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Supplementary Figure 6. Expression of AKT1E17K promotes the dysregulation of integrins.

Supplementary Materials and Methods

Supplementary Table 1. Summary of tumor formation in Dct::TVA mice. .

Pharmacological inhibition of KRAS>RAF>MEK1/2>ERK1/2 signaling has provided no clinical benefit to patients with pancreatic ductal adenocarcinoma (PDAC). Interestingly, combined inhibition of MEK1/2 (with trametinib [T]) plus autophagy (with chloroquine [CQ] or hydroxychloroquine [HCQ]) demonstrated striking anti-tumor effects in preclinical models and in a patient (Patient 1). However, not all patients respond to the T/HCQ regimen, and Patient 1 eventually developed resistant disease. Here we report that primary or acquired resistance is associated with focal DNA copy number gains encompassing c-MYC. Furthermore, ectopic expression of c-MYC in PDAC cell lines rendered them T/HCQ resistant. Interestingly, a CDK4/6 inhibitor, palbociclib (P), also induced autophagy and overrode c-MYC–mediated T/HCQ resistance, such that P/HCQ promoted regression of T/HCQ-resistant PDAC tumors with elevated c-MYC expression. Finally, P/HCQ treatment of Patient 1 resulted in a biochemical disease response. These data suggest that elevated c-MYC expression is both a marker and a mediator of T/HCQ resistance, which may be overcome by the use of P/HCQ.

Alterations in the PI3K/AKT pathway occur in up to 70% of melanomas and are associated with disease progression. The three AKT paralogs are highly conserved but data suggest they have distinct functions. Activating mutations of AKT1 and AKT3 occur in human melanoma but their role in melanoma formation and metastasis remains unclear. Using an established melanoma mouse model, we evaluated E17K, E40K, and Q79K mutations in AKT1, AKT2, and AKT3 and show that mice harboring tumors expressing AKT1E17K had the highest incidence of brain metastasis and lowest mean survival. Tumors expressing AKT1E17K displayed elevated levels of focal adhesion factors and enhanced phosphorylation of focal adhesion kinase (FAK). AKT1E17K expression in melanoma cells increased invasion and this was reduced by pharmacologic inhibition of either AKT or FAK. These data suggest that the different AKT paralogs have distinct roles in melanoma brain metastasis and that AKT and FAK may be promising therapeutic targets. Implications: This study suggests that AKT1E17K promotes melanoma brain metastasis through activation of FAK and provides a rationale for the therapeutic targeting of AKT and/or FAK to reduce melanoma metastasis.

Targeted therapies have revolutionized cancer care, but the development of resistance remains a challenge in the clinic. To identify rational targets for combination strategies, we used an established melanoma mouse model and selected for resistant tumors following genetic suppression of NRAS expression. Complete tumor regression was observed in all mice, but 40% of tumors recurred. Analysis of resistant tumors showed that the most common mechanism of resistance was overexpression and activation of receptor tyrosine kinases (RTKs). Interestingly, the most commonly overexpressed RTK was Met and inhibition of Met overcame NRAS resistance in this context. Analysis of NRAS mutant human melanoma cells showed enhanced efficacy of cytotoxicity with combined RTK and mitogen-activated protein kinase kinase inhibition. In this study, we establish the importance of adaptive RTK signaling in the escape of NRAS mutant melanoma from inhibition of RAS and provide the rationale for combined blockade of RAS and RTK signaling in this context.

Alterations in the PI3K/AKT pathway occur in up to 70% of melanomas and are associated with disease progression. The three AKT paralogs are highly conserved but data suggest they have distinct functions. Activating mutations of AKT1 and AKT3 occur in human melanoma but their role in melanoma formation and metastasis remains unclear. Using an established melanoma mouse model, we evaluated E17K, E40K, and Q79K mutations in AKT1, AKT2, and AKT3 and show that mice harboring tumors expressing AKT1(E17K) had the highest incidence of brain metastasis and lowest mean survival. Tumors expressing AKT1(E17K) displayed elevated levels of focal adhesion factors and enhanced phosphorylation of focal adhesion kinase. AKT1(E17K) expression in melanoma cells increased invasion and this was reduced by pharmacological inhibition of either AKT or FAK. These data suggest that the different AKT paralogs have distinct roles in melanoma brain metastasis and that AKT and FAK may be promising therapeutic targets.

