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Purpose: In preclinical models, glucocorticoid receptor (GR) signaling drives resistance to taxane chemotherapy in multiple solid tumors via upregulation of antiapoptotic pathways. ORIC-101 is a potent and selective GR antagonist that was investigated in combination with taxane chemotherapy as an anticancer regimen preclinically and in a phase 1 clinical trial., Patients and Methods: The ability of ORIC-101 to reverse taxane resistance was assessed in cell lines and xenograft models, and a phase 1 study (NCT03928314) was conducted in patients with advanced solid tumors to determine the dose, safety, and antitumor activity of ORIC-101 with nab-paclitaxel., Results: ORIC-101 reversed chemoprotection induced by glucocorticoids in vitro and achieved tumor regressions when combined with paclitaxel in both taxane-naïve and -resistant xenograft models. In the phase 1 study, 21 patients were treated in dose escalation and 62 patients were treated in dose expansion. All patients in dose expansion had previously progressed on a taxane-based regimen. In dose escalation, five objective responses were observed. A preplanned futility analysis in dose expansion showed a 3.2% (95% confidence interval, 0.4-11.2) objective response rate with a median progression-free survival of 2 months (95% confidence interval, 1.8-2.8) across all four cohorts, leading to study termination. Pharmacodynamic analysis of tissue and plasma showed GR pathway downregulation in most patients in cycle 1., Conclusions: ORIC-101 with nab-paclitaxel showed limited clinical activity in taxane-resistant solid tumors. Despite clear inhibition of GR pathway signaling, the insufficient clinical signal underscores the challenges of targeting a single resistance pathway when multiple mechanisms of resistance may be in play., Significance: Glucocorticoid receptor (GR) upregulation is a mechanism of resistance to taxane chemotherapy in preclinical cancer models. ORIC-101 is a small molecule GR inhibitor. In this phase 1 study, ORIC-101 plus nab-paclitaxel did not show meaningful clinical benefit in patients who previously progressed on taxanes despite successful GR pathway downregulation., (©2024 The Authors; Published by the American Association for Cancer Research.)

Purpose: Increased glucocorticoid receptor (GR) signaling is a proposed compensatory mechanism of resistance to androgen receptor (AR) inhibition in metastatic castration-resistant prostate cancer (mCRPC). ORIC-101 is a potent and selective orally-bioavailable GR antagonist., Patients and Methods: Safety, pharmacokinetic/pharmacodynamic, and antitumor activity of ORIC-101 in combination with enzalutamide were studied in patients with mCRPC progressing on enzalutamide. ORIC-101 doses ranging from 80 to 240 mg once daily were tested in combination with enzalutamide 160 mg once daily. Pharmacokinetics/pharmacodynamics was assessed after a single dose and at steady state. Disease control rate (DCR) at 12 weeks was evaluated at the recommended phase 2 dose (RP2D)., Results: A total of 41 patients were enrolled. There were no dose-limiting toxicities and the RP2D was selected as 240 mg of ORIC-101 and 160 mg of enzalutamide daily. At the RP2D, the most common treatment-related adverse events were fatigue (38.7%), nausea (29.0%), decreased appetite (19.4%), and constipation (12.9%). Pharmacokinetic/pharmacodynamic data confirmed ORIC-101 achieved exposures necessary for GR target engagement. Overall, for 31 patients treated at the RP2D, there was insufficient clinical benefit based on DCR (25.8%; 80% confidence interval: 15.65-38.52) which did not meet the prespecified target rate, leading to termination of the study. Exploratory subgroup analyses based on baseline GR expression, presence of AR resistance variants, and molecular features of aggressive variant prostate cancer suggested possible benefit in patients with high GR expression and no other resistance markers, although this would require confirmation., Conclusions: Although the combination of ORIC-101 and enzalutamide demonstrated an acceptable tolerability profile, GR target inhibition with ORIC-101 did not produce clinical benefit in men with metastatic prostate cancer resistant to enzalutamide., (©2024 American Association for Cancer Research.)

