Fluorescent Biosensor for Measuring Ras Activity in Living Cells.

The small GTPase Ras is a critical regulator of cell growth and proliferation. Its activity is frequently dysregulated in cancers, prompting decades of work to pharmacologically target Ras. Understanding Ras biology and developing effective Ras therapeutics both require probing Ras activity in its native context, yet tools to measure its activities in cellulo are limited. Here, we developed a ratiometric Ras activity reporter (RasAR) that provides quantitative measurement of Ras activity in living cells with high spatiotemporal resolution. We demonstrated that RasAR can probe live-cell activities of all the primary isoforms of Ras. Given that the functional roles of different isoforms of Ras are intimately linked to their subcellular distribution and regulation, we interrogated the spatiotemporal regulation of Ras utilizing subcellularly targeted RasAR and uncovered the role of Src kinase as an upstream regulator to inhibit HRas. Furthermore, we showed that RasAR enables capture of KRasG12C inhibition dynamics in living cells upon treatment with KRasG12C covalent inhibitors, including ARS1620, Sotorasib, and Adagrasib. We found in living cells a residual Ras activity lingers for hours in the presence of these inhibitors. Together, RasAR represents a powerful molecular tool to enable live-cell interrogation of Ras activity and facilitate the development of Ras inhibitors.

MAP kinase and autophagy pathways cooperate to maintain RAS mutant cancer cell survival.

Oncogenic mutations in the small GTPase KRAS are frequently found in human cancers, and, currently, there are no effective targeted therapies for these tumors. Using a combinatorial siRNA approach, we analyzed a panel of KRAS mutant colorectal and pancreatic cancer cell lines for their dependency on 28 gene nodes that represent canonical RAS effector pathways and selected stress response pathways. We found that RAF node knockdown best differentiated KRAS mutant and KRAS WT cancer cells, suggesting RAF kinases are key oncoeffectors for KRAS addiction. By analyzing all 376 pairwise combination of these gene nodes, we found that cotargeting the RAF, RAC, and autophagy pathways can improve the capture of KRAS dependency better than targeting RAF alone. In particular, codepletion of the oncoeffector kinases BRAF and CRAF, together with the autophagy E1 ligase ATG7, gives the best therapeutic window between KRAS mutant cells and normal, untransformed cells. Distinct patterns of RAS effector dependency were observed across KRAS mutant cell lines, indicative of heterogeneous utilization of effector and stress response pathways in supporting KRAS addiction. Our findings revealed previously unappreciated complexity in the signaling network downstream of the KRAS oncogene and suggest rational target combinations for more effective therapeutic intervention.

Introduction.

The phosphoinositide-3-kinase (PI3K) family of lipid kinases has been well conserved from yeast to mammals. In this evolutionary perspective on the PI3K family, we discuss the prototypical properties of PI3Ks: 1) the utilization of sparse but specifically localized lipid substrates; 2) the nucleation signaling complexes at membrane-targeted sites; and 3) the integration of intracellular signaling with extracellular cues. Together, these three core properties serve to establish order within the entropic environment of the cell. Many human diseases, including cancer and diabetes, are the direct result of loss or defects in one or more of these core properties, putting much hope in the clinical use of PI3K inhibitors singly and in combination to restore order within diseased tissues.

Class 1A PI3K regulates vessel integrity during development and tumorigenesis.

PI3K is important in the regulation of growth, proliferation, and survival of tumor cells. We show that class 1A PI3K is also critical in the tumor microenvironment by regulating the integrity of the tumor vasculature. Using Tie2Cre-mediated deletion of the PI3K regulatory subunits (p85alpha, p55alpha, p50alpha, and p85beta), we generated mice with endothelial cell-specific loss of class 1A PI3K. Complete loss of all subunits caused acute embryonic lethality at E11.5 due to hemorrhaging, whereas retention of a single p85alpha allele yielded viable mice that survived to adulthood. These heterozygous mice exhibited no vascular defects until challenged with a pathological insult, such as tumor cells or high levels of VEGF. Under these pathological conditions, heterozygous mice exhibited localized vascular abnormalities, including vessel leakage and the inability to maintain large vessels, which caused a deceleration of tumorigenesis. Furthermore, we show that a PI3K inhibitor can mimic the effects of class 1A PI3K loss, which suggests that targeting class 1A PI3K may be a promising therapy for blocking tumor angiogenesis.

E2F4 cooperates with pRB in the development of extra-embryonic tissues.