Hyper-activation of the PI3K/AKT signaling pathway occurs in most metastatic melanomas and increased PI3K/AKT pathway activity correlates with disease progression. The serine/threonine kinase, AKT, represents a major signaling hub within the pathway and consists of three highly conserved paralogs that have both distinct and overlapping functions. Activating mutations of AKT1 and AKT3 occur in human melanoma but their role in melanoma formation and metastasis remains unclear. Using an established melanoma mouse model, we evaluated the ability of constitutively active E17K, E40K, or Q79K mutants of each AKT paralog to promote tumor progression and metastasis in the context of BRAFV600E expression and loss of Cdkn2a and Pten. Expression of AKT1E17K promoted highly aggressive melanomas that metastasized to the lungs and brain. This metastatic phenotype was not significantly observed in the case of other mutant AKT-positive tumors, suggesting that the AKT paralogs have distinct, non-overlapping roles in the development of melanoma metastases. AKT1E17K-positive tumors showed AKT1E17K-dependent up-regulation of multiple focal adhesion (FA) factors, which are key components of focal adhesions and established stimulators of cell motility, as well as phosphorylation of focal adhesion kinase (FAK). Ectopic expression of AKT1E17K in non-metastatic melanoma cells increased cell invasion, a phenotype abrogated by pharmacological inhibition of AKT or FAK. These findings strongly suggest that one mechanism by which AKT1 promotes melanoma metastasis is through regulation and activation of proteins involved in focal adhesions. This has important implications for the development of therapeutic strategies aimed at preventing or treating disseminated disease. Citation Format: David A. Kircher, Kirby A. Trombetti, Mark R. Silvis, Gennie L. Parkman, Grant M. Fischer, Stephanie N. Angel, Christopher M. Stehn, Sean C. Strain, Allie H. Grossmann, Keith L. Duffy, Martin McMahon, Michael A. Davies, Michelle C. Mendoza, Matthew W. VanBrocklin, Sheri L. Holmen. AKT1E17K activates focal adhesion kinase and promotes melanoma brain metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2736.

Pancreatic ductal adenocarcinoma (PDAC) is a recalcitrant disease responsible for ~43,000 deaths in the USA in 2017. Despite an advanced understanding of the genetics, biochemistry, and biology of pancreatic cancer, there is no effective pathway-targeted therapy for PDAC such that the standard-of-care treatment for most patients remains conventional cytotoxic chemotherapy. Although the clinical picture remains grim, progress has been made in understanding how alterations in tumor suppressors and proto-oncogenes contribute to PDAC initiation and progression. Whereas initiating mutations in KRAS promote the growth of premalignant pancreatic intraepithelial neoplasia, progression to cancer malignancy requires cooperating alterations of tumor suppressors such as TP53, CDKN2A, and/or SMAD4. Downstream of KRAS oncoproteins, the RAF®MEK®ERK MAP kinase signaling pathway plays a central role in the genesis of PDAC. However, to date, pharmacologic inhibition of this pathway has demonstrated little clinical benefit in PDAC patients. We have previously shown that combined inhibition of MEK1/2 and autophagy with chloroquine/hydroxychloroquine (CQ/HCQ) displays synergistic antiproliferative effects against PDAC cell lines in vitro, in xenografted patient-derived PDAC tumors, and treatment of a pancreatic cancer patient with trametinib/HCQ resulted in dramatic tumor regression. Unfortunately, this patient did go on to progress on trametinib/HCQ therapy and a second patient who was treated with the combination of trametinib and hydroxychloroquine was refractory to treatment. Strikingly, the only genetic difference between these two patients’ tumors at the start of trametinib/HCQ treatment was an MYC amplification, and our first patient demonstrated an acquired MYC amplification upon development of resistance. Here we demonstrate in preclinical models that overexpression of MYC in Mia-PaCa2 and PDX220 cells lines resulted in both refractory and resistant tumors. We have identified that the CDK4/6 inhibitor, palbociclib, also induces protective autophagy, and treating xenografted Mia-PaCa2 tumors overexpressing MYC with palbociclib/CQ resulted in striking tumor regression. We therefore tested the combination of palbociclib/CQ on PDX derived from our first patient upon resistance to trametinib/HCQ, which demonstrated regression and suppression of tumors when treated with palbociclib/CQ, but not palbociclib alone or trametinib/CQ. Finally, we treated our first patient with the combination of palbociclib/HCQ and saw a dramatic decline in CA19-9. These data suggest that MYC amplification may be a negative biomarker for response and also a resistance mechanism that can be overcome with combined inhibition of CDK4/6 and autophagy. Citation Format: Conan Kinsey, Dilru Silva, Mark Silvis, Sophia Schuman, Kajsa Affolter, Martin McMahon. Myc amplification is a negative biomarker and a resistance mechanism to trametinib/HCQ treatment, but can be overcome by combined palbociclib/HCQ 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 C30.