The FOXA1 pioneer factor is an essential mediator of steroid receptor function in multiple hormone-dependent cancers, including breast and prostate cancers, enabling nuclear receptors such as estrogen receptor (ER) and androgen receptor (AR) to activate lineage-specific growth programs. FOXA1 is also highly expressed in non-small cell lung cancer (NSCLC), but whether and how it regulates tumor growth in this context is not known. Analyzing data from loss-of-function screens, we identified a subset of NSCLC tumor lines where proliferation is FOXA1 dependent. Using rapid immunoprecipitation and mass spectrometry of endogenous protein, we identified chromatin-localized interactions between FOXA1 and glucocorticoid receptor (GR) in these tumor cells. Knockdown of GR inhibited proliferation of FOXA1-dependent, but not FOXA1-independent NSCLC cells. In these FOXA1-dependent models, FOXA1 and GR cooperate to regulate gene targets involved in EGF signaling and G 1 -S cell-cycle progression. To investigate the therapeutic potential for targeting this complex, we examined the effects of highly selective inhibitors of the GR ligand-binding pocket and found that GR antagonism with ORIC-101 suppressed FOXA1/GR target expression, activation of EGF signaling, entry into the S-phase, and attendant proliferation in vitro and in vivo . Taken together, our findings point to a subset of NSCLCs harboring a dependence on the FOXA1/GR growth program and provide rationale for its therapeutic targeting., Significance: NSCLC is the leading cause of cancer deaths worldwide. There is a need to identify novel druggable dependencies. We identify a subset of NSCLCs dependent on FOXA1-GR and sensitive to GR antagonism., (© 2023 The Authors; Published by the American Association for Cancer Research.)

Here, we have developed an automated image processing algorithm for segmenting lungs and individual lung tumors in in vivo micro-computed tomography (micro-CT) scans of mouse models of non-small cell lung cancer and lung fibrosis. Over 3000 scans acquired across multiple studies were used to train/validate a 3D U-net lung segmentation model and a Support Vector Machine (SVM) classifier to segment individual lung tumors. The U-net lung segmentation algorithm can be used to estimate changes in soft tissue volume within lungs (primarily tumors and blood vessels), whereas the trained SVM is able to discriminate between tumors and blood vessels and identify individual tumors. The trained segmentation algorithms (1) significantly reduce time required for lung and tumor segmentation, (2) reduce bias and error associated with manual image segmentation, and (3) facilitate identification of individual lung tumors and objective assessment of changes in lung and individual tumor volumes under different experimental conditions., Competing Interests: All authors are current or former employees of Genentech. G.Z.F, K.H.B., S.J., A.L., J.E.L., J.H.C., M.R.J., and R.A.D.C. are current or past stockholders in Genentech, a Roche company., (© 2022 The Author(s).)

Lung development, integrity and repair rely on precise Wnt signaling, which is corrupted in diverse diseases, including cancer. Here, we discover that EHMT2 methyltransferase regulates Wnt signaling in the lung by controlling the transcriptional activity of chromatin-bound β-catenin, through a non-histone substrate in mouse lung. Inhibition of EHMT2 induces transcriptional, morphologic, and molecular changes consistent with alveolar type 2 (AT2) lineage commitment. Mechanistically, EHMT2 activity functions to support regenerative properties of Kras G12D tumors and normal AT2 cells-the predominant cell of origin of this cancer. Consequently, EHMT2 inhibition prevents Kras G12D lung adenocarcinoma (LUAD) tumor formation and propagation and disrupts normal AT2 cell differentiation. Consistent with these findings, low gene EHMT2 expression in human LUAD correlates with enhanced AT2 gene expression and improved prognosis. These data reveal EHMT2 as a critical regulator of Wnt signaling, implicating Ehmt2 as a potential target in lung cancer and other AT2-mediated lung pathologies., Competing Interests: AP, AD, AL, EJ, MJ was an employee of Genentech when the work was performed and may hold stock, XW, MH, CP, ZM, AK, OF, JE, JH, BH, JG holds shares in the company, (© 2022, Pribluda et al.)

KRAS, which is mutated in ∼30% of all cancers, activates the RAF-MEK-ERK signaling cascade. CRAF is required for growth of KRAS mutant lung tumors, but the requirement for CRAF kinase activity is unknown. Here, we show that subsets of KRAS mutant tumors are dependent on CRAF for growth. Kinase-dead but not dimer-defective CRAF rescues growth inhibition, suggesting that dimerization but not kinase activity is required. Quantitative proteomics demonstrates increased levels of CRAF:ARAF dimers in KRAS mutant cells, and depletion of both CRAF and ARAF rescues the CRAF-loss phenotype. Mechanistically, CRAF depletion causes sustained ERK activation and induction of cell-cycle arrest, while treatment with low-dose MEK or ERK inhibitor rescues the CRAF-loss phenotype. Our studies highlight the role of CRAF in regulating MAPK signal intensity to promote tumorigenesis downstream of mutant KRAS and suggest that disrupting CRAF dimerization or degrading CRAF may have therapeutic benefit., Competing Interests: Declaration of interests All authors of the paper are or were employees of Genentech/Roche during the execution of this work. A patent related to this study has been submitted: US Patent Application No. 63/108009, Recombinant Proteins for Quantification of Protein Levels., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Recent progress in understanding the molecular basis of cellular processes, identification of promising therapeutic targets and evolution of the regulatory landscape makes this an exciting and unprecedented time to be in the field of oncology drug development. However, high costs, long development timelines and steep rates of attrition continue to afflict the drug development process. Lack of predictive preclinical models is considered one of the key reasons for the high rate of attrition in oncology. Generating meaningful and predictive results preclinically requires a firm grasp of the relevant biological questions and alignment of the model systems that mirror the patient context. In doing so, the ability to conduct both forward translation, the process of implementing basic research discoveries into practice, as well as reverse translation, the process of elucidating the mechanistic basis of clinical observations, greatly enhances our ability to develop effective anticancer treatments. In this Review, we outline issues in preclinical-to-clinical translatability of molecularly targeted cancer therapies, present concepts and examples of successful reverse translation, and highlight the need to better align tumour biology in patients with preclinical model systems including tracking of strengths and weaknesses of preclinical models throughout programme development., (© 2021. Springer Nature Limited.)

