Determining the ERK-regulated phosphoproteome driving KRAS-mutant cancer.

To delineate the mechanisms by which the ERK1 and ERK2 mitogen-activated protein kinases support mutant KRAS-driven cancer growth, we determined the ERK-dependent phosphoproteome in KRAS-mutant pancreatic cancer. We determined that ERK1 and ERK2 share near-identical signaling and transforming outputs and that the KRAS-regulated phosphoproteome is driven nearly completely by ERK. We identified 4666 ERK-dependent phosphosites on 2123 proteins, of which 79 and 66%, respectively, were not previously associated with ERK, substantially expanding the depth and breadth of ERK-dependent phosphorylation events and revealing a considerably more complex function for ERK in cancer. We established that ERK controls a highly dynamic and complex phosphoproteome that converges on cyclin-dependent kinase regulation and RAS homolog guanosine triphosphatase function (RHO GTPase). Our findings establish the most comprehensive molecular portrait and mechanisms by which ERK drives KRAS-dependent pancreatic cancer growth.

Defining the KRAS- and ERK-dependent transcriptome in KRAS-mutant cancers.

How the KRAS oncogene drives cancer growth remains poorly understood. Therefore, we established a systemwide portrait of KRAS- and extracellular signal-regulated kinase (ERK)-dependent gene transcription in KRAS-mutant cancer to delineate the molecular mechanisms of growth and of inhibitor resistance. Unexpectedly, our KRAS-dependent gene signature diverges substantially from the frequently cited Hallmark KRAS signaling gene signature, is driven predominantly through the ERK mitogen-activated protein kinase (MAPK) cascade, and accurately reflects KRAS- and ERK-regulated gene transcription in KRAS-mutant cancer patients. Integration with our ERK-regulated phospho- and total proteome highlights ERK deregulation of the anaphase promoting complex/cyclosome (APC/C) and other components of the cell cycle machinery as key processes that drive pancreatic ductal adenocarcinoma (PDAC) growth. Our findings elucidate mechanistically the critical role of ERK in driving KRAS-mutant tumor growth and in resistance to KRAS-ERK MAPK targeted therapies.

VCP/p97, a pleiotropic protein regulator of the DNA damage response and proteostasis, is a potential therapeutic target in KRAS-mutant pancreatic cancer.

We and others have recently shown that proteins involved in the DNA damage response (DDR) are critical for KRAS-mutant pancreatic ductal adenocarcinoma (PDAC) cell growth in vitro. However, the CRISPR-Cas9 library that enabled us to identify these key proteins had limited representation of DDR-related genes. To further investigate the DDR in this context, we performed a comprehensive, DDR-focused CRISPR-Cas9 loss-of-function screen. This screen identified valosin-containing protein (VCP) as an essential gene in KRAS-mutant PDAC cell lines. We observed that genetic and pharmacologic inhibition of VCP limited cell growth and induced apoptotic death. Addressing the basis for VCP-dependent growth, we first evaluated the contribution of VCP to the DDR and found that loss of VCP resulted in accumulation of DNA double-strand breaks. We next addressed its role in proteostasis and found that loss of VCP caused accumulation of polyubiquitinated proteins. We also found that loss of VCP increased autophagy. Therefore, we reasoned that inhibiting both VCP and autophagy could be an effective combination. Accordingly, we found that VCP inhibition synergized with the autophagy inhibitor chloroquine. We conclude that concurrent targeting of autophagy can enhance the efficacy of VCP inhibitors in KRAS-mutant PDAC.

Oncogenic RAS promotes MYC protein stability by upregulating the expression of the inhibitor of apoptosis protein family member Survivin.

