Channing Der - Profile on Academia.edu (original) (raw)
Papers by Channing Der
Supplementary Data and Materials from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
Figure S5 page2 from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
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Figure S6 page1 from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
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Figure S5 page3 from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
Figure S5 page1 from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
Figure S5 page1
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2013
Background: HDAC6 plays an important role in cell migration. Results: ERK interacts with and phos... more Background: HDAC6 plays an important role in cell migration. Results: ERK interacts with and phosphorylates HDAC6 to promote cell migration. Conclusion: ERK signaling pathway promotes cell migration, in part, through phosphorylating HDAC6. Significance: Inhibition of HDAC6 activity as well as the EGFR-Ras-Raf-MEK-ERK signaling pathway may cooperatively reduce cell migration.
Combination Therapies with CDK4/6 Inhibitors to Treat KRAS-Mutant Pancreatic Cancer
Cancer Research, Nov 8, 2022
Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step tha... more 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.
Cancer Discovery, 2020
Allele-specifi c signaling by different KRAS alleles remains poorly understood. The KRAS G12R mut... more Allele-specifi c 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-specifi c properties. We evaluated whether KRAS G12R is functionally distinct from the more common KRAS G12D -or KRAS G12V -mutant proteins (KRAS G12D/V ). We found that KRAS G12D/V but not KRAS G12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRAS G12D/V -but not KRAS G12R -mutant PDAC. Surprisingly, we found that KRAS G12R 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 KRAS G12R -mutant PDAC. Finally, we determined that KRAS G12R -mutant PDAC displayed a distinct drug sensitivity profi le compared with KRAS G12D -mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy. We determined that KRAS G12R is impaired in activating a key effector, p110α PI3K. As such, KRAS G12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this defi ciency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers.
Data from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step tha... more 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.
The aggressive nature of pancreatic ductal adenocarcinoma (PDAC) mandates the development of impr... more 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.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2016
Recently we identified that PREX1 overexpression is critical for metastatic but not tumorigenic g... more Recently we identified that PREX1 overexpression is critical for metastatic but not tumorigenic growth in a mouse model of NRAS-driven melanoma. In addition, a PREX1 gene signature correlated with and was dependent on ERK mitogen-activated protein kinase (MAPK) activation in human melanoma cell lines. In the current study, the underlying mechanism of PREX1 overexpression in human melanoma was assessed. PREX1 protein levels were increased in melanoma tumor tissues and cell lines compared with benign nevi and normal melanocytes, respectively. Suppression of PREX1 by siRNA impaired invasion but not proliferation in vitro. PREX1-dependent invasion was attributable to PREX1-mediated activation of the small GTPase RAC1 but not the related small GTPase CDC42. Pharmacologic inhibition of ERK signaling reduced PREX1 gene transcription and additionally regulated PREX1 protein stability. This ERKdependent upregulation of PREX1 in melanoma, due to both increased gene transcription and protein stability, contrasts with the mechanisms identified in breast and prostate cancers, where PREX1 overexpression was driven by gene amplification and HDAC-mediated gene transcription, respectively. Thus, although PREX1 expression is aberrantly upregulated and regulates RAC1
Farnesylated proteins: how do they get to where they need to go, and how does location regulate their ability to control proliferation, death, transformation and aging?
The FASEB Journal, 2006
Filling in the GAPs in understanding RAS
Science, 2021
A newly identified regulator increases the efficacy of a new class of targeted anti-RAS drugs
Abstract 4660: Inhibition of p38 enhances ERK inhibitor efficacy in pancreatic ductal adenocarcinoma
Cancer Research, 2016
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the Unite... more Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States, with a poor prognosis and limited treatment options. Oncogenic mutation of KRAS in greater than 90% of PDAC leads to aberrant activation of multiple effector pathways including the extra cellular related kinase (ERK)/mitogen activated protein kinase (MAPK) cascade. Hyperactivation of the ERK MAPK cascade has been correlated with poorer prognosis in PDAC patients. We recently showed that direct pharmacological inhibition of ERK1/2 kinases with the ERK1/2-selective inhibitor SCH772984 inhibits the growth of PDAC cell lines both in vitro and in vivo. However, much like the response to ERK/MAPK pathway inhibitors acting at upstream nodes RAF or MEK, resistance to direct inhibition at the level of ERK will also inevitably arise. We performed a novel gain-of-function “Cancer Toolkit” (CTK) genetic screen to identify mechanisms of resistance to the ERK inhibitor SCH772984 in a panel o...
