Survival of pancreatic cancer cells lacking KRAS function
ABSTRACT: Activating mutations in the proto-oncogene KRAS are a hallmark of pancreatic ductal adenocarcinoma (PDAC), an aggressive malignancy with few effective therapeutic options. Despite efforts to develop KRAS-targeted drugs, the absolute dependence of PDAC cells on KRAS remains incompletely understood. Here we modeled complete KRAS inhibition using CRISPR/Cas-mediated genome editing. While KRAS knockout led to decreased in vitro proliferation and impaired in vivo tumorigenic growth, KRAS was dispensable in a subset of human and mouse PDAC cells. KRAS knockout cells exhibited hyperactivation of the PI3K pathway and induced sensitivity to phosphoinositide 3-kinase (PI3K) inhibitors. Mechanistically, PI3K inhibition in KRAS knockout cells led to transient mitogen-activated protein kinase (MAPK) blockade while impeding AKT-dependent 4EBP1 phosphorylation and cap-dependent translation. Furthermore, comparison of gene expression profiles of cells retaining or lacking KRAS revealed a novel functional role of KRAS in the suppression of metastasis-related genes. Accordingly, KRAS knockout gene expression signatures correlated with PDAC circulating tumor cell (CTC) signatures, and human PDAC tumors with gene expression patterns enriched in signatures from KRAS knockout cells were associated with worse survival in patients. Together, these data underscore the potential for resistance of PDAC to even the very best of KRAS inhibitors and suggest combination therapies with PI3K inhibitors as a viable strategy to circumvent resistance. Overall design: KRAS intact and knockout subclones from human 8988T and mouse A13 parental cell lines. Biologic replicates include 4 intact and 3 knockout clones from 8988T and 2 intact and 2 knockout clones from A13.
Project description:Activating Kras mutations are the hallmark genetic alterations in pancreatic ductal adenocarcinoma (PDAC) and key drivers of PDAC initiation and progression. Despite increased efforts to develop novel Kras inhibitors, the degree of Kras oncogene addiction in PDAC cells remains unclear. Here, we analyzed the requirement of endogenous Kras for the maintenance of murine PDAC cells using an inducible shRNA-based system that enables temporal control of endogenous Kras expression. Surprisingly, the majority of murine PDAC cells analyzed tolerated acute and sustained Kras knockdown by adapting to a reversible cell state, characterized by differences in cell morphology, proliferative kinetics, and tumor-initiating capacity. While significant mutational or transcriptional changes were not observed in the Kras-inhibited state, global phosphoproteomic profiling revealed alterations in cell signaling, including increased phosphorylation of focal adhesion pathway components. Accordingly, Kras-inhibited cells displayed prominent focal adhesion plaque structures, enhanced adherence properties, and increased dependency on adhesion for viability in vitro. Our analyses highlight the possibility of adaptive non-genetic and non-transcriptional mechanisms of resistance to Kras inhibition. Furthermore, we have identified candidate proteins whose signaling activities are altered in the Kras-inhibited state, providing a basis for the rational design of combination therapeutic strategies with novel Kras inhibitors. Overall design: 6 DOX-inducible shKras-expressing clones derived from 3 parental pancreatic tumor cell lines (A, B, and D; 2 clones per cell line) were treated with doxycycline (DOX) to induce Kras knockdown. One control DOX-inducible shLuc-expressing line was used as a control. RNA-sequencing was performed on long-term DOX-treated (LT DOX) or untreated (No DOX) cells. Technical replicates for specific samples are noted.
Project description:Almost all human pancreatic ductal adenocarcinomas (PDACs) are driven by oncogenic Kras and the progression of the disease is characterized by the serial appearance of certain genetic lesions. Mouse models have convincingly shown that Kras mutation induces classical PanIN lesions that can progress to PDAC in the appropriate tumor suppressor background. However, the cooperative mechanism between mutant Kras-dependent signaling surrogates and other oncogenic pathways remains to be fully elucidated in order to devise better therapeutic strategy. Mounting evidence PTEN/PI3K perturbation on PDAC tumorigenesis, we observed frequent PTEN inactivation at both genomic and histopathological levels in primary human PDAC samples. The importance of PTEN/PI3K pathway during the development of PDAC was further supported by genetic studies demonstrating that Pten deficiency in cooperation with Kras activation accelerated the formation of invasive PDAC. Mechanistically, combined Kras mutation and Pten inactivation leads to NFkB activation and subsequent induction of cytokine pathways, accompanied with strong stromal activation and immune cell infiltration. Therefore, PTEN/PI3K pathway dictates the activity of NFkB network and serves as a major surrogate during Kras-mediated pancreatic tumorigenesis. Primary pancreatic ductal epithelial cell cultures were established from 6 week old Pdx1-Cre;LSL-KrasG12D L/+ (n=3) or Pdx1-Cre;LSL-KrasG12D L/+;Pten L/+ (n=5) mice. Total RNA was collected from early passage cells.