The small GTPase KRAS is frequently mutated in pancreatic cancer and its cooperation with the transcription factor MYC is essential for malignant transformation. The key to oncogenic KRAS and MYC working together is the stabilization of MYC expression due to KRAS activating the extracellular signal-regulated kinase 1/2, which phosphorylates MYC at serine 62 (Ser 62). This prevents the proteasomal degradation of MYC while enhancing its transcriptional activity. Here, we identify how this essential signaling connection between oncogenic KRAS and MYC expression is mediated by the inhibitor of apoptosis protein family member Survivin. This discovery stemmed from our finding that Survivin expression is downregulated upon treatment of pancreatic cancer cells with the KRASG12C inhibitor Sotorasib. We went on to show that oncogenic KRAS increases Survivin expression by activating extracellular signal-regulated kinase 1/2 in pancreatic cancer cells and that treating the cells either with siRNAs targeting Survivin or with YM155, a small molecule that potently blocks Survivin expression, downregulates MYC and strongly inhibited their growth. We further determined that Survivin protects MYC from degradation by blocking autophagy, which then prevents cellular inhibitor of protein phosphatase 2A from undergoing autophagic degradation. Cellular inhibitor of protein phosphatase 2A, by inhibiting protein phosphatase 2A, helps to maintain MYC phosphorylation at Ser 62, thereby ensuring its cooperation with oncogenic KRAS in driving cancer progression. Overall, these findings highlight a novel role for Survivin in mediating the cooperative actions of KRAS and MYC during malignant transformation and raise the possibility that targeting Survivin may offer therapeutic benefits against KRAS-driven cancers.

Combination Therapies with CDK4/6 Inhibitors to Treat KRAS-Mutant Pancreatic Cancer.

Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step that complements activation of KRAS in promoting the development and malignant growth of pancreatic ductal adenocarcinoma (PDAC). However, pharmacologic restoration of p16INK4A function with inhibitors of CDK4 and CDK6 (CDK4/6) has shown limited clinical efficacy in PDAC. Here, we found that concurrent treatment with both a CDK4/6 inhibitor (CDK4/6i) and an ERK-MAPK inhibitor (ERKi) synergistically suppresses the growth of PDAC cell lines and organoids by cooperatively blocking CDK4/6i-induced compensatory upregulation of ERK, PI3K, antiapoptotic signaling, and MYC expression. On the basis of these findings, a Phase I clinical trial was initiated to evaluate the ERKi ulixertinib in combination with the CDK4/6i palbociclib in patients with advanced PDAC (NCT03454035). As inhibition of other proteins might also counter CDK4/6i-mediated signaling changes to increase cellular CDK4/6i sensitivity, a CRISPR-Cas9 loss-of-function screen was conducted that revealed a spectrum of functionally diverse genes whose loss enhanced CDK4/6i growth inhibitory activity. These genes were enriched around diverse signaling nodes, including cell-cycle regulatory proteins centered on CDK2 activation, PI3K-AKT-mTOR signaling, SRC family kinases, HDAC proteins, autophagy-activating pathways, chromosome regulation and maintenance, and DNA damage and repair pathways. Novel therapeutic combinations were validated using siRNA and small-molecule inhibitor-based approaches. In addition, genes whose loss imparts a survival advantage were identified (e.g., RB1, PTEN, FBXW7), suggesting possible resistance mechanisms to CDK4/6 inhibition. In summary, this study has identified novel combinations with CDK4/6i that may have clinical benefit to patients with PDAC. SIGNIFICANCE: CRISPR-Cas9 screening and protein activity mapping reveal combinations that increase potency of CDK4/6 inhibitors and overcome drug-induced compensations in pancreatic cancer.

Susceptibility to autophagy inhibition is enhanced by dual IGF1R and MAPK/ERK inhibition in pancreatic cancer.