Farnesyltransferase and Geranylgeranyltransferase Inhibitors
Since 1982, when mutated and oncogenic forms of ras genes were first identified in human tumor ce... more Since 1982, when mutated and oncogenic forms of ras genes were first identified in human tumor cells, their protein products have attracted considerable interest as a target for anticancer drug development. Researchers were inspired to delineate the functions of Ras proteins in normal cells and to determine how mutated Ras proteins were altered in these functions. The impressive accumulation of information about the genetics, biochemistry, biology, and structure of Ras proteins over the last 17 years has provided important clues to how anti-Ras drugs may be developed.
Farnesyltransferase inhibitors for treatment of laminopathies, cellular aging and atherosclerosis
[23] Transcriptional activation analysis of oncogene function
Heterotrimeric G-Protein Effectors, 1994
ABSTRACT
Abstract LB-217: CK2 protein kinase promotes resistance to MAPK pathway inhibition
Cancer Research, 2014
Small molecule kinase inhibitors have opened potential new avenues for treating cancers dependent... more Small molecule kinase inhibitors have opened potential new avenues for treating cancers dependent on the RAS-RAF-MEK-ERK MAPK pathway, yet identification of both de novo/innate/intrinsic and acquired resistance mechanisms will be critical for the successful application of these inhibitors in the clinic. We interrogated the kinome to identify resistance mechanisms towards the novel ERK1/2-selective inhibitor SCH772984. We first utilized a kinome-focused RNAi screen to identify genes that, when silenced, sensitized KRAS-dependent pancreatic cancer cells to SCH772984. In our drug dose-response screen of 711 kinases (QIAGEN library), we used 4 independent siRNA duplexes to knock down each gene, treated at 5 different drug doses, then evaluated viability with a standard CellTiter-Glo assay. Nineteen kinases enhanced sensitivity to SCH772984 (where at least 2 siRNAs for each target decreased IC50) at least 5-fold, indicating that they could drive ERK1/2 inhibitor resistance. Among these w...
Abstract B69: Evaluation of Src-mediated signaling events in pancreatic cancer
Cancer Research, 2012
The importance of the Src tyrosine kinase in pancreatic ductal adenocarcinoma (PDAC) has been val... more The importance of the Src tyrosine kinase in pancreatic ductal adenocarcinoma (PDAC) has been validated by substantial evidence derived from preclinical genetic and pharmacological studies in PDAC cell lines and mouse models. In particular, a key role has been established for Src modulation of invasion and metastasis. Further, Src overexpression and activation has been correlated with poorer PDAC patient survival. Nevertheless, successful clinical application of Src inhibitors will requre a better understanding of the specific signaling events that contribute to Src-mediated transformation in PDAC and of appropriate biomarkers for its inhibition. Here we have applied Src-specific Rapamycin-regulated (RapR) allosteric activation of Src kinase activity, a novel technology that is highly controllable in a temporally and spatially regulated manner, to the analysis of Src-regulated functions. Most studies of Src-mediated events and Src substrates have utilized cells that have already been transformed by constitutively active Src, although mutational activation of Src is rare. We have engineered RapR-Src using the wildtype kinase that is still regulated by other signaling events. Our studies focus primarily on Src activation in the authentic aberrant signaling environment of PDAC cells that harbor the multitude of genetic alterations characteristic of patient tumors. In addition, essentially all PDAC tumors harbor mutationally active K-Ras. In model studies of K-Ras-driven pancreatic tumorigenesis, concurrent activation of Src dramatically facilitated formation of invasive PDAC and Src-mediated signaling was still required for tumor growth. Also, a recent study showed synergistic cooperation of K-Ras and Src activation in PDAC progression and growth. Therefore, to address how K-Ras may influence Src function, we also wished to evaluate RapR-Src in matched pair sets of control and KRAS-transformed human pancreatic ductal epithelial cells (HPDE). We first examined Src expression and activation by western blotting for total and phosphorylated Y416 Src, and determined that Src is strongly overexpressed and highly activated in a subset of our PDAC cell lines compared to HPDE cells. We then knocked down Src by using lentiviral delivery of short hairpin RNAs and examined the consequences to properties of transformed growth. Stable lentiviral knockdown of Src in PDAC cell lines robustly decreased cell motility and invasion, validating that endogenous Src is essential for these functions. In normal HPDE cells, we observed that activation of RapR-Src caused immediate cell spreading. In HPDE cells transformed by KRAS, activation of RapR-Src resulted not only in cell spreading but also in long filopodial protrusions terminating in highly dynamic ends. These results indicate that the application of RapR-Src technology to PDAC models can begin to provide a detailed characterization of cell behavior and identification of the signaling pathways that are specifically mediated by Src, required for the maintenance of pancreatic cancer, and inhibited by Src-directed therapeutics. CFPAC cells have been selected for further study, and results of our ongoing experiments to address these questions will be presented. Future studies will assess RapR-Src-mediated immediate and long-term phosphorylation events in Src-dependent PDAC cells and will assess resistance mechanisms in response to pharmacologic inhibition of Src. Citation Format: Leanna R. Gentry, Andrei V. Karginov, James J. Fiordalisi, Channing J. Der, Adrienne D. Cox. Evaluation of Src-mediated signaling events in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B69.
