Peng, Sen

Of the thousands of genes and substrates identified in KRAS-mutant signaling networks in pancreatic ductal adenocarcinoma (PDAC), more than 200 are transcription factors, implying extensive and complex transcriptional regulation. However, we observed that genetic suppression of the transcription factor MYC alone was sufficient to phenocopy the effect of KRAS suppression in signaling, growth, and metabolic processes in PDAC cells. We determined the gene transcription changes caused by acute suppression of MYC function in KRAS -mutant PDAC cell lines and performed dependency map and pathway analyses on the affected gene sets. The expression of 1685 genes was increased upon suppression of MYC, and this gene set may comprise the bulk of the MYC-regulated genes essential for PDAC growth. In contrast, the 1325 genes whose expression was inhibited may comprise a compensatory response to oncogenic stress, mediated in part by the GTPase RHO. MYC-dependent transcriptional activity was largely ERK dependent, and almost one-third of ERK-regulated genes were also regulated by MYC in PDAC cells. Furthermore, chemical proteomic profiling revealed MYC-regulated protein kinases that can be targeted therapeutically. Together, these data provide a molecular portrait of MYC-dependent signaling that encompasses potentially exploitable mechanisms for treating PDAC.

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.

AbstractHigh‐grade gliomas (HGGs) are aggressive, treatment‐resistant, and often fatal human brain cancers. The TNF‐like weak inducer of apoptosis (TWEAK)/fibroblast growth factor‐inducible 14 (Fn14) signaling axis is involved in tissue repair after injury and constitutive signaling has been implicated in the pathogenesis of numerous solid cancers. The Fn14 gene is expressed at low levels in the normal, uninjured brain but is highly expressed in primary isocitrate dehydrogenase wild‐type and recurrent HGGs. Fn14 signaling is implicated in numerous aspects of glioma biology including brain invasion and chemotherapy resistance, but whether Fn14 overexpression can directly promote tumor malignancy has not been reported. Here, we used the replication‐competent avian sarcoma‐leukosis virus/tumor virus A system to examine the impact of Fn14 expression on glioma development and pathobiology. We found that the sole addition of Fn14 to an established oncogenic cocktail previously shown to generate proneural‐like gliomas led to the development of highly invasive and lethal brain cancer with striking biological features including extensive pseudopalisading necrosis, constitutive canonical and noncanonical NF‐κB pathway signaling, and high plasminogen activator inhibitor‐1 (PAI‐1) expression. Analyses of HGG patient datasets revealed that high human PAI‐1 gene (SERPINE1) expression correlates with shorter patient survival, and that the SERPINE1 and Fn14 (TNFRSF12A) genes are frequently co‐expressed in bulk tumor tissues, in tumor subregions, and in malignant cells residing in the tumor microenvironment. These findings provide new insights into the potential importance of Fn14 in human HGG pathobiology and designate both the NF‐κB signaling node and PAI‐1 as potential targets for therapeutic intervention.Main PointsThis work demonstrates that elevated levels of the TWEAK receptor Fn14 in tumor‐initiating, neural progenitor cells leads to the transformation of proneural‐like gliomas into more aggressive and lethal tumors that exhibit constitutive NF‐κB pathway activation and plasminogen activator inhibitor‐1 overexpression.

Abstract Background Tumor heterogeneity underlies resistance and disease progression in glioblastoma (GBM), and tumors most commonly recur adjacent to the surgical resection margins in contrast non-enhancing (NE) regions. To date, no targeted therapies have meaningfully altered overall patient survival in the up-front setting. The aim of this study was to characterize intratumoral heterogeneity in recurrent GBM using bulk samples from primary resection and recurrent samples taken from contrast-enhancing (EN) and contrast NE regions. Methods Whole exome and RNA sequencing were performed on matched bulk primary and multiple recurrent EN and NE tumor samples from 16 GBM patients who received standard of care treatment alone or in combination with investigational clinical trial regimens. Results Private mutations emerge across multi-region sampling in recurrent tumors. Genomic clonal analysis revealed increased enrichment in gene alterations regulating the G2M checkpoint, Kras signaling, Wnt signaling, and DNA repair in recurrent disease. Subsequent functional studies identified augmented PI3K/AKT transcriptional and protein activity throughout progression, validated by phospho-protein levels. Moreover, a mesenchymal transcriptional signature was observed in recurrent EN regions, which differed from the proneural signature in recurrent NE regions. Conclusions Subclonal populations observed within bulk resected primary GBMs transcriptionally evolve across tumor recurrence (EN and NE regions) and exhibit aberrant gene expression of common signaling pathways that persist despite standard or targeted therapy. Our findings provide evidence that there are both adaptive and clonally mediated dependencies of GBM on key pathways, such as the PI3K/AKT axis, for survival across recurrences.