Macroautophagy/autophagy is upregulated in pancreatic ductal adenocarcinoma (PDAC) and PDAC growth is reliant on autophagy. However, autophagy inhibitors as monotherapy have shown limited clinical efficacy. To identify targets that sensitize PDAC cells to autophagy inhibition, we performed a CRISPR-Cas9 genetic loss-of-function screen in cells treated with the lysosomal inhibitor chloroquine (CQ) and identified IGF1R as a sensitizer. IGF1R inhibition increases autophagic flux and sensitivity to CQ-mediated growth suppression both in vitro and in vivo. Importantly, sensitization is further enhanced with the concurrent inhibition of MAPK1/ERK2 (mitogen-activated protein kinase 1)-MAPK3/ERK1. IGF1R and MAPK/ERK inhibition converge on suppression of glycolysis. In summary, IGF1R and MAPK/ERK signaling promotes resistance to CQ/HCQ in PDAC, and their dual inhibition increases sensitivity to autophagy inhibitors.

Unraveling and targeting RAS-driven metabolic signaling for therapeutic gain.

RAS mutations are among the most frequent oncogenic drivers observed in human cancers. With a lack of available treatment options, RAS-mutant cancers account for many of the deadliest cancers in the United States. Recent studies established that altered metabolic requirements are a hallmark of cancer, and many of these alterations are driven by aberrant RAS signaling. Specifically, RAS-driven cancers are characterized by upregulated glycolysis, the differential channeling of glycolytic intermediates, upregulated nutrient scavenging pathways such as autophagy and macropinocytosis, and altered glutamine utilization and mitochondrial function. This unique metabolic landscape promotes tumorigenesis, proliferation, survival in nutrient deficient environments and confers resistance to conventional cytotoxic and targeted therapies. Emerging work demonstrates how these dependencies can be therapeutically exploited in vitro and in vivo with many metabolic inhibitors currently in clinical trials. This review aims to outline the unique metabolic requirements induced by aberrant RAS signaling and how these altered dependencies present opportunities for therapeutic intervention.

Concurrent Inhibition of IGF1R and ERK Increases Pancreatic Cancer Sensitivity to Autophagy Inhibitors.

The aggressive nature of pancreatic ductal adenocarcinoma (PDAC) mandates the development of improved therapies. As KRAS mutations are found in 95% of PDAC and are critical for tumor maintenance, one promising strategy involves exploiting KRAS-dependent metabolic perturbations. The macrometabolic process of autophagy is upregulated in KRAS-mutant PDAC, and PDAC growth is reliant on autophagy. However, inhibition of autophagy as monotherapy using the lysosomal inhibitor hydroxychloroquine (HCQ) has shown limited clinical efficacy. To identify strategies that can improve PDAC sensitivity to HCQ, we applied a CRISPR-Cas9 loss-of-function screen and found that a top sensitizer was the receptor tyrosine kinase (RTK) insulin-like growth factor 1 receptor (IGF1R). Additionally, reverse phase protein array pathway activation mapping profiled the signaling pathways altered by chloroquine (CQ) treatment. Activating phosphorylation of RTKs, including IGF1R, was a common compensatory increase in response to CQ. Inhibition of IGF1R increased autophagic flux and sensitivity to CQ-mediated growth suppression both in vitro and in vivo. Cotargeting both IGF1R and pathways that antagonize autophagy, such as ERK-MAPK axis, was strongly synergistic. IGF1R and ERK inhibition converged on suppression of glycolysis, leading to enhanced dependence on autophagy. Accordingly, concurrent inhibition of IGF1R, ERK, and autophagy induced cytotoxicity in PDAC cell lines and decreased viability in human PDAC organoids. In conclusion, targeting IGF1R together with ERK enhances the effectiveness of autophagy inhibitors in PDAC. SIGNIFICANCE: Compensatory upregulation of IGF1R and ERK-MAPK signaling limits the efficacy of autophagy inhibitors chloroquine and hydroxychloroquine, and their concurrent inhibition synergistically increases autophagy dependence and chloroquine sensitivity in pancreatic ductal adenocarcinoma.

CHK1 protects oncogenic KRAS-expressing cells from DNA damage and is a target for pancreatic cancer treatment.