Isoprenoid modification and plasma membrane association: critical factors for ras oncogenicity
Cancer cells (Cold Spring Harbor, N.Y. : 1989), 1991
Association of ras protein with the plasma membrane is critical for its transforming activity. Th... more Association of ras protein with the plasma membrane is critical for its transforming activity. This association is promoted by a series of post-translational modifications that are signaled by the consensus C-terminal CAAX motif present in all ras proteins. The recent discovery that a 15-carbon isoprenoid (farnesyl) group, derived from an essential intermediate in cholesterol biosynthesis, is attached covalently to ras proteins has stimulated considerable interest and has suggested several important new directions for ras studies. In particular, one promising pharmacologic approach for antagonizing oncogenic ras activity in human malignancies would be to design specific inhibitors of the enzymes that catalyze ras processing and thereby interfere with ras protein association with the plasma membrane.
Supplementary Data and Materials from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
Figure S5 page2 from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
Figure S5 page2
Figure S6 page1 from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
Figure S6 page1
Figure S5 page3 from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
Figure S5 page1 from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
Figure S5 page1
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2013
Background: HDAC6 plays an important role in cell migration. Results: ERK interacts with and phos... more Background: HDAC6 plays an important role in cell migration. Results: ERK interacts with and phosphorylates HDAC6 to promote cell migration. Conclusion: ERK signaling pathway promotes cell migration, in part, through phosphorylating HDAC6. Significance: Inhibition of HDAC6 activity as well as the EGFR-Ras-Raf-MEK-ERK signaling pathway may cooperatively reduce cell migration.
Combination Therapies with CDK4/6 Inhibitors to Treat KRAS-Mutant Pancreatic Cancer
Cancer Research, Nov 8, 2022
Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step tha... more 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.
Cancer Discovery, 2020
Allele-specifi c signaling by different KRAS alleles remains poorly understood. The KRAS G12R mut... more Allele-specifi c 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-specifi c properties. We evaluated whether KRAS G12R is functionally distinct from the more common KRAS G12D -or KRAS G12V -mutant proteins (KRAS G12D/V ). We found that KRAS G12D/V but not KRAS G12R drives macropinocytosis and that MYC is essential for macropinocytosis in KRAS G12D/V -but not KRAS G12R -mutant PDAC. Surprisingly, we found that KRAS G12R 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 KRAS G12R -mutant PDAC. Finally, we determined that KRAS G12R -mutant PDAC displayed a distinct drug sensitivity profi le compared with KRAS G12D -mutant PDAC but is still responsive to the combined inhibition of ERK and autophagy. We determined that KRAS G12R is impaired in activating a key effector, p110α PI3K. As such, KRAS G12R is impaired in driving macropinocytosis. However, overexpression of PI3Kγ in PDAC compensates for this defi ciency, providing one basis for the prevalence of this otherwise rare KRAS mutant in pancreatic cancer but not other cancers.
Data from Combination Therapies with CDK4/6 Inhibitors to Treat <i>KRAS-</i>Mutant Pancreatic Cancer
Mutational loss of CDKN2A (encoding p16INK4A) tumor-suppressor function is a key genetic step tha... more 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.
The aggressive nature of pancreatic ductal adenocarcinoma (PDAC) mandates the development of impr... more 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.