Project description:Almost all human pancreatic ductal adenocarcinomas (PDACs) are driven by oncogenic Kras and the progression of the disease is characterized by the serial appearance of certain genetic lesions. Mouse models have convincingly shown that Kras mutation induces classical PanIN lesions that can progress to PDAC in the appropriate tumor suppressor background. However, the cooperative mechanism between mutant Kras-dependent signaling surrogates and other oncogenic pathways remains to be fully elucidated in order to devise better therapeutic strategy. Mounting evidence PTEN/PI3K perturbation on PDAC tumorigenesis, we observed frequent PTEN inactivation at both genomic and histopathological levels in primary human PDAC samples. The importance of PTEN/PI3K pathway during the development of PDAC was further supported by genetic studies demonstrating that Pten deficiency in cooperation with Kras activation accelerated the formation of invasive PDAC. Mechanistically, combined Kras mutation and Pten inactivation leads to NFkB activation and subsequent induction of cytokine pathways, accompanied with strong stromal activation and immune cell infiltration. Therefore, PTEN/PI3K pathway dictates the activity of NFkB network and serves as a major surrogate during Kras-mediated pancreatic tumorigenesis. Overall design: Primary pancreatic ductal epithelial cell cultures were established from 6 week old Pdx1-Cre;LSL-KrasG12D L/+ (n=3) or Pdx1-Cre;LSL-KrasG12D L/+;Pten L/+ (n=5) mice. Total RNA was collected from early passage cells.
Project description:Objective Oncogenic Kras-activated robust Mek/Erk signals phosphorylate to the tuberous sclerosis complex (Tsc) and deactivates mammalian target of rapamycin (mTOR) suppression in pancreatic ductal adenocarcinoma (PDAC); however, Mek and mTOR inhibitors alone have demonstrated minimal clinical antitumor activity. Design We generated transgenic mouse models in which mTOR was hyperactivated either through the Kras/Mek/Erk cascade, by loss of Pten or through Tsc1 haploinsufficiency. Primary cancer cells were isolated from mouse tumours. Oncogenic signalling was assessed in vitro and in vivo, with and without single or multiple targeted molecule inhibition. Transcriptional profiling was used to identify biomarkers predictive of the underlying pathway alterations and of therapeutic response. Results from the preclinical models were confirmed on human material. Results Reduction of Tsc1 function facilitated activation of Kras/Mek/Erk-mediated mTOR signalling, which promoted the development of metastatic PDACs. Single inhibition of mTOR or Mek elicited strong feedback activation of Erk or Akt, respectively. Only dual inhibition of Mek and PI3K reduced mTOR activity and effectively induced cancer cell apoptosis. Analysis of downstream targets demonstrated that oncogenic activity of the Mek/Erk/Tsc/mTOR axis relied on Aldh1a3 function. Moreover, in clinical PDAC samples, ALDH1A3 specifically labelled an aggressive subtype. Conclusions These results advance our understanding of Mek/Erk-driven mTOR activation and its downstream targets in PDAC, and provide a mechanistic rationale for effective therapeutic matching for Aldh1a3-positive PDACs. Overall design: We performed gene expression microarray analysis in duplicates on 11 cell lines representing three experimental models
Project description:There are currently no effective targeted therapies for KRAS mutant cancers. Therapeutic strategies that combine MEK inhibitors with agents that target apoptotic pathways may be a promising therapeutic approach. We investigated combining MEK and MDM2 inhibitors as a potential treatment strategy for KRAS mutant non-small cell lung cancers and colorectal carcinomas that harbor wild-type TP53. The combination of pimasertib (MEK inhibitor) + SAR405838 (MDM2 inhibitor) was synergistic and induced the expression of PUMA and BIM, led to apoptosis and growth inhibition in vitro, and tumor regression in vivo. Acquired resistance to the combination commonly resulted from the acquisition of TP53 mutations, conferring complete resistance to MDM2 inhibition. In contrast, resistant clones exhibited marked variability in sensitivity to MEK inhibition, which significantly impacted sensitivity to subsequent treatment with alternative MEK inhibitor-based combination therapies. These results highlight both the potential promise and limitations of combining MEK and MDM2 inhibitors for treatment of KRAS mutant NSCLC and CRC. Overall design: A427, DV-90, H1944, H358, LU-99A, SW1573 cells treated with 10 nM, 100 nM, 1000 nM pimasertib, SAR405838 or combination (3 replicates each)
Project description:Global mRNA expression profiles of murine primary PDAC cells following JQ1 or SAHA monotherapy as well as JQ1-SAHA combination therapy were collected using Affymetix mouse whole genome array (Mouse Genome 430A 2.0 Array) . Primary PDAC cells isolated from Ptf1aCre/+;Kras+/LSL-G12D;p53lox/lox (Kras;p53) mice were treated either with JQ1 (100 nM) or SAHA (2000 nM) or vehicle 10% (2-Hydroxypropyl)-β-cyclodextrin (Sigma-Aldrich) or as combination therapy with the indicated dosage for monotherapy. Total RNA isolation was performed after 6 hours of treatment. Primary PDAC cells from Ptf1aCre/+;Kras+/LSL-G12D;p53lox/lox (Kras;p53) mice treated either with JQ1, SAHA, vehicle or JQ1-SAHA combination were analyzed by global gene expression analysis.