Breast cancer is now globally the most frequent cancer and leading cause of women's death. Two thirds of breast cancers express the luminal estrogen receptor-positive (ERα + ) phenotype that is initially responsive to antihormonal therapies, but drug resistance emerges. A major barrier to the understanding of the ERα-pathway biology and therapeutic discoveries is the restricted repertoire of luminal ERα + breast cancer models. The ERα + phenotype is not stable in cultured cells for reasons not fully understood. We examine 400 patient-derived breast epithelial and breast cancer explant cultures (PDECs) grown in various three-dimensional matrix scaffolds, finding that ERα is primarily regulated by the matrix stiffness. Matrix stiffness upregulates the ERα signaling via stress-mediated p38 activation and H3K27me3-mediated epigenetic regulation. The finding that the matrix stiffness is a central cue to the ERα phenotype reveals a mechanobiological component in breast tissue hormonal signaling and enables the development of novel therapeutic interventions. Subject terms: ER-positive (ER + ), breast cancer, ex vivo model, preclinical model, PDEC, stiffness, p38 SAPK., (© 2021. The Author(s).)

Purpose: Lung adenocarcinomas comprise the largest fraction of non-small cell lung cancer, which is the leading cause of cancer-related deaths. Seventy-five percent of adenocarcinomas lack targeted therapies because of scarcity of druggable drivers. Here, we classified tumors on the basis of signaling similarities and discovered subgroups within this unmet patient population., Experimental Design: We leveraged transcriptional data from >800 early- and advanced-stage patients., Results: We identified three robust subtypes dubbed mucinous, proliferative, and mesenchymal with respective pathway phenotypes. These transcriptional states lack discrete and causative mutational etiology as evidenced by similarly distributed oncogenic drivers, including KRAS and EGFR . The subtypes capture heterogeneity even among tumors lacking known oncogenic drivers. Paired multi-regional intratumoral biopsies demonstrated unified subtypes despite divergently evolved prooncogenic mutations, indicating subtype stability during selective pressure. Heterogeneity among in vitro and in vivo preclinical models is expounded by the human lung adenocarcinoma subtypes and can be leveraged to discover subtype-specific vulnerabilities. As proof of concept, we identified differential subtype response to MEK pathway inhibition in a chemical library screen of 89 lung cancer cell lines, which reproduces across model systems and a clinical trial., Conclusions: Our findings support forward translational relevance of transcriptional subtypes, where further exploration therein may improve lung adenocarcinoma treatment. See related commentary by Skoulidis, p. 913 ., (©2020 American Association for Cancer Research.)

The adenosinergic pathway represents an attractive new therapeutic approach in cancer immunotherapy. In this pathway, ecto-5-nucleotidase CD73 has the unique function of regulating production of immunosuppressive adenosine (ADO) through the hydrolysis of AMP. CD73 is overexpressed in many cancers, resulting in elevated levels of ADO that correspond to poor patient prognosis. Therefore, reducing the level of ADO via inhibition of CD73 is a potential strategy for treating cancers. Based on the binding mode of adenosine 5'-(α,β-methylene)diphosphate (AOPCP) with human CD73, we designed a series of novel monophosphonate small-molecule CD73 inhibitors. Among them, OP-5244 ( 35 ) proved to be a highly potent and orally bioavailable CD73 inhibitor. In preclinical studies, 35 completely inhibited ADO production in both human cancer cells and CD8 + T cells. Furthermore, 35 lowered the ratio of ADO/AMP significantly and reversed immunosuppression in mouse models, indicating its potential as an in vivo tool compound for further development.