We apply genetic screens to delineate modulators of KRAS mutant pancreatic ductal adenocarcinoma (PDAC) sensitivity to ERK inhibitor treatment, and we identify components of the ATR-CHK1 DNA damage repair (DDR) pathway. Pharmacologic inhibition of CHK1 alone causes apoptotic growth suppression of both PDAC cell lines and organoids, which correlates with loss of MYC expression. CHK1 inhibition also activates ERK and AMPK and increases autophagy, providing a mechanistic basis for increased efficacy of concurrent CHK1 and ERK inhibition and/or autophagy inhibition with chloroquine. To assess how CHK1 inhibition-induced ERK activation promotes PDAC survival, we perform a CRISPR-Cas9 loss-of-function screen targeting direct/indirect ERK substrates and identify RIF1. A key component of non-homologous end joining repair, RIF1 suppression sensitizes PDAC cells to CHK1 inhibition-mediated apoptotic growth suppression. Furthermore, ERK inhibition alone decreases RIF1 expression and phenocopies RIF1 depletion. We conclude that concurrent DDR suppression enhances the efficacy of ERK and/or autophagy inhibitors in KRAS mutant PDAC.

KRAS-dependent cancer cells promote survival by producing exosomes enriched in Survivin.

Mutations in KRAS frequently occur in human cancer and are especially prevalent in pancreatic ductal adenocarcinoma (PDAC), where they have been shown to promote aggressive phenotypes. However, targeting this onco-protein has proven to be challenging, highlighting the need to further identify the various mechanisms used by KRAS to drive cancer progression. Here, we considered the role played by exosomes, a specific class of extracellular vesicles (EVs) derived from the endocytic cellular trafficking machinery, in mediating the ability of KRAS to promote cell survival. We found that exosomes isolated from the serum of PDAC patients, as well as from KRAS-transformed fibroblasts and pancreatic cancer cells, were all highly enriched in the cell survival protein Survivin. Exosomes containing Survivin, upon engaging serum-starved cells, strongly enhanced their survival. Moreover, they significantly compromised the effectiveness of the conventional chemotherapy drug paclitaxel, as well as a novel therapy that combines an ERK inhibitor with chloroquine, which is currently in clinical trials for PDAC. The survival benefits provided by oncogenic KRAS-derived exosomes were markedly reduced when depleted of Survivin using siRNA or upon treatment with the Survivin inhibitor YM155. Taken together, these findings demonstrate how KRAS mutations give rise to exosomes that provide a unique form of intercellular communication to promote cancer cell survival and therapy resistance, as well as raise interesting possibilities regarding their potential for serving as therapeutic targets and diagnostic markers for KRAS-dependent cancers.

Author Correction: Combination of ERK and autophagy inhibition as a treatment approach for pancreatic cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Genome-wide DNA methylation analysis of KRAS mutant cell lines.

Oncogenic RAS mutations are associated with DNA methylation changes that alter gene expression to drive cancer. Recent studies suggest that DNA methylation changes may be stochastic in nature, while other groups propose distinct signaling pathways responsible for aberrant methylation. Better understanding of DNA methylation events associated with oncogenic KRAS expression could enhance therapeutic approaches. Here we analyzed the basal CpG methylation of 11 KRAS-mutant and dependent pancreatic cancer cell lines and observed strikingly similar methylation patterns. KRAS knockdown resulted in unique methylation changes with limited overlap between each cell line. In KRAS-mutant Pa16C pancreatic cancer cells, while KRAS knockdown resulted in over 8,000 differentially methylated (DM) CpGs, treatment with the ERK1/2-selective inhibitor SCH772984 showed less than 40 DM CpGs, suggesting that ERK is not a broadly active driver of KRAS-associated DNA methylation. KRAS G12V overexpression in an isogenic lung model reveals >50,600 DM CpGs compared to non-transformed controls. In lung and pancreatic cells, gene ontology analyses of DM promoters show an enrichment for genes involved in differentiation and development. Taken all together, KRAS-mediated DNA methylation are stochastic and independent of canonical downstream effector signaling. These epigenetically altered genes associated with KRAS expression could represent potential therapeutic targets in KRAS-driven cancer.

