provide analysis of the preclinical rationale for combining the MAPK pathway and autophagy and highlight the most recent clinical trials that have been launched to capitalize on this potentially synthetic lethal approach to cancer therapy.

An infectious Rous Sarcoma Virus Gag mutant that is defective in nuclear cycling

During retroviral replication, unspliced viral genomic RNA (gRNA) must escape the nucleus for translation into viral proteins and packaging into virions. “Complex” retroviruses such as Human Immunodeficiency Virus (HIV) use cis-acting elements on the unspliced gRNA in conjunction with trans-acting viral proteins to facilitate this escape. “Simple” retroviruses such as Mason-Pfizer Monkey Virus (MPMV) and Murine Leukemia Virus (MLV) exclusively use cis-acting elements on the gRNA in conjunction with host nuclear export proteins for nuclear escape. Uniquely, the simple retrovirus Rous Sarcoma Virus (RSV) has a Gag structural protein that cycles through the nucleus prior to plasma membrane binding. This trafficking has been implicated in facilitating gRNA nuclear export and is thought to be a required mechanism. Previously described mutants that abolish nuclear cycling displayed enhanced plasma membrane binding, enhanced virion release, and a significant loss in genome incorporation resulting in loss of infectivity. Here, we describe a nuclear cycling deficient RSV Gag mutant that has similar plasma membrane binding and genome incorporation to WT virus and surprisingly, is replication competent albeit with a slower rate of spread compared to WT. This mutant suggests that RSV Gag nuclear cycling is not strictly required for RSV replication. Importance While mechanisms for retroviral Gag assembly at the plasma membrane are beginning to be characterized, characterization of intermediate trafficking locales remain elusive. This is in part due to the difficulty of tracking individual proteins from translation to plasma membrane binding. RSV Gag nuclear cycling is a unique phenotype that may provide comparative insight to viral trafficking evolution and may present a model intermediate to cis- and trans-acting mechanisms for gRNA export.

Bifunctional Role of CrkL during Bone Remodeling

Coupled signaling between bone-forming osteoblasts and bone-resorbing osteoclasts is crucial to the maintenance of bone homeostasis. We previously reported that v-crk avian sarcoma virus CT10 oncogene homolog-like (CrkL), which belongs to the Crk family of adaptors, inhibits bone morphogenetic protein 2 (BMP2)-mediated osteoblast differentiation, while enhancing receptor activator of nuclear factor kappa-B ligand (RANKL)-induced osteoclast differentiation. In this study, we investigated whether CrkL can also regulate the coupling signals between osteoblasts and osteoclasts, facilitating bone homeostasis. Osteoblastic CrkL strongly decreased RANKL expression through its inhibition of runt-related transcription factor 2 (Runx2) transcription. Reduction in RANKL expression by CrkL in osteoblasts resulted in the inhibition of not only osteoblast-dependent osteoclast differentiation but also osteoclast-dependent osteoblast differentiation, suggesting that CrkL participates in the coupling signals between osteoblasts and osteoclasts via its regulation of RANKL expression. Therefore, CrkL bifunctionally regulates osteoclast differentiation through both a direct and indirect mechanism while it inhibits osteoblast differentiation through its blockade of both BMP2 and RANKL reverse signaling pathways. Collectively, these data suggest that CrkL is involved in bone homeostasis, where it helps to regulate the complex interactions of the osteoblasts, osteoclasts, and their coupling signals.

Immunohistochemical and Molecular Studies of p53 and KRAS Protein and Their Relations to Colorectal Carcinoma

The study inc1uded 50 tissue blocks embedded in paraffin wax (16 females and 34 males), obtained from a patients group with (CRC) colorectal cancer , as well as 35 Tissue blocks that were embedded in paraffin wax from norma1 co1on (ulcerative co1itis) as controls. A relatively few oncogenes and most prominently tumor-suppressing genes, Kirastien rat sarcoma virus (KRAS), and P53 genes have been mutated into a significant part of CRCs, and a broad collection of mutated genes has been defined in CRC subsets. Current findings showed very significant differences between patients and control subjects in the p53 positive rate (P<0.001). TP53 Pro/Pro genotype positivity was higher in the contro1 group I than in the patient group I and this was a significant difference (Pi<0.001) with an odd ratio of less than one. The genotype Pro/Pro was considered to be protective against colorectal carcinoma preventively fractured 0.767. The positive rate of p53 Arg/Arg genotype in patients was more frequent and statistically significant (P <0.01), because the odd ratio was more than one. The genotype Arg/Arg would be considered a colorectal carcinoma risk factor. We conclude that p53 over expression is used as an indicator of p53 mutation (as identified by immuno-historic chemistry) and KRAS protein expression was negatively impaired for all the patients in the current study.

Structure of the mature Rous sarcoma virus lattice reveals a role for IP6 in the formation of the capsid hexamer

Inositol hexakisphosphate (IP6) is an assembly cofactor for HIV-1. We report here that IP6 is also used for assembly of Rous sarcoma virus (RSV), a retrovirus from a different genus. IP6 is ~100-fold more potent at promoting RSV mature capsid protein (CA) assembly than observed for HIV-1 and removal of IP6 in cells reduces infectivity by 100-fold. Here, visualized by cryo-electron tomography and subtomogram averaging, mature capsid-like particles show an IP6-like density in the CA hexamer, coordinated by rings of six lysines and six arginines. Phosphate and IP6 have opposing effects on CA in vitro assembly, inducing formation of T = 1 icosahedrons and tubes, respectively, implying that phosphate promotes pentamer and IP6 hexamer formation. Subtomogram averaging and classification optimized for analysis of pleomorphic retrovirus particles reveal that the heterogeneity of mature RSV CA polyhedrons results from an unexpected, intrinsic CA hexamer flexibility. In contrast, the CA pentamer forms rigid units organizing the local architecture. These different features of hexamers and pentamers determine the structural mechanism to form CA polyhedrons of variable shape in mature RSV particles.

Visualizing Rous Sarcoma Virus Genomic RNA Dimerization in the Nucleus, Cytoplasm, and at the Plasma Membrane

Retroviruses are unique in that they package their RNA genomes as non-covalently linked dimers. Failure to dimerize their genomes results in decreased infectivity and reduced packaging of genomic RNA into virus particles. Two models of retrovirus genome dimerization have been characterized: in murine leukemia virus (MLV), genomic RNA dimerization occurs co-transcriptionally in the nucleus, resulting in the preferential formation of genome homodimers; whereas in human immunodeficiency virus (HIV-1), genomic RNA dimerization occurs in the cytoplasm and at the plasma membrane, with a random distribution of heterodimers and homodimers. Although in vitro studies have identified the genomic RNA sequences that facilitate dimerization in Rous sarcoma virus (RSV), in vivo characterization of the location and preferences of genome dimerization has not been performed. In this study, we utilized three single molecule RNA imaging approaches to visualize genome dimers of RSV in cultured quail fibroblasts. The formation of genomic RNA heterodimers within cells was dependent on the presence of the dimerization initiation site (DIS) sequence in the L3 stem. Subcellular localization analysis revealed that heterodimers were present the nucleus, cytoplasm, and at the plasma membrane, indicating that genome dimers can form in the nucleus. Furthermore, single virion analysis revealed that RSV preferentially packages genome homodimers into virus particles. Therefore, the mechanism of RSV genomic RNA dimer formation appears more similar to MLV than HIV-1.