Systemic cytokine release and on-target/off-tumor toxicity to normal tissues are the main adverse effects limiting the clinical utility of T cell-redirecting therapies. This study was designed to determine how binding affinity for CD3 and tumor target HER2 impact the efficacy and nonclinical safety of anti-HER2/CD3 T cell-dependent antibodies (TDBs). Affinity was found to be a major determinant for the overall tolerability. Higher affinity for CD3 associated with rapidly elevated peripheral cytokine concentrations, weight loss in mice, and poor tolerability in cynomolgus monkeys. A TDB with lower CD3 affinity was better tolerated in cynomolgus monkeys compared with a higher CD3-affinity TDB. In contrast to tolerability, T cell binding affinity had only limited impact on in vitro and in vivo antitumor activity. High affinity for HER2 was critical for the tumor-killing activity of anti-HER2/CD3 TDBs, but higher HER2 affinity also associated with a more severe toxicity profile, including cytokine release and damage to HER2-expressing tissues. The tolerability of the anti-HER2/CD3 was improved by implementing a dose-fractionation strategy. Fine-tuning the affinities for both the tumor target and CD3 is likely a valuable strategy for achieving maximal therapeutic index of CD3 bispecific antibodies.

With only a fraction of patients responding to cancer immunotherapy, a better understanding of the entire tumor microenvironment is needed. Using single-cell transcriptomics, we chart the fibroblastic landscape during pancreatic ductal adenocarcinoma (PDAC) progression in animal models. We identify a population of carcinoma-associated fibroblasts (CAF) that are programmed by TGFβ and express the leucine-rich repeat containing 15 (LRRC15) protein. These LRRC15 + CAFs surround tumor islets and are absent from normal pancreatic tissue. The presence of LRRC15 + CAFs in human patients was confirmed in >80,000 single cells from 22 patients with PDAC as well as by using IHC on samples from 70 patients. Furthermore, immunotherapy clinical trials comprising more than 600 patients across six cancer types revealed elevated levels of the LRRC15 + CAF signature correlated with poor response to anti-PD-L1 therapy. This work has important implications for targeting nonimmune elements of the tumor microenvironment to boost responses of patients with cancer to immune checkpoint blockade therapy. SIGNIFICANCE: This study describes the single-cell landscape of CAFs in pancreatic cancer during in vivo tumor evolution. A TGFβ-driven, LRRC15 + CAF lineage is associated with poor outcome in immunotherapy trial data comprising multiple solid-tumor entities and represents a target for combinatorial therapy. This article is highlighted in the In This Issue feature, p. 161 ., (©2019 American Association for Cancer Research.)

The kinase RIP1 acts in multiple signaling pathways to regulate inflammatory responses and it can trigger both apoptosis and necroptosis. Its kinase activity has been implicated in a range of inflammatory, neurodegenerative, and oncogenic diseases. Here, we explore the effect of inhibiting RIP1 genetically, using knock-in mice that express catalytically inactive RIP1 D138N, or pharmacologically, using the murine-potent inhibitor GNE684. Inhibition of RIP1 reduced collagen antibody-induced arthritis, and prevented skin inflammation caused by mutation of Sharpin, or colitis caused by deletion of Nemo from intestinal epithelial cells. Conversely, inhibition of RIP1 had no effect on tumor growth or survival in pancreatic tumor models driven by mutant Kras, nor did it reduce lung metastases in a B16 melanoma model. Collectively, our data emphasize a role for the kinase activity of RIP1 in certain inflammatory disease models, but question its relevance to tumor progression and metastases.

Emerging research suggests that multiple tumor compartments can influence treatment responsiveness and relapse, yet the search for therapeutic resistance mechanisms remains largely focused on acquired genomic alterations in cancer cells. Here we show how treatment-induced changes occur in multiple tumor compartments during tumor relapse and can reduce benefit of follow-on therapies. By using serial biopsies, next-generation sequencing, and single-cell transcriptomics, we tracked the evolution of multiple cellular compartments within individual lesions during first-line treatment response, relapse, and second-line therapeutic interventions in an autochthonous model of melanoma. We discovered that although treatment-relapsed tumors remained genetically stable, they converged on a shared resistance phenotype characterized by dramatic changes in tumor cell differentiation state, immune infiltration, and extracellular matrix (ECM) composition. Similar alterations in tumor cell differentiation were also observed in more than half of our treatment-relapsed patient tumors. Tumor cell-state changes were coincident with ECM remodeling and increased tumor stiffness, which alone was sufficient to alter tumor cell fate and reduce treatment responses in melanoma cell lines in vitro. Despite the absence of acquired mutations in the targeted pathway, resistant tumors showed significantly decreased responsiveness to second-line therapy intervention within the same pathway. The ability to preclinically model relapse and refractory settings-while capturing dynamics within and crosstalk between all relevant tumor compartments-provides a unique opportunity to better design and sequence appropriate clinical interventions.