The amount of food waste generated by the U.S. is staggering, both expensive in economic cost and environmental side effects. Surplus food, which could be used to feed people facing food insecurity, is instead discarded and placed in landfills. Institutions, universities, and non-profits have noticed this issue and are beginning to take action to reduce surplus food waste, typically by redirecting it to food banks and other organizations or having students transport or eat the food. These approaches present challenges such as transportation, volunteer availability, and lack of prioritization of those in need. In this paper, we introduce PittGrub, a notification system to intelligently select users to invite to events that have leftover food. PittGrub was invented to help reduce food waste at the University of Pittsburgh. We use reinforcement learning to determine how many notifications to send out and a valuation model to determine whom to prioritize in the notifications. Our goal is to produce a system that prioritizes feeding students in need while simultaneously eliminating food waste and maintaining a fair distribution of notifications. As far as we are aware, PittGrub is unique in its approach to eliminating surplus food waste while striving for social good. We compare our proposed techniques to multiple baselines on simulated datasets to demonstrate effectiveness. Experimental results among various algorithms show promise in eliminating food waste while helping those facing food insecurity and treating users fairly. Our prototype is currently in beta and coming soon to the Apple App Store.

SUMMARY Isocitrate dehydrogenase 1 (IDH1) is the most commonly mutated gene in grade II–III glioma and secondary glioblastoma (GBM). A causal role for IDH1R132H in gliomagenesis has been proposed, but functional validation in vivo has not been demonstrated. In this study, we assessed the role of IDH1R132H in glioma development in the context of clinically relevant cooperating genetic alterations in vitro and in vivo. Immortal astrocytes expressing IDH1R132H exhibited elevated (R)-2-hydroxyglutarate levels, reduced NADPH, increased proliferation, and anchorage-independent growth. Although not sufficient on its own, IDH1R132H cooperated with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote glioma development in vivo. These tumors resembled pro-neural human mutant IDH1 GBM genetically, histologically, and functionally. Our findings support the hypothesis that IDH1R132H promotes glioma development. This model enhances our understanding of the biology of IDH1R132H-driven gliomas and facilitates testing of therapeutic strategies designed to combat this deadly disease., In Brief Philip et al. show that mutant IDH1 cooperates with PDGFA and loss of Cdkn2a, Atrx, and Pten to promote gliomagenesis in vivo in a mouse model of glioma. These tumors resemble proneural human mutant IDH1 glioblastoma and exhibit enhanced sensitivity to PARP inhibition in combination with chemotherapy.

SummaryThe significance of ERG in human prostate cancer is unclear because mouse prostate is resistant to ERG-mediated transformation. We determined that ERG activates the transcriptional program regulated by YAP1 of the Hippo signaling pathway and found that prostate-specific activation of either ERG or YAP1 in mice induces similar transcriptional changes and results in age-related prostate tumors. ERG binds to chromatin regions occupied by TEAD/YAP1 and transactivates Hippo target genes. In addition, in human luminal-type prostate cancer cells, ERG binds to the promoter of YAP1 and is necessary for YAP1 expression. These results provide direct genetic evidence of a causal role for ERG in prostate cancer and reveal a connection between ERG and the Hippo signaling pathway.