Project description:Somatic NOTCH1 mutations are found in ~60% of T lineage acute lymphoblastic leukemias (T-ALLs). Notch1 is cleaved by γ secretase to generate activated Notch intracellular domain (NICD) proteins. The NOTCH1 mutations found in T-ALL constitutively activate Notch1 signaling by increasing NICD levels. Genetic alterations in components of the Ras/PI3 kinase (PI3K)/Akt pathway are also highly prevalent in T-ALL, and often coexist with NOTCH1 mutations. Exposing a T-ALL cell line to the PI3 kinase (PI3K) inhibitor GDC-0941 generated drug resistant clones that down-regulated NICD expression. To address the in vivo relevance of this unexpected observation, we transplanted primary wild-type (WT) and KrasG12D mutant T-ALLs into recipient mice, and treated them with GDC-0941 alone and in combination with the MEK inhibitor PD0325901 (PD901). Although many leukemias responded dramatically to these targeted agents in vivo, drug-resistant clones invariably emerged. Multiple resistant T-ALLs lost NICD expression through mechanisms that included loss of Notch1 mutations found in the parental T-ALL. These GDC-0941-resistant leukemias exhibited reduced expression of many Notch1 target genes, elevated levels of phosphorylated Akt (pAkt), and displayed cross-resistance to γ secretase inhibitors (GSIs). Consistent with these data, inhibiting Notch1 activity in T-ALL cells enhanced PI3K signaling, providing a likely mechanism for in vivo selection against clones with Notch1 pathway activation. Thus, oncogenic Notch1 mutations that promote clonal outgrowth during malignant transformation unexpectedly “switch” to become deleterious during treatment with a PI3K inhibitor. These data advance our understanding of T-ALL pathogenesis and have implications for implementing new therapeutic regimens. We analyzed 28 mouse T-ALL samples obtained after in vivo treatment with GDC-0941 alone or GDC-0941 + PD0325901. These T-ALL samples are either Kras wild type or harbor a KrasG12D mutations.
Project description:The highest frequencies of KRAS mutations occur in colorectal carcinoma (CRC) and pancreatic ductal adenocarcinoma (PDAC). Therapeutically targeting downstream pathways mediating oncogenic properties of KRAS mutant cancers is limited by an incomplete understanding of the contextual cues modulating the signaling output of activated KRAS. We performed mass spectrometry on mouse tissues expressing wild-type or mutant KRAS to determine how tissue context and genetic background modulate oncogenic signaling. Mutant KRAS dramatically altered the proteomes and phosphoproteomes of pre-neoplastic and neoplastic colons and pancreases in a largely context-specific manner. We developed an approach to humanize the mouse networks with data from human cancer and identified genes within the CRC and PDAC networks synthetically lethal with mutant KRAS. Our studies demonstrate the context-dependent plasticity of oncogenic signaling, identify non-canonical mediators of KRAS oncogenicity within the KRAS-regulated signaling network, and demonstrate how statistical integration of mouse and human datasets can reveal cross-species therapeutic insights.
Project description:Copy number profiling of 92 human lung tumors on Affymetrix 100K SNP arrays was conducted in order to assess the interaction of common genomic alterations with response to targeted anti-cancer therapeutics. Class 1 phosphatidylinositol 3' kinase (PI3K) plays a major role in cell proliferation and survival in a wide variety of human cancers. Here we investigate biomarker strategies for PI3K pathway inhibitors in non-small-cell lung cancer (NSCLC). Molecular profiling of NSCLC tumor samples showed that copy number gains in PIK3CA and total loss of PTEN protein were common in squamous cell carcinoma samples, whereas LKB1 loss and mutations in KRAS and EGFR were common in adenocarcinomas. A panel of NSCLC cell lines characterized for alterations in the PI3K pathway was screened with PI3K and dual PI3K/mTOR inhibitors to assess the preclinical predictive value of candidate biomarkers. Cell lines harboring pathway alterations (RTK activation, PI3K mutation or amplification, PTEN loss) were exquisitely sensitive to the PI3K inhibitor GDC-0941. A dual PI3K/mTOR inhibitor had broader activity across the cell line panel and in tumor xenografts. The combination of GDC-0941 with paclitaxel, erlotinib, or a MEK inhibitor had greater effects on cell viability than PI3K inhibition alone. CONCLUSIONS: Candidate biomarkers for PI3K inhibitors have predictive value in preclinical models and show histology-specific alterations in primary tumors, suggesting that distinct biomarker strategies may be required in squamous compared with non-squamous NSCLC patient populations. Lung tumors were profiled on Affymetrix GeneChip Mapping 100K Set Arrays Tumor samples were profiled for copy number without any treatment of the tumor.