T cell-retargeting therapies have transformed the therapeutic landscape of oncology. Regardless of the modality, T cell activating therapies are commonly accompanied by systemic cytokine release, which can progress to deadly cytokine release syndrome (CRS). Because of incomplete mechanistic understanding of the relationship between T cell activation and systemic cytokine release, optimal toxicity management that retains full therapeutic potential remains unclear. Here, we report the cell type-specific cellular mechanisms that link CD3 bispecific antibody-mediated killing to toxic cytokine release. The immunologic cascade is initiated by T cell triggering, whereas monocytes and macrophages are the primary source of systemic toxic cytokine release. We demonstrate that T cell-generated tumor necrosis factor-α (TNF-α) is the primary mechanism mediating monocyte activation and systemic cytokine release after CD3 bispecific treatment. Prevention of TNF-α release is sufficient to impair systemic release of monocyte cytokines without affecting antitumor efficacy. Systemic cytokine release is only observed upon initial exposure to CD3 bispecific antibody not subsequent doses, indicating a biological distinction between doses. Despite impaired cytokine release after second exposure, T cell cytotoxicity remained unaffected, demonstrating that cytolytic activity of T cells can be achieved in the absence of cytokine release. The mechanistic uncoupling of toxic cytokines and T cell cytolytic activity in the context of CD3 bispecifics provides a biological rationale to clinically explore preventative treatment approaches to mitigate toxicity., (Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Tissue-resident macrophages require specific milieus for the maintenance of defining gene-expression programs. Expression of the transcription factor GATA6 is required for the homeostasis, function and localization of peritoneal cavity-resident macrophages. Gata6 expression is maintained in a non-cell autonomous manner and is elicited by the vitamin A metabolite, retinoic acid. Here, we found that the GATA6 transcriptional program is a common feature of macrophages residing in all visceral body cavities. Retinoic acid-dependent and -independent hallmark genes of GATA6 + macrophages were induced by mesothelial and fibroblastic stromal cells that express the transcription factor Wilms' Tumor 1 (WT1), which drives the expression of two rate-limiting enzymes in retinol metabolism. Depletion of Wt1 + stromal cells reduced the frequency of GATA6 + macrophages in the peritoneal, pleural and pericardial cavities. Thus, Wt1 +  mesothelial and fibroblastic stromal cells constitute essential niche components supporting the tissue-specifying transcriptional landscape and homeostasis of cavity-resident macrophages., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Purpose: The response to cancer immune therapy is dependent on endogenous tumor-reactive T cells. To bypass this requirement, CD3-bispecific antibodies have been developed to induce a polyclonal T-cell response against the tumor. Anti-HER2/CD3 T-cell-dependent bispecific (TDB) antibody is highly efficacious in the treatment of HER2-overexpressing tumors in mice. Efficacy and immunologic effects of anti-HER2/CD3 TDB were investigated in mammary tumor model with very few T cells prior treatment. We further describe the mechanism for TDB-induced T-cell recruitment to tumors., Experimental Design: The immunologic effects and the mechanism of CD3-bispecific antibody-induced T-cell recruitment into spontaneous HER2-overexpressing mammary tumors was studied using human HER2 transgenic, immunocompetent mouse models., Results: Anti-HER2/CD3 TDB treatment induced an inflammatory response in tumors converting them from poorly infiltrated to an inflamed, T-cell abundant, phenotype. Multiple mechanisms accounted for the TDB-induced increase in T cells within tumors. TDB treatment induced CD8 + T-cell proliferation. T cells were also actively recruited post-TDB treatment by IFNγ-dependent T-cell chemokines mediated via CXCR3. This active T-cell recruitment by TDB-induced chemokine signaling was the dominant mechanism and necessary for the therapeutic activity of anti-HER2/CD3 TDB., Conclusions: In summary, we demonstrate that the activity of anti-HER2/CD3 TDB was not dependent on high-level baseline T-cell infiltration. Our results suggest that anti-HER2/CD3 TDB may be efficacious in patients and indications that respond poorly to checkpoint inhibitors. An active T-cell recruitment mediated by TDB-induced chemokine signaling was the major mechanism for T-cell recruitment., (©2018 American Association for Cancer Research.)