The retinoblastoma gene, RB-1, was the first identified tumor suppressor. Rb(-/-) mice die in mid-gestation with defects in proliferation, differentiation and apoptosis. The activating E2F transcription factors, E2F1-3, contribute to these embryonic defects, indicating that they are key downstream targets of the retinoblastoma protein, pRB. E2F4 is the major pRB-associated E2F in vivo, yet its role in Rb(-/-) embryos is unknown. Here we establish that E2f4 deficiency reduced the lifespan of Rb(-/-) embryos by exacerbating the Rb mutant placental defect. We further show that this reflects the accumulation of trophectoderm-like cells in both Rb and Rb;E2f4 mutant placentas. Thus, Rb and E2f4 play cooperative roles in placental development. We used a conditional mouse model to allow Rb(-/-);E2f4(-/-) embryos to develop in the presence of Rb wild-type placentas. Under these conditions, Rb(-/-);E2f4(-/-) mutants survived to birth. These Rb(-/-);E2f4(-/-) embryos exhibited all of the defects characteristic of the Rb and E2f4 single mutants and had no novel defects. Taken together, our data show that pRB and E2F4 cooperate in placental development, but play largely non-overlapping roles in the development of many embryonic tissues.

Selective requirements for E2f3 in the development and tumorigenicity of Rb-deficient chimeric tissues.

The tumor suppressor function of the retinoblastoma protein pRB is largely dependent upon its capacity to inhibit the E2F transcription factors and thereby cell proliferation. Attempts to study the interplay between pRB and the E2Fs have been hampered by the prenatal death of Rb; E2f nullizygous mice. In this study, we isolated Rb; E2f3 mutant embryonic stem cells and generated Rb(-/-); E2f3(-/-) chimeric mice, thus bypassing the lethality of the Rb(-/-); E2f3(-/-) germ line mutant mice. We show that loss of E2F3 has opposing effects on two of the known developmental defects arising in Rb(-/-) chimeras; it suppresses the formation of cataracts while aggravating the retinal dysplasia. This model system also allows us to assess how E2f3 status influences tumor formation in Rb(-/-) tissues. We find that E2f3 is dispensable for the development of pRB-deficient pituitary and thyroid tumors. In contrast, E2f3 inactivation completely suppresses the pulmonary neuroendocrine hyperplasia arising in Rb(-/-) chimeric mice. This hyperproliferative state is thought to represent the preneoplastic lesion of small-cell lung carcinoma. Therefore, our observation highlights a potential role for E2F3 in the early stages of this tumor type.

The retinoblastoma protein tumor suppressor is important for appropriate osteoblast differentiation and bone development.

Mutation of the retinoblastoma (RB) tumor suppressor gene is strongly linked to osteosarcoma formation. This observation and the documented interaction between the retinoblastoma protein (pRb) and Runx2 suggests that pRb is important in bone development. To assess this hypothesis, we used a conditional knockout strategy to generate pRb-deficient embryos that survive to birth. Analysis of these embryos shows that Rb inactivation causes the abnormal development and impaired ossification of several bones, correlating with an impairment in osteoblast differentiation. We further show that Rb inactivation acts to promote osteoblast differentiation in vitro and, through conditional analysis, establish that this occurs in a cell-intrinsic manner. Although these in vivo and in vitro differentiation phenotypes seem paradoxical, we find that Rb-deficient osteoblasts have an impaired ability to exit the cell cycle both in vivo and in vitro that can explain the observed differentiation defects. Consistent with this observation, we show that the cell cycle and the bone defects in Rb-deficient embryos can be suppressed by deletion of E2f1, a known proliferation inducer that acts downstream of Rb. Thus, we conclude that pRb plays a key role in regulating osteoblast differentiation by mediating the inhibition of E2F and consequently promoting cell cycle exit.

Synthetic Lethal Interaction of SHOC2 Depletion with MEK Inhibition in RAS-Driven Cancers.

The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy.

Differential Effector Engagement by Oncogenic KRAS.

KRAS can bind numerous effector proteins, which activate different downstream signaling events. The best known are RAF, phosphatidylinositide (PI)-3' kinase, and RalGDS families, but many additional direct and indirect effectors have been reported. We have assessed how these effectors contribute to several major phenotypes in a quantitative way, using an arrayed combinatorial siRNA screen in which we knocked down 41 KRAS effectors nodes in 92 cell lines. We show that every cell line has a unique combination of effector dependencies, but in spite of this heterogeneity, we were able to identify two major subtypes of KRAS mutant cancers of the lung, pancreas, and large intestine, which reflect different KRAS effector engagement and opportunities for therapeutic intervention.

Development of siRNA payloads to target KRAS-mutant cancer.

