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The hypoxia-inducible factor 1-α (HIF-1α) enables cells to adapt and respond to hypoxia (Hx), and the activity of this transcription factor is regulated by several oncogenic signals and cellular stressors. While the pathways controlling normoxic degradation of HIF-1α are well understood, the mechanisms supporting the sustained stabilization and activity of HIF-1α under Hx are less clear. We report that ABL kinase activity protects HIF-1α from proteasomal degradation during Hx. Using a fluorescence-activated cell sorting (FACS)-based CRISPR/Cas9 screen, we identified HIF-1α as a substrate of the cleavage and polyadenylation specificity factor-1 (CPSF1), an E3-ligase which targets HIF-1α for degradation in the presence of an ABL kinase inhibitor in Hx. We show that ABL kinases phosphorylate and interact with CUL4A, a cullin ring ligase adaptor, and compete with CPSF1 for CUL4A binding, leading to increased HIF-1α protein levels. Further, we identified the MYC proto-oncogene protein as a second CPSF1 substrate and show that active ABL kinase protects MYC from CPSF1-mediated degradation. These studies uncover a role for CPSF1 in cancer pathobiology as an E3-ligase antagonizing the expression of the oncogenic transcription factors, HIF-1α and MYC.

Resistance to second-generation androgen receptor (AR) antagonists and CYP17 inhibitors in patients with castration-resistant prostate cancer (CRPC) develops rapidly through reactivation of the androgen signaling axis and has been attributed to AR overexpression, production of constitutively active AR splice variants, or the selection for AR mutants with altered ligand-binding specificity. It has been established that androgens induce cell-cycle progression, in part, through upregulation of cyclin D1 (CCND1) expression and subsequent activation of cyclin-dependent kinases 4 and 6 (CDK4/6). Thus, the efficacy of the newly described CDK4/6 inhibitors (G1T28 and G1T38), docetaxel and enzalutamide, was evaluated as single agents in clinically relevant in vitro and in vivo models of hormone-sensitive and treatment-resistant prostate cancer. CDK4/6 inhibition (CDK4/6i) was as effective as docetaxel in animal models of treatment-resistant CRPC but exhibited significantly less toxicity. The in vivo effects were durable and importantly were observed in prostate cancer cells expressing wild-type AR, AR mutants, and those that have lost AR expression. CDK4/6i was also effective in prostate tumor models expressing the AR-V7 variant or the AR F876L mutation, both of which are associated with treatment resistance. Furthermore, CDK4/6i was effective in prostate cancer models where AR expression was lost. It is concluded that CDK4/6 inhibitors are a viable alternative to taxanes as therapeutic interventions in endocrine therapy–refractory CRPC.Implications: The preclinical efficacy of CDK4/6 monotherapy observed here suggests the need for near-term clinical studies of these agents in advanced prostate cancer. Mol Cancer Res; 15(6); 660–9. ©2017 AACR.

Contains legends to supplementary figures.

Supplemental Figure 1. The effects of G1T28 are due to CDK4/6 inhibition. Supplemental Figure 2. Analysis of pharmacodynamics endpoints indicates on-target activity in 22Rv1 tumors. A. Supplemental Figure 3. G1T38 and G128 have similar in vitro efficacy. Supplemental Figure 4. G1T28 inhibits anti-androgen stimulated prostate cancer cell growth.

Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in > 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell–cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC.Significance:A high-throughput pipeline using multiplex immunofluorescence in pancreatic cancer reveals striking expression subtype intratumoral heterogeneity with implications for therapy selection and identifies co-expressor cells that may serve as intermediates during subtype switching.

Supplementary Figures

High-throughput phenotypic screens leveraging biochemical perturbations, high-content readouts, and complex multicellular models could advance therapeutic discovery yet remain constrained by limitations of scale. To address this, we establish a method for compressing screens by pooling perturbations followed by computational deconvolution. Conducting controlled benchmarks with a highly bioactive small molecule library and a high-content imaging readout, we demonstrate increased efficiency for compressed experimental designs compared to conventional approaches. To prove generalizability, we apply compressed screening to examine transcriptional responses of patient-derived pancreatic cancer organoids to a library of tumor-microenvironment (TME)-nominated recombinant protein ligands. Using single-cell RNA-seq as a readout, we uncover reproducible phenotypic shifts induced by ligands that correlate with clinical features in larger datasets and are distinct from reference signatures available in public databases. In sum, our approach enables phenotypic screens that interrogate complex multicellular models with rich phenotypic readouts to advance translatable drug discovery as well as basic biology.

Targeted therapies have revolutionized cancer chemotherapy. Unfortunately, most patients develop multifocal resistance to these drugs within a matter of months. Here, we used a high-throughput phenotypic small molecule screen to identify MCB-613 as a compound that selectively targetsEGFR-mutant, EGFR inhibitor-resistant non-small cell lung cancer (NSCLC) cells harboring diverse resistance mechanisms. Subsequent proteomic and functional genomic screens involving MCB-613 identified its target in this context to be KEAP1, revealing that this gene is selectively essential in the setting of EGFR inhibitor resistance. In-depth molecular characterization demonstrated that (1) MCB-613 binds KEAP1 covalently; (2) a single molecule of MCB-613 is capable of bridging two KEAP1 monomers together; and, (3) this modification interferes with the degradation of canonical KEAP1 substrates such as NRF2. Surprisingly, NRF2 knockout sensitizes cells to MCB-613, suggesting that the drug functions through modulation of an alternative KEAP1 substrate. Together, these findings advance MCB-613 as a new tool for exploiting the selective essentiality of KEAP1 in drug-resistant,EGFR-mutant NSCLC cells.