Carolina Digital Repository (University of North Carolina at Chapel Hill), 2016
Recently we identified that PREX1 overexpression is critical for metastatic but not tumorigenic g... more Recently we identified that PREX1 overexpression is critical for metastatic but not tumorigenic growth in a mouse model of NRAS-driven melanoma. In addition, a PREX1 gene signature correlated with and was dependent on ERK mitogen-activated protein kinase (MAPK) activation in human melanoma cell lines. In the current study, the underlying mechanism of PREX1 overexpression in human melanoma was assessed. PREX1 protein levels were increased in melanoma tumor tissues and cell lines compared with benign nevi and normal melanocytes, respectively. Suppression of PREX1 by siRNA impaired invasion but not proliferation in vitro. PREX1-dependent invasion was attributable to PREX1-mediated activation of the small GTPase RAC1 but not the related small GTPase CDC42. Pharmacologic inhibition of ERK signaling reduced PREX1 gene transcription and additionally regulated PREX1 protein stability. This ERKdependent upregulation of PREX1 in melanoma, due to both increased gene transcription and protein stability, contrasts with the mechanisms identified in breast and prostate cancers, where PREX1 overexpression was driven by gene amplification and HDAC-mediated gene transcription, respectively. Thus, although PREX1 expression is aberrantly upregulated and regulates RAC1
Farnesylated proteins: how do they get to where they need to go, and how does location regulate their ability to control proliferation, death, transformation and aging?
The FASEB Journal, 2006
Filling in the GAPs in understanding RAS
Science, 2021
A newly identified regulator increases the efficacy of a new class of targeted anti-RAS drugs
Abstract 4660: Inhibition of p38 enhances ERK inhibitor efficacy in pancreatic ductal adenocarcinoma
Cancer Research, 2016
Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the Unite... more Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths in the United States, with a poor prognosis and limited treatment options. Oncogenic mutation of KRAS in greater than 90% of PDAC leads to aberrant activation of multiple effector pathways including the extra cellular related kinase (ERK)/mitogen activated protein kinase (MAPK) cascade. Hyperactivation of the ERK MAPK cascade has been correlated with poorer prognosis in PDAC patients. We recently showed that direct pharmacological inhibition of ERK1/2 kinases with the ERK1/2-selective inhibitor SCH772984 inhibits the growth of PDAC cell lines both in vitro and in vivo. However, much like the response to ERK/MAPK pathway inhibitors acting at upstream nodes RAF or MEK, resistance to direct inhibition at the level of ERK will also inevitably arise. We performed a novel gain-of-function “Cancer Toolkit” (CTK) genetic screen to identify mechanisms of resistance to the ERK inhibitor SCH772984 in a panel o...
Farnesyltransferase and Geranylgeranyltransferase Inhibitors
Since 1982, when mutated and oncogenic forms of ras genes were first identified in human tumor ce... more Since 1982, when mutated and oncogenic forms of ras genes were first identified in human tumor cells, their protein products have attracted considerable interest as a target for anticancer drug development. Researchers were inspired to delineate the functions of Ras proteins in normal cells and to determine how mutated Ras proteins were altered in these functions. The impressive accumulation of information about the genetics, biochemistry, biology, and structure of Ras proteins over the last 17 years has provided important clues to how anti-Ras drugs may be developed.
Farnesyltransferase inhibitors for treatment of laminopathies, cellular aging and atherosclerosis
[23] Transcriptional activation analysis of oncogene function
Heterotrimeric G-Protein Effectors, 1994
ABSTRACT
Abstract LB-217: CK2 protein kinase promotes resistance to MAPK pathway inhibition
Cancer Research, 2014
Small molecule kinase inhibitors have opened potential new avenues for treating cancers dependent... more Small molecule kinase inhibitors have opened potential new avenues for treating cancers dependent on the RAS-RAF-MEK-ERK MAPK pathway, yet identification of both de novo/innate/intrinsic and acquired resistance mechanisms will be critical for the successful application of these inhibitors in the clinic. We interrogated the kinome to identify resistance mechanisms towards the novel ERK1/2-selective inhibitor SCH772984. We first utilized a kinome-focused RNAi screen to identify genes that, when silenced, sensitized KRAS-dependent pancreatic cancer cells to SCH772984. In our drug dose-response screen of 711 kinases (QIAGEN library), we used 4 independent siRNA duplexes to knock down each gene, treated at 5 different drug doses, then evaluated viability with a standard CellTiter-Glo assay. Nineteen kinases enhanced sensitivity to SCH772984 (where at least 2 siRNAs for each target decreased IC50) at least 5-fold, indicating that they could drive ERK1/2 inhibitor resistance. Among these w...