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent tumorigenic growth. We determined the role of mutationally activated KRAS, found in ~95% of PDAC, in supporting autophagy. Surprisingly, acute KRAS suppression, which blocks proliferation, was associated with increased rather than decreased autophagic flux. Pharmacologic inhibition of ERK MAPK phenocopied the genetic silencing of KRAS and also increased autophagic flux. We speculated that the loss of ERK-driven metabolic processes may induce compensatory mechanisms to increase autophagy. Addressing a mechanism for ERK suppression-increased autophagy, we describe three mechanisms: 1) ERK inhibition-mediated increased transcription of autophagy and lysosomal genes, 2) ERK inhibitor-induced AMPK activation and suppression of mTOR signaling, and 3) ERK inhibition-facilitated decreased glycolytic flux. We then addressed whether ERK inhibition increased PDAC dependence on autophagy. Supporting this possibility, we found that cotreatment with the autophagy inhibitor chloroquine (CQ) synergistically enhanced ERK inhibitor-mediated antiproliferative activity. Similarly, genetic or pharmacologic inhibition of specific regulators of autophagy also enhanced ERK inhibitor activity. Encouraged by the synergistic relationship between ERK and autophagy inhibition, we performed a CRISPR/Cas-9 mediated genetic loss of function screen in the presence of CQ to determine additional sensitizers as well as mediators of resistance to autophagy inhibition. Top sensitizers included multiple mediators of the DNA damage response. One such sensitizing gene was CHEK1, which encodes the CHK1 serine/threonine kinase, required for checkpoint-mediated cell cycle arrest and activation of DNA repair in response to DNA damage. Accordingly, we showed that treatment with prexasertib, a clinical candidate CHK1 inhibitor, increased autophagic flux in PDAC cells and synergized with CQ to decrease PDAC cell proliferation and increase apoptosis. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. This abstract is also being presented as Poster B07. Citation Format: Kirsten L. Bryant, Jennifer E. Klomp, Ye S. Lee, Clint A. Stalnecker, Kajal R. Grover, A. Cole Edwards, Sen Peng, Mariaelena Pierobon, Emanuel F. Petricoin III, Nhan Tran, Alec C. Kimmelman, Adrienne D. Cox, Channing J. Der. Enhancing the effect of autophagy inhibition for pancreatic cancer treatment [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr PR10.

DART nanoparticles overcome critical drug delivery barriers for primary tumors and brain metastases.

Abstract Purpose: Glioblastoma is the most frequent and lethal primary brain tumor. Development of novel therapies relies on the availability of relevant preclinical models. We have established a panel of 96 glioblastoma patient-derived xenografts (PDX) and undertaken its genomic and phenotypic characterization. Experimental Design: PDXs were established from glioblastoma, IDH-wildtype (n = 93), glioblastoma, IDH-mutant (n = 2), diffuse midline glioma, H3 K27M-mutant (n = 1), and both primary (n = 60) and recurrent (n = 34) tumors. Tumor growth rates, histopathology, and treatment response were characterized. Integrated molecular profiling was performed by whole-exome sequencing (WES, n = 83), RNA-sequencing (n = 68), and genome-wide methylation profiling (n = 76). WES data from 24 patient tumors was compared with derivative models. Results: PDXs recapitulate many key phenotypic and molecular features of patient tumors. Orthotopic PDXs show characteristic tumor morphology and invasion patterns, but largely lack microvascular proliferation and necrosis. PDXs capture common and rare molecular drivers, including alterations of TERT, EGFR, PTEN, TP53, BRAF, and IDH1, most at frequencies comparable with human glioblastoma. However, PDGFRA amplification was absent. RNA-sequencing and genome-wide methylation profiling demonstrated broad representation of glioblastoma molecular subtypes. MGMT promoter methylation correlated with increased survival in response to temozolomide. WES of 24 matched patient tumors showed preservation of most genetic driver alterations, including EGFR amplification. However, in four patient–PDX pairs, driver alterations were gained or lost on engraftment, consistent with clonal selection. Conclusions: Our PDX panel captures the molecular heterogeneity of glioblastoma and recapitulates many salient genetic and phenotypic features. All models and genomic data are openly available to investigators.