The development of brain metastases in patients with advanced stage melanoma is common, but the molecular mechanisms responsible for their development are poorly understood. Melanoma brain metastases cause significant morbidity and mortality and confer a poor prognosis; traditional therapies including whole brain radiation, stereotactic radiotherapy, or chemotherapy yield only modest increases in overall survival (OS) for these patients. While recently approved therapies have significantly improved OS in melanoma patients, only a small number of studies have investigated their efficacy in patients with brain metastases. Preliminary data suggest that some responses have been observed in intracranial lesions, which has sparked new clinical trials designed to evaluate the efficacy in melanoma patients with brain metastases. Simultaneously, recent advances in our understanding of the mechanisms of melanoma cell dissemination to the brain have revealed novel and potentially therapeutic targets. In this review, we provide an overview of newly discovered mechanisms of melanoma spread to the brain, discuss preclinical models that are being used to further our understanding of this deadly disease and provide an update of the current clinical trials for melanoma patients with brain metastases.

Cell–cell adhesion protein αE-catenin inhibits skin squamous cell carcinoma (SCC) development; however, the mechanisms responsible for this function are not completely understood. We report here that αE-catenin inhibits β4 integrin-mediated activation of SRC tyrosine kinase. SRC is the first discovered oncogene, but the protein substrate critical for SRC-mediated transformation has not been identified. We found that YAP1, the pivotal effector of the Hippo signaling pathway, is a direct SRC phosphorylation target, and YAP1 phosphorylation at three sites in its transcription activation domain is necessary for SRC–YAP1-mediated transformation. We uncovered a marked increase in this YAP1 phosphorylation in human and mouse SCC tumors with low/negative expression of αE-catenin. We demonstrate that the tumor suppressor function of αE-catenin involves negative regulation of the β4 integrin–SRC signaling pathway and that SRC-mediated phosphorylation and activation of YAP1 are an alternative to the canonical Hippo signaling pathway that directly connect oncogenic tyrosine kinase signaling with YAP1.

Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na+) transport. The greatest reduction in Na+transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na+transport in mpkCCD cells.

Oncogene-induced senescence, e.g., in melanocytic nevi, terminates the expansion of pre-malignant cells via transcriptional silencing of proliferation-related genes due to decoration of their promoters with repressive tri-methylated histone H3 lysine 9 (H3K9) marks. We show here that structurally distinct H3K9-active demethylases—the lysine-specific demethylase-1 (LSD1) and several Jumonji C domain-containing moieties (such as JMJD2C)—disable senescence and permit Ras/Braf-evoked transformation. In mouse and zebrafish models, enforced LSD1 or JMJD2C expression promoted Braf-V600E-driven melanomagenesis. A large subset of established melanoma cell lines and primary human melanoma samples presented with a collective upregulation of related and unrelated H3K9 demethylase activities, whose targeted inhibition restored senescence, even in Braf inhibitor-resistant melanomas, evoked secondary immune effects and controlled tumor growth in vivo.

The octameric exocyst complex is associated with the junctional complex and recycling endosomes and is proposed to selectively tether cargo vesicles directed toward the basolateral surface of polarized Madin-Darby canine kidney (MDCK) cells. We observed that the exocyst subunits Sec6, Sec8, and Exo70 were localized to early endosomes, transferrin-positive common recycling endosomes, and Rab11a-positive apical recycling endosomes of polarized MDCK cells. Consistent with its localization to multiple populations of endosomes, addition of function-blocking Sec8 antibodies to streptolysin-O–permeabilized cells revealed exocyst requirements for several endocytic pathways including basolateral recycling, apical recycling, and basolateral-to-apical transcytosis. The latter was selectively dependent on interactions between the small GTPase Rab11a and Sec15A and was inhibited by expression of the C-terminus of Sec15A or down-regulation of Sec15A expression using shRNA. These results indicate that the exocyst complex may be a multipurpose regulator of endocytic traffic directed toward both poles of polarized epithelial cells and that transcytotic traffic is likely to require Rab11a-dependent recruitment and modulation of exocyst function, likely through interactions with Sec15A.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl-selective anion channel expressed in epithelial tissues. Mutations in CFTR lead to the debilitating genetic disease cystic fibrosis (CF). Within each epithelial cell, CFTR interacts with a large number of transient macromolecular complexes, many of which are involved in the trafficking and targeting of CFTR. Understanding how these complexes regulate the trafficking and fate of CFTR, provides a singular insight not only into the patho-physiology of cystic fibrosis, but also provides potential drug targets to help cure this debilitating disease.