Atypical KRASG12R Mutant Is Impaired in PI3K Signaling and Macropinocytosis in Pancreatic Cancer.

Allele-specific signaling by different KRAS alleles remains poorly understood. The KRAS G12R mutation displays uneven prevalence among cancers that harbor the highest occurrence of KRAS mutations: It is rare (∼1%) in lung and colorectal cancers, yet relatively common (∼20%) in pancreatic ductal adenocarcinoma (PDAC), suggesting context-specific properties. We evaluated whether KRASG12R is functionally distinct from the more common KRASG12D- or KRASG12V-mutant proteins (KRASG12D/V). We found that KRASG12D/V but not KRASG12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRASG12D/V- but not KRASG12R-mutant PDAC. Surprisingly, we found that KRASG12R is defective for interaction with a key effector, p110α PI3K (PI3Kα), due to structural perturbations in switch II. Instead, upregulated KRAS-independent PI3Kγ activity was able to support macropinocytosis in KRASG12R-mutant PDAC. Finally, we determined that KRASG12R-mutant PDAC displayed a distinct drug sensitivity profile compared with KRASG12D-mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy. SIGNIFICANCE: We determined that KRASG12R is impaired in activating a key effector, p110α PI3K. As such, KRASG12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this deficiency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers.See related commentary by Falcomatà et al., p. 23.This article is highlighted in the In This Issue feature, p. 1.

Combination of ERK and autophagy inhibition as a treatment approach for pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent tumorigenic growth, but the role of KRAS in supporting autophagy has not been established. We show that, to our surprise, suppression of KRAS increased autophagic flux, as did pharmacological inhibition of its effector ERK MAPK. Furthermore, we demonstrate that either KRAS suppression or ERK inhibition decreased both glycolytic and mitochondrial functions. We speculated that ERK inhibition might thus enhance PDAC dependence on autophagy, in part by impairing other KRAS- or ERK-driven metabolic processes. Accordingly, we found that the autophagy inhibitor chloroquine and genetic or pharmacologic inhibition of specific autophagy regulators synergistically enhanced the ability of ERK inhibitors to mediate antitumor activity in KRAS-driven PDAC. We conclude that combinations of pharmacologic inhibitors that concurrently block both ERK MAPK and autophagic processes that are upregulated in response to ERK inhibition may be effective treatments for PDAC.

KRAS Suppression-Induced Degradation of MYC Is Antagonized by a MEK5-ERK5 Compensatory Mechanism.

Our recent ERK1/2 inhibitor analyses in pancreatic ductal adenocarcinoma (PDAC) indicated ERK1/2-independent mechanisms maintaining MYC protein stability. To identify these mechanisms, we determined the signaling networks by which mutant KRAS regulates MYC. Acute KRAS suppression caused rapid proteasome-dependent loss of MYC protein, through both ERK1/2-dependent and -independent mechanisms. Surprisingly, MYC degradation was independent of PI3K-AKT-GSK3ß signaling and the E3 ligase FBWX7. We then established and applied a high-throughput screen for MYC protein degradation and performed a kinome-wide proteomics screen. We identified an ERK1/2-inhibition-induced feedforward mechanism dependent on EGFR and SRC, leading to ERK5 activation and phosphorylation of MYC at S62, preventing degradation. Concurrent inhibition of ERK1/2 and ERK5 disrupted this mechanism, synergistically causing loss of MYC and suppressing PDAC growth.

Mutant RAS Calms Stressed-Out Cancer Cells.

Mutant RAS-driven cancers are infamously resistant to chemotherapeutics. Reporting in Cell, Grabocka and Bar-Sagi (2016) demonstrate that when subjected to stress, mutant KRAS-dependent lipid production leads to upregulated stress granule formation. This confers not only cell-autonomous cytoprotection but also paracrine establishment of a stress-resistant tumor niche.