Purpose: The tumor microenvironment presents with altered extracellular matrix (ECM) and stroma composition, which may affect treatment efficacy and contribute to tissue stiffness. Ultrasound (US) elastography can visualize and quantify tissue stiffness noninvasively. However, the contributions of ECM and stromal components to stiffness are poorly understood. We therefore set out to quantify ECM and stroma density and their relation to tumor stiffness. Experimental Design: A modified clinical ultrasound system was used to measure tumor stiffness and perfusion during tumor growth in preclinical tumor models. In vivo measurements were compared with collagen mass spectroscopy and automatic analysis of matrix and stromal markers derived from immunofluorescence images. Results: US elastography estimates of tumor stiffness were positively correlated with tumor volume in collagen and myofibroblast-rich tumors, while no correlations were found for tumors with low collagen and myofibroblast content. US elastography measurements were strongly correlated with ex vivo mechanical testing and mass spectroscopy-based measurements of total collagen and immature collagen crosslinks. Registration of ultrasound and confocal microscopy data showed strong correlations between blood vessel density and T-cell density in syngeneic tumors, while no correlations were found for genetic tumor models. In contrast to collagen density, which was positively correlated with stiffness, no significant correlations were observed for hyaluronic acid density. Finally, localized delivery of collagenase led to a significant reduction in tumor stiffness without changes in perfusion 24 hours after treatment. Conclusions: US elastography can be used as a potential biomarker to assess changes in the tumor microenvironment, particularly changes affecting the ECM. Clin Cancer Res; 24(18); 4455-67. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Delta ligands regulate Notch signaling in normal intestinal stem cells, while Jagged1 activates Notch in intestinal adenomas carrying active β-catenin. We used the Apc Min/+ mouse model, tumor spheroid cultures, and patient-derived orthoxenografts to address this divergent ligand-dependent Notch function and its implication in disease. We found that intestinal-specific Jag1 deletion or antibody targeting Jag1 prevents tumor initiation in mice. Addiction to Jag1 is concomitant with the absence of Manic Fringe (MFNG) in adenoma cells, and its ectopic expression reverts Jag1 dependence. In 239 human colorectal cancer patient samples, MFNG imposes a negative correlation between Jag1 and Notch, being high Jag1 in the absence of MFNG predictive of poor prognosis. Jag1 antibody treatment reduces patient-derived tumor orthoxenograft growth without affecting normal intestinal mucosa. Our data provide an explanation to Jag1 dependence in cancer, and reveal that Jag1-Notch1 interference provides therapeutic benefit in a subset of colorectal cancer and FAP syndrome patients.

[This corrects the article DOI: 10.1371/journal.pone.0185862.].

KRAS mutant tumors are largely recalcitrant to targeted therapies. Genetically engineered mouse models (GEMMs) of Kras mutant cancer recapitulate critical aspects of this disease and are widely used for preclinical validation of targets and therapies. Through comprehensive profiling of exomes and matched transcriptomes of >200 Kras G12D-initiated GEMM tumors from one lung and two pancreatic cancer models, we discover that significant intratumoral and intertumoral genomic heterogeneity evolves during tumorigenesis. Known oncogenes and tumor suppressor genes, beyond those engineered, are mutated, amplified, and deleted. Unlike human tumors, the GEMM genomic landscapes are dominated by copy number alterations, while protein-altering mutations are rare. However, interspecies comparative analyses of the genomic landscapes demonstrate fidelity between genes altered in KRAS mutant human and murine tumors. Genes that are spontaneously altered during murine tumorigenesis are also among the most prevalent found in human indications. Using targeted therapies, we also demonstrate that this inherent tumor heterogeneity can be exploited preclinically to discover cancer-specific and genotype-specific therapeutic vulnerabilities. Focusing on Kras allelic imbalance, a feature shared by all three models, we discover that MAPK pathway inhibition impinges uniquely on this event, indicating distinct susceptibility and fitness advantage of Kras -mutant cells. These data reveal previously unknown genomic diversity among KrasG12D -initiated GEMM tumors, places them in context of human patients, and demonstrates how to exploit this inherent tumor heterogeneity to discover therapeutic vulnerabilities., Competing Interests: Conflict of interest statement: All authors were employees of Genentech when the work was performed and A.D., J.H.C., J.E.L., B.-e.W., Z.M., O.F., and M.R.J. hold Roche stock., (Copyright © 2017 the Author(s). Published by PNAS.)

Mitogen-activated protein kinase (MAPK) pathway dysregulation is implicated in >30% of all cancers, rationalizing the development of RAF, MEK and ERK inhibitors. While BRAF and MEK inhibitors improve BRAF mutant melanoma patient outcomes, these inhibitors had limited success in other MAPK dysregulated tumors, with insufficient pathway suppression and likely pathway reactivation. In this study we show that inhibition of either MEK or ERK alone only transiently inhibits the MAPK pathway due to feedback reactivation. Simultaneous targeting of both MEK and ERK nodes results in deeper and more durable suppression of MAPK signaling that is not achievable with any dose of single agent, in tumors where feedback reactivation occurs. Strikingly, combined MEK and ERK inhibition is synergistic in RAS mutant models but only additive in BRAF mutant models where the RAF complex is dissociated from RAS and thus feedback productivity is disabled. We discovered that pathway reactivation in RAS mutant models occurs at the level of CRAF with combination treatment resulting in a markedly more active pool of CRAF. However, distinct from single node targeting, combining MEK and ERK inhibitor treatment effectively blocks the downstream signaling as assessed by transcriptional signatures and phospho-p90RSK. Importantly, these data reveal that MAPK pathway inhibitors whose activity is attenuated due to feedback reactivation can be rescued with sufficient inhibition by using a combination of MEK and ERK inhibitors. The MEK and ERK combination significantly suppresses MAPK pathway output and tumor growth in vivo to a greater extent than the maximum tolerated doses of single agents, and results in improved anti-tumor activity in multiple xenografts as well as in two Kras mutant genetically engineered mouse (GEM) models. Collectively, these data demonstrate that combined MEK and ERK inhibition is functionally unique, yielding greater than additive anti-tumor effects and elucidates a highly effective combination strategy in MAPK-dependent cancer, such as KRAS mutant tumors.