Abstract B69: Evaluation of Src-mediated signaling events in pancreatic cancer
Cancer Research, 2012
The importance of the Src tyrosine kinase in pancreatic ductal adenocarcinoma (PDAC) has been val... more The importance of the Src tyrosine kinase in pancreatic ductal adenocarcinoma (PDAC) has been validated by substantial evidence derived from preclinical genetic and pharmacological studies in PDAC cell lines and mouse models. In particular, a key role has been established for Src modulation of invasion and metastasis. Further, Src overexpression and activation has been correlated with poorer PDAC patient survival. Nevertheless, successful clinical application of Src inhibitors will requre a better understanding of the specific signaling events that contribute to Src-mediated transformation in PDAC and of appropriate biomarkers for its inhibition. Here we have applied Src-specific Rapamycin-regulated (RapR) allosteric activation of Src kinase activity, a novel technology that is highly controllable in a temporally and spatially regulated manner, to the analysis of Src-regulated functions. Most studies of Src-mediated events and Src substrates have utilized cells that have already been transformed by constitutively active Src, although mutational activation of Src is rare. We have engineered RapR-Src using the wildtype kinase that is still regulated by other signaling events. Our studies focus primarily on Src activation in the authentic aberrant signaling environment of PDAC cells that harbor the multitude of genetic alterations characteristic of patient tumors. In addition, essentially all PDAC tumors harbor mutationally active K-Ras. In model studies of K-Ras-driven pancreatic tumorigenesis, concurrent activation of Src dramatically facilitated formation of invasive PDAC and Src-mediated signaling was still required for tumor growth. Also, a recent study showed synergistic cooperation of K-Ras and Src activation in PDAC progression and growth. Therefore, to address how K-Ras may influence Src function, we also wished to evaluate RapR-Src in matched pair sets of control and KRAS-transformed human pancreatic ductal epithelial cells (HPDE). We first examined Src expression and activation by western blotting for total and phosphorylated Y416 Src, and determined that Src is strongly overexpressed and highly activated in a subset of our PDAC cell lines compared to HPDE cells. We then knocked down Src by using lentiviral delivery of short hairpin RNAs and examined the consequences to properties of transformed growth. Stable lentiviral knockdown of Src in PDAC cell lines robustly decreased cell motility and invasion, validating that endogenous Src is essential for these functions. In normal HPDE cells, we observed that activation of RapR-Src caused immediate cell spreading. In HPDE cells transformed by KRAS, activation of RapR-Src resulted not only in cell spreading but also in long filopodial protrusions terminating in highly dynamic ends. These results indicate that the application of RapR-Src technology to PDAC models can begin to provide a detailed characterization of cell behavior and identification of the signaling pathways that are specifically mediated by Src, required for the maintenance of pancreatic cancer, and inhibited by Src-directed therapeutics. CFPAC cells have been selected for further study, and results of our ongoing experiments to address these questions will be presented. Future studies will assess RapR-Src-mediated immediate and long-term phosphorylation events in Src-dependent PDAC cells and will assess resistance mechanisms in response to pharmacologic inhibition of Src. Citation Format: Leanna R. Gentry, Andrei V. Karginov, James J. Fiordalisi, Channing J. Der, Adrienne D. Cox. Evaluation of Src-mediated signaling events in pancreatic cancer. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Progress and Challenges; Jun 18-21, 2012; Lake Tahoe, NV. Philadelphia (PA): AACR; Cancer Res 2012;72(12 Suppl):Abstract nr B69.
Isoprenoid modification and plasma membrane association: critical factors for ras oncogenicity
Cancer cells (Cold Spring Harbor, N.Y. : 1989), 1991
Association of ras protein with the plasma membrane is critical for its transforming activity. Th... more Association of ras protein with the plasma membrane is critical for its transforming activity. This association is promoted by a series of post-translational modifications that are signaled by the consensus C-terminal CAAX motif present in all ras proteins. The recent discovery that a 15-carbon isoprenoid (farnesyl) group, derived from an essential intermediate in cholesterol biosynthesis, is attached covalently to ras proteins has stimulated considerable interest and has suggested several important new directions for ras studies. In particular, one promising pharmacologic approach for antagonizing oncogenic ras activity in human malignancies would be to design specific inhibitors of the enzymes that catalyze ras processing and thereby interfere with ras protein association with the plasma membrane.