Abstract Glioblastoma (GBM) is increasingly recognized as a disease involving dysfunctional cellular metabolism. GBMs are known to be complex heterogeneous systems containing multiple distinct cell populations and are supported by an aberrant network of blood vessels. A better understanding of GBM metabolism, its variation with respect to the tumor microenvironment, and resulting regional changes in chemical composition is required. This may shed light on the observed heterogeneous drug distribution, which cannot be fully described by limited or uneven disruption of the blood–brain barrier. In this work, we used mass spectrometry imaging (MSI) to map metabolites and lipids in patient-derived xenograft models of GBM. A data analysis workflow revealed that distinctive spectral signatures were detected from different regions of the intracranial tumor model. A series of long-chain acylcarnitines were identified and detected with increased intensity at the tumor edge. A 3D MSI dataset demonstrated that these molecules were observed throughout the entire tumor/normal interface and were not confined to a single plane. mRNA sequencing demonstrated that hallmark genes related to fatty acid metabolism were highly expressed in samples with higher acylcarnitine content. These data suggest that cells in the core and the edge of the tumor undergo different fatty acid metabolism, resulting in different chemical environments within the tumor. This may influence drug distribution through changes in tissue drug affinity or transport and constitute an important consideration for therapeutic strategies in the treatment of GBM. Significance: GBM tumors exhibit a metabolic gradient that should be taken into consideration when designing therapeutic strategies for treatment. See related commentary by Tan and Weljie, p. 1231

Abstract Pancreatic ductal adenocarcinoma (PDAC) cell growth is dependent on altered biochemical and cellular activities that facilitate increased metabolic dependencies. We determined the role of mutationally activated KRAS, found in ~95% of PDAC, in supporting autophagy. Surprisingly, acute KRAS suppression, which blocks proliferation, was associated with increased rather than decreased autophagic flux. Similarly, KRAS transformation of immortalized human pancreatic epithelial cells showed decreased autophagic flux. Pharmacologic inhibition of ERK MAPK phenocopied the genetic silencing of KRAS and also increased autophagic flux. Addressing a mechanism for ERK suppression increased autophagy, we found increased AMPK activity, decreased mTOR activity, and increased expression of autophagic proteins. We speculated that increased autophagy may be a compensatory mechanism caused by the loss of KRAS- and ERK-dependent metabolic processes. To address this possibility, we performed RNA-seq analyses to monitor gene expression changes in response to ERK inhibition. We observed increased transcription of genes responsible for the metabolic processes of autophagy and β-oxidation, with concurrent decreased transcription of glycolysis and mitochondrial-associated genes. These changes were validated in metabolomic analyses following either genetic silencing of KRAS or ERK inhibition. Thus, the loss of ERK-driven metabolic processes may induce compensatory mechanisms to increase autophagy. We then addressed whether ERK inhibition increased PDAC dependence on autophagy. Supporting this possibility, we found that cotreatment with the autophagy inhibitor chloroquine synergistically enhanced ERK inhibitor mediated antiproliferative activity. Similarly, genetic or pharmacologic inhibition of specific regulators of autophagy also enhanced ERK inhibitor activity. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Kirsten Bryant, Sen Peng, Andrey Tikunov, Mariaelena Pierobon, Venugopal Gunda, Garima Tomar, Pankaj Singh, Emanuel Petricoin, Jeffrey Macdonald, Nhan Tran, Alec Kimmelman, Channing Der. Inhibition of ERK MAPK signaling increases pancreatic cancer dependency on autophagy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5492.