Alveolar type II (AT2) cell dysfunction contributes to a number of significant human pathologies including respiratory distress syndrome, lung adenocarcinoma, and debilitating fibrotic diseases, but the critical transcription factors that maintain AT2 cell identity are unknown. Here we show that the E26 transformation-specific (ETS) family transcription factor Etv5 is essential to maintain AT2 cell identity. Deletion of Etv5 from AT2 cells produced gene and protein signatures characteristic of differentiated alveolar type I (AT1) cells. Consistent with a defect in the AT2 stem cell population, Etv5 deficiency markedly reduced recovery following bleomycin-induced lung injury. Lung tumorigenesis driven by mutant KrasG12D was also compromised by Etv5 deficiency. ERK activation downstream of Ras was found to stabilize Etv5 through inactivation of the cullin-RING ubiquitin ligase CRL4 COP1/DET1 that targets Etv5 for proteasomal degradation. These findings identify Etv5 as a critical output of Ras signaling in AT2 cells, contributing to both lung homeostasis and tumor initiation.

Purpose: Sonic hedgehog (SHH), an activating ligand of smoothened (SMO), is overexpressed in > 70% of pancreatic cancers (PCs). We investigated the impact of vismodegib, an SHH antagonist, plus gemcitabine (GV) or gemcitabine plus placebo (GP) in a multicenter phase Ib/randomized phase II trial and preclinical PC models., Patients and Methods: Patients with PC not amenable to curative therapy who had received no prior therapy for metastatic disease and had Karnofsky performance score ≥ 80 were enrolled. Patients were randomly assigned in a one-to-one ratio to GV or GP. The primary end point was progression-free-survival (PFS). Exploratory correlative studies included serial SHH serum levels and contrast perfusion computed tomography imaging. To further investigate putative biologic mechanisms of SMO inhibition, two autochthonous pancreatic cancer models (Kras(G12D); p16/p19(fl/fl); Pdx1-Cre and Kras(G12D); p53(R270H/wt); Pdx1-Cre) were studied., Results: No safety issues were identified in the phase Ib portion (n = 7), and the phase II study enrolled 106 evaluable patients (n = 53 in each arm). Median PFS was 4.0 and 2.5 months for GV and GP arms, respectively (95% CI, 2.5 to 5.3 and 1.9 to 3.8, respectively; adjusted hazard ratio, 0.81; 95% CI, 0.54 to 1.21; P = .30). Median overall survival (OS) was 6.9 and 6.1 months for GV and GP arms, respectively (95% CI, 5.8 to 8.0 and 5.0 to 8.0, respectively; adjusted hazard ratio, 1.04; 95% CI, 0.69 to 1.58; P = .84). Response rates were not significantly different. There were no significant associations between correlative markers and overall response rate, PFS, or OS. Preclinical trials revealed no significant differences with vismodegib in drug delivery, tumor growth rate, or OS in either model., Conclusion: The addition of vismodegib to gemcitabine in an unselected cohort did not improve overall response rate, PFS, or OS in patients with metastatic PC. Our preclinical and clinical results revealed no statistically significant differences with respect to drug delivery or treatment efficacy using vismodegib., (© 2015 by American Society of Clinical Oncology.)

Cancer stem cells (CSCs) are hypothesized to actively maintain tumors similarly to how their normal counterparts replenish differentiated cell types within tissues, making them an attractive therapeutic target for the treatment of cancer. Because most CSC markers also label normal tissue stem cells, it is unclear how to selectively target them without compromising normal tissue homeostasis. We evaluated a strategy that targets the cell surface leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5), a well-characterized tissue stem cell and CSC marker, with an antibody conjugated to distinct cytotoxic drugs. One antibody-drug conjugate (ADC) demonstrated potent tumor efficacy and safety in vivo. Furthermore, the ADC decreased tumor size and proliferation, translating to improved survival in a genetically engineered model of intestinal tumorigenesis. These data demonstrate that ADCs can be leveraged to exploit differences between normal and cancer stem cells to successfully target gastrointestinal cancers., (Copyright © 2015, American Association for the Advancement of Science.)