UNLABELLED: RNAi is a powerful tool for target identification and can lead to novel therapies for pharmacologically intractable targets such as KRAS. RNAi therapy must combine potent siRNA payloads with reliable in vivo delivery for efficient target inhibition. We used a functional "Sensor" assay to establish a library of potent siRNAs against RAS pathway genes and to show that they efficiently suppress their targets at low dose. This reduces off-target effects and enables combination gene knockdown. We administered Sensor siRNAs in vitro and in vivo and validated the delivery of KRAS siRNA alone and siRNA targeting the complete RAF effector node (A/B/CRAF) as promising strategies to treat KRAS-mutant colorectal cancer. We further demonstrate that improved therapeutic efficacy is achieved by formulating siRNA payloads that combine both single-gene siRNA and node-targeted siRNAs (KRAS + PIK3CA/B). The customizable nature of Sensor siRNA payloads offers a universal platform for the combination target identification and development of RNAi therapeutics. SIGNIFICANCE: To advance RNAi therapy for KRAS-mutant cancer, we developed a validated siRNA library against RAS pathway genes that enables combination gene silencing. Using an in vivo model for real-time siRNA delivery tracking, we show that siRNA-mediated inhibition of KRAS as well as RAF or PI3K combinations can impair KRAS-mutant colorectal cancer in xenograft models.

Killing tumors by keeping ras and PI3' kinase apart.

Ras proteins mediate PI3K activation through direct binding to p110 catalytic subunits. However, it is unclear when and where this interaction occurs. In this issue of Cancer Cell, Castellano and colleagues report that KRAS-driven lung cancers require the Ras-p110α interaction for full activation of PI3K and tumor maintenance.

Cell-to-cell variability in PI3K protein level regulates PI3K-AKT pathway activity in cell populations.

BACKGROUND: Cell-to-cell variability in populations has been widely observed in mammalian cells. This heterogeneity can result from random stochastic events or can be deliberately maintained through regulatory processes. In the latter case, heterogeneity should confer a selective advantage that benefits the entire population. RESULTS: Using multicolor flow cytometry, we have uncovered robust heterogeneity in phosphoinositide 3-kinase (PI3K) activity in MCF10A cell populations, which had been previously masked by techniques that only measure population averages. We show that AKT activity is bimodal in response to EGF stimulation and correlates with PI3K protein level, such that only cells with high PI3K protein can activate AKT. We further show that heterogeneity in PI3K protein levels is invariably maintained in cell populations through a degradation/resynthesis cycle that can be regulated by cell density. CONCLUSIONS: Given that the PI3K pathway is one of the most frequently upregulated pathways in cancer, we propose that heterogeneity in PI3K activity is beneficial to normal tissues by restricting PI3K activation to only a subset of cells. This may serve to protect the population as a whole from overactivating the pathway, which can lead to cellular senescence or cancer. Consistent with this, we show that oncogenic mutations in p110α (H1047R and E545K) partially evade this negative regulation, resulting in increased AKT activity in the population.

Disruption of calvarial ossification in E2f4 mutant embryos correlates with increased proliferation and progenitor cell populations.

The E2F family of transcription factors, in association with pocket protein family members, are important for regulating genes required for cellular proliferation. The most abundant E2F, E2F4, is implicated in maintaining the G(0)/G(1) cell cycle state via transcriptional repression of genes that encode proteins required for S-phase progression. Here, we investigate E2F4's role in bone development using E2f4 germline mutant mice. We find that mutation of E2f4 impairs the formation of several bones that arise through intramembranous or endochondral ossification. The most severe defect occurred in the calvarial bones of the skull where we observed a striking delay in their ossification. In vivo and in vitro analyses established that E2F4 loss did not block the intrinsic differentiation potential of calvarial osteoblast progenitors. However, our data showed that E2f4 mutation elevated proliferation in the developing calvaria in vivo and it increased the endogenous pool of undifferentiated progenitor cells. These data suggest that E2F4 plays an important role in enabling osteoblast progenitors to exit the cell cycle and subsequently differentiate thereby contributing to the commitment of these cells to the bone lineage.

The role of E2F4 in adipogenesis is independent of its cell cycle regulatory activity.

The E2F and pocket protein families are known to play an important role in the regulation of both cellular proliferation and terminal differentiation. In this study, we have used compound E2F and pocket protein mutant mouse embryonic fibroblasts to dissect the role of these proteins in adipogenesis. This analysis shows that loss of E2F4 allows cells to undergo spontaneous differentiation. The ability of E2F4 to prevent adipogenesis seems to be quite distinct from the known properties of E2F. First, it can be separated from any change in either E2F-responsive gene expression or cell cycle regulation. Second, it is a specific property of E2F4, and not other E2Fs, and it occurs independently of E2F4's ability to interact with pocket proteins. In addition, E2F4 loss does not override the differentiation defect resulting from pRB loss even though it completely suppresses the proliferation defect of Rb(-/-) mouse embryonic fibroblasts. This finding definitively separates the known, positive role of pRB in adipogenesis from its cell cycle function and shows that this pocket protein is required to act downstream of E2F4 in the differentiation process.