The Hippo pathway regulates contact inhibition of cell proliferation and, ultimately, organ size in diverse multicellular organisms. Inactivation of the Hippo pathway promotes nuclear localization of the transcriptional coactivator Yap1, a Hippo pathway effector, and can cause cancer. Here, we show that deletion of αE (α epithelial) catenin in the hair follicle stem cell compartment resulted in the development of skin squamous cell carcinoma in mice. Tumor formation was accelerated by simultaneous deletion of αE-catenin and the tumor suppressor-encoding gene p53. A small interfering RNA screen revealed a functional connection between αE-catenin and Yap1. By interacting with Yap1, αE-catenin promoted its cytoplasmic localization, and Yap1 showed constitutive nuclear localization in αE-catenin-null cells. We also found an inverse correlation between αE-catenin abundance and Yap1 activation in human squamous cell carcinoma tumors. These findings identify αE-catenin as a tumor suppressor that inhibits Yap1 activity and sequesters it in the cytoplasm.

This Podcast features conversations with authors of articles related to Hippo signaling that appear in Science and Science Signaling . James Martin discusses his group’s finding that Hippo signaling regulates the size of the mouse heart by antagonizing Wnt signaling through a mechanism that involves cooperation between the transcription factors Yap and β-catenin, as described in a Research Report published in the 22 April 2011 issue of Science . Valeri Vasioukhin and Mark Silvis discuss their findings related to Hippo signaling in tumorigenesis, as reported in the 24 May 2011 issue of Science Signaling . They report that the adhesion protein αE-catenin acts as a tumor suppressor by regulating the localization and activity of the transcriptional coactivator Yap, which is also regulated by Hippo signaling.

The cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP/PKA-activated anion channel, undergoes efficient apical recycling in polarized epithelia. The regulatory mechanisms underlying CFTR recycling are understood poorly, yet this process is required for proper channel copy number at the apical membrane, and it is defective in the common CFTR mutant, ΔF508. Herein, we investigated the function of Rab11 isoforms in regulating CFTR trafficking in T84 cells, a colonic epithelial line that expresses CFTR endogenously. Western blotting of immunoisolated Rab11a or Rab11b vesicles revealed localization of endogenous CFTR within both compartments. CFTR function assays performed on T84 cells expressing the Rab11a or Rab11b GDP-locked S25N mutants demonstrated that only the Rab11b mutant inhibited 80% of the cAMP-activated halide efflux and that only the constitutively active Rab11b-Q70L increased the rate constant for stimulated halide efflux. Similarly, RNAi knockdown of Rab11b, but not Rab11a, reduced by 50% the CFTR-mediated anion conductance response. In polarized T84 monolayers, adenoviral expression of Rab11b-S25N resulted in a 70% inhibition of forskolin-stimulated transepithelial anion secretion and a 50% decrease in apical membrane CFTR as assessed by cell surface biotinylation. Biotin protection assays revealed a robust inhibition of CFTR recycling in polarized T84 cells expressing Rab11b-S25N, demonstrating the selective requirement for the Rab11b isoform. This is the first report detailing apical CFTR recycling in a native expression system and to demonstrate that Rab11b regulates apical recycling in polarized epithelial cells.

Cystic fibrosis is a common lethal genetic disease among Caucasians. The cystic fibrosis gene encodes a cyclic adenosine monophosphate-activated chloride channel (cystic fibrosis transmembrane conductance regulator (CFTR)) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. Mutations in CFTR fall into two broad categories; those that affect protein biosynthesis/stability and traffic to the cell surface and those that cause altered channel kinetics in proteins that reach the cell surface. Here we report a novel mechanism by which mutations in CFTR give rise to disease. N287Y, a mutation within an intracellular loop of CFTR, increases channel endocytosis from the cell surface without affecting either biosynthesis or channel gating. The sole consequence of this novel mutation is to generate a novel tyrosine-based endocytic sequence within an intracellular loop in CFTR leading to increased removal from the cell surface and a reduction in the steady-state level of CFTR at the cell surface.