The adult prostate possesses a significant regenerative capacity that is of great interest for understanding adult stem cell biology. We demonstrate that leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) is expressed in a rare population of prostate epithelial progenitor cells, and a castration-resistant Lgr5(+) population exists in regressed prostate tissue. Genetic lineage tracing revealed that Lgr5(+) cells and their progeny are primarily luminal. Lgr5(+) castration-resistant cells are long lived and upon regeneration, both luminal Lgr5(+) cells and basal Lgr5(+) cells expand. Moreover, single Lgr5(+) cells can generate multilineage prostatic structures in renal transplantation assays. Additionally, Lgr5(+) cell depletion revealed that the regenerative potential of the castrated adult prostate depends on Lgr5(+) cells. Together, these data reveal insights into the cellular hierarchy of castration-resistant Lgr5+ cells, indicate a requirement for Lgr5(+) cells during prostatic regeneration, and identify an Lgr5(+) adult stem cell population in the prostate., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Much has been written about the advantages and disadvantages of various oncology model systems, with the overall finding that these models lack the predictive power required to translate preclinical efficacy into clinical activity. Despite assertions that some preclinical model systems are superior to others, no single model can suffice to inform preclinical target validation and molecule selection. This perspective provides a balanced albeit critical view of these claims of superiority and outlines a framework for the proper use of existing preclinical models for drug testing and discovery. We also highlight gaps in oncology mouse models and discuss general and pervasive model-independent shortcomings in preclinical oncology work, and we propose ways to address these issues.

Genetically engineered mouse models (GEMMs) of lung cancer closely recapitulate the human disease but suffer from the difficulty of evaluating tumor growth by conventional methods. Herein, a novel automated image analysis method for estimating the lung tumor burden from in vivo micro-computed tomography (micro-CT) data is described. The proposed tumor burden metric is the segmented soft tissue volume contained within a chest space region of interest, excluding an estimate of the heart volume. The method was validated by comparison with previously published manual analysis methods and applied in two therapeutic studies in a mutant K-ras GEMM of non-small cell lung carcinoma. Mice were imaged by micro-CT pre-treatment and stratified into four treatment groups: an antibody inhibiting vascular endothelial growth factor (anti-VEGF), chemotherapy, combination of anti-VEGF and chemotherapy, or control antibody. In the first study, post-treatment imaging was performed 4 weeks later. In the second study, mice were scanned serially on a high-throughput scanner every 2 weeks for 8 weeks during treatment. In both studies, the automated tumor burden estimates were well correlated with manual metrics (r value range: 0.83-0.93, P < .0001) and showed a similar, significant reduction in tumor growth in mice treated with anti-VEGF alone or in combination with chemotherapy. Given the fully automated nature of this technique, the proposed analysis method can provide a valuable tool in preclinical drug research for screening and randomizing animals into treatment groups and evaluating treatment efficacy in mouse models of lung cancer in a highly robust and efficient manner., (Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.)

Activating mutations in the KRAS oncogene occur in approximately 90% of pancreatic cancers, resulting in aberrant activation of the MAPK and the PI3K pathways, driving malignant progression. Significant efforts to develop targeted inhibitors of nodes within these pathways are underway and several are currently in clinical trials for patients with KRAS-mutant tumors, including patients with pancreatic cancer. To model MEK and PI3K inhibition in late-stage pancreatic cancer, we conducted preclinical trials with a mutant Kras-driven genetically engineered mouse model that faithfully recapitulates human pancreatic ductal adenocarcinoma development. Treatment of advanced disease with either a MEK (GDC-0973) or PI3K inhibitor (GDC-0941) alone showed modest tumor growth inhibition and did not significantly enhance overall survival. However, combination of the two agents resulted in a significant survival advantage as compared with control tumor-bearing mice. To model the clinical scenario, we also evaluated the combination of these targeted agents with gemcitabine, the current standard-of-care chemotherapy for pancreatic cancer. The addition of MEK or PI3K inhibition to gemcitabine, or the triple combination regimen, incrementally enhanced overall survival as compared with gemcitabine alone. These results are reminiscent of the survival advantage conferred in this model and in patients by the combination of gemcitabine and erlotinib, an approved therapeutic regimen for advanced nonresectable pancreatic cancer. Taken together, these data indicate that inhibition of MEK and PI3K alone or in combination with chemotherapy do not confer a dramatic improvement as compared with currently available therapies for patients with pancreatic cancer., (©2014 American Association for Cancer Research.)