KRAS Mutant Pancreatic Cancer: No Lone Path to an Effective Treatment.

Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest cancers with a dismal 7% 5-year survival rate and is projected to become the second leading cause of cancer-related deaths by 2020. KRAS is mutated in 95% of PDACs and is a well-validated driver of PDAC growth and maintenance. However, despite comprehensive efforts, an effective anti-RAS drug has yet to reach the clinic. Different paths to inhibiting RAS signaling are currently under investigation in the hope of finding a successful treatment. Recently, direct RAS binding molecules have been discovered, challenging the perception that RAS is an "undruggable" protein. Other strategies currently being pursued take an indirect approach, targeting proteins that facilitate RAS membrane association or downstream effector signaling. Unbiased genetic screens have identified synthetic lethal interactors of mutant RAS. Most recently, metabolic targets in pathways related to glycolytic signaling, glutamine utilization, autophagy, and macropinocytosis are also being explored. Harnessing the patient's immune system to fight their cancer is an additional exciting route that is being considered. The "best" path to inhibiting KRAS has yet to be determined, with each having promise as well as potential pitfalls. We will summarize the state-of-the-art for each direction, focusing on efforts directed toward the development of therapeutics for pancreatic cancer patients with mutated KRAS.

Long-Term ERK Inhibition in KRAS-Mutant Pancreatic Cancer Is Associated with MYC Degradation and Senescence-like Growth Suppression.

Induction of compensatory mechanisms and ERK reactivation has limited the effectiveness of Raf and MEK inhibitors in RAS-mutant cancers. We determined that direct pharmacologic inhibition of ERK suppressed the growth of a subset of KRAS-mutant pancreatic cancer cell lines and that concurrent phosphatidylinositol 3-kinase (PI3K) inhibition caused synergistic cell death. Additional combinations that enhanced ERK inhibitor action were also identified. Unexpectedly, long-term treatment of sensitive cell lines caused senescence, mediated in part by MYC degradation and p16 reactivation. Enhanced basal PI3K-AKT-mTOR signaling was associated with de novo resistance to ERK inhibitor, as were other protein kinases identified by kinome-wide siRNA screening and a genetic gain-of-function screen. Our findings reveal distinct consequences of inhibiting this kinase cascade at the level of ERK.

A novel fluorescence-based biosynthetic trafficking method provides pharmacologic evidence that PI4-kinase IIIα is important for protein trafficking from the endoplasmic reticulum to the plasma membrane.

BACKGROUND: Biosynthetic trafficking of receptors and other membrane-associated proteins from the endoplasmic reticulum (ER) to the plasma membrane (PM) underlies the capacity of these proteins to participate in crucial cellular roles. Phosphoinositides have been shown to mediate distinct biological functions in cells, and phosphatidylinositol 4-phosphate (PI4P), in particular, has emerged as a key regulator of biosynthetic trafficking. RESULTS: To investigate the source of PI4P that orchestrates trafficking events, we developed a novel flow cytometry based method to monitor biosynthetic trafficking of transiently transfected proteins. We demonstrated that our method can be used to assess the trafficking of both type-1 transmembrane and GPI-linked proteins, and that it can accurately monitor the pharmacological disruption of biosynthetic trafficking with brefeldin A, a well-documented inhibitor of early biosynthetic trafficking. Furthermore, utilizing our newly developed method, we applied pharmacological inhibition of different isoforms of PI 4-kinase to reveal a role for a distinct pool of PI4P, synthesized by PI4KIIIα, in ER-to-PM trafficking. CONCLUSIONS: Taken together, these findings provide evidence that a specific pool of PI4P plays a role in biosynthetic trafficking of two different classes of proteins from the ER to the Golgi complex. Furthermore, our simple, flow cytometry-based biosynthetic trafficking assay can be widely applied to the study of multiple classes of proteins and varied pharmacological and genetic perturbations.