Pancreatic ductal adenocarcinoma (PDAC) has been classified into classical and basal-like transcriptional subtypes by bulk RNA measurements. However, recent work has uncovered greater complexity to transcriptional subtypes than was initially appreciated using bulk RNA expression profiling. To provide a deeper understanding of PDAC subtypes, we developed a multiplex immunofluorescence (mIF) pipeline that quantifies protein expression of six PDAC subtype markers (CLDN18.2, TFF1, GATA6, KRT17, KRT5, and S100A2) and permits spatially resolved, single-cell interrogation of pancreatic tumors from resection specimens and core needle biopsies. Both primary and metastatic tumors displayed striking intratumoral subtype heterogeneity that was associated with patient outcomes, existed at the scale of individual glands, and was significantly reduced in patient-derived organoid cultures. Tumor cells co-expressing classical and basal markers were present in > 90% of tumors, existed on a basal-classical polarization continuum, and were enriched in tumors containing a greater admixture of basal and classical cell populations. Cell–cell neighbor analyses within tumor glands further suggested that co-expressor cells may represent an intermediate state between expression subtype poles. The extensive intratumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC. Significance: A high-throughput pipeline using multiplex immunofluorescence in pancreatic cancer reveals striking expression subtype intratumoral heterogeneity with implications for therapy selection and identifies co-expressor cells that may serve as intermediates during subtype switching.

Metastases arise from a subset of cancer cells that disseminate from the primary tumor; however, the factors that contribute to proliferation of cancer cells in a secondary site are incompletely understood. The ability of cancer cells to thrive in a new tissue site is influenced by genetic and epigenetic changes that are important for disease initiation and progression, but these factors alone do not predict if and where cancers metastasize. Specific cancer types metastasize to consistent subsets of tissues, suggesting that factors within the primary tumor influence the tissue environments where cancers can grow. Using pancreatic cancer as a model, we find that primary and metastatic tumors are metabolically similar to each other and that the tumor initiating capacity and proliferation of both primary- and metastasis-derived cells is favored in the primary site relative to the metastatic site. Moreover, propagating lung or liver metastatic cells in vivo to enrich for tumor cells adapted to grow in the lung or the liver does not enhance their relative ability to form large tumors in those sites, change their preference to grow in the primary site, nor stably alter their metabolism relative to primary tumors. To assess whether this preference for the primary site is specific to pancreatic cancer, we analyzed liver and lung cancer cells and find that these cells also best form tumors in the tissue that corresponds to their primary site. Together, these data suggest that the cancer tissue-of-origin influences the metabolism of both primary and metastatic tumors and may impact whether cancer cells can thrive in a metastatic site.One-Sentence SummaryTissue-of-origin is a major determinant of metastatic tumor metabolism and accessing the right metabolic environment may contribute to why cancers metastasize to specific tissues.

Immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex class I (MHC-I)(1,2,3,4,5). Current approaches to profile MHC-I associated peptides, collectively known as the “immunopeptidome”, are limited to in vitro investigation or bulk tumor lysates, limiting our understanding of cancer-specific patterns of antigen presentation in vivo(6). To overcome these limitations, we engineered an inducible affinity tag into the mouse MHC-I gene (H2-K1) and targeted this allele to the Kras(LSL-G12D/+); p53(fl/fl) (KP) mouse model (KP/K(b)Strep)(7). This approach allowed us to precisely isolate MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAD) in vivo. In addition, we profiled the LUAD immunopeptidome from the alveolar type 2 cell-of-origin through late-stage disease. Differential peptide presentation in LUAD was not predictable by mRNA expression or translation efficiency and is likely driven by post-translational mechanisms. Vaccination with peptides presented by LUAD in vivo provoked CD8(+) T cell responses in naïve and tumor-bearing mice. Many peptides unique to LUAD, including immunogenic peptides, exhibited minimal expression of the cognate mRNA, provoking reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance(8). Beyond cancer, the K(b)Strep allele is compatible with other Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease, and autoimmunity.

Local adaptation directs populations towards environment-specific fitness maxima through acquisition of positively selected traits. However, rapid environmental changes can identify hidden fitness trade-offs that turn adaptation into maladaptation, resulting in evolutionary traps. Cancer, a disease that is prone to drug resistance, is in principle susceptible to such traps. We therefore performed pooled CRISPR-Cas9 knockout screens in acute myeloid leukemia (AML) cells treated with various chemotherapies to map the drug-dependent genetic basis of fitness trade-offs, a concept known as antagonistic pleiotropy (AP). We identified a PRC2-NSD2/3-mediated MYC regulatory axis as a drug-induced AP pathway whose ability to confer resistance to bromodomain inhibition and sensitivity to BCL-2 inhibition templates an evolutionary trap. Across diverse AML cell-line and patient-derived xenograft models, we find that acquisition of resistance to bromodomain inhibition through this pathway exposes coincident hypersensitivity to BCL-2 inhibition. Thus, drug-induced AP can be leveraged to design evolutionary traps that selectively target drug resistance in cancer.

Significance Despite the usefulness of genetically engineered mouse models in cancer research, their small total blood volume and the rarity of circulating tumor cells (CTCs) preclude the use of existing liquid biopsy techniques for longitudinal CTC studies in mice. We have devised a method for collecting CTCs from an unanesthetized mouse longitudinally, spanning multiple days or weeks, to study acute perturbations (e.g., drug treatment) or potentially long-term phenotypes (e.g., tumor progression) within the same mouse. Here, we show that our optofluidic-based approach eliminates confounding biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice., Circulating tumor cells (CTCs) play a fundamental role in cancer progression. However, in mice, limited blood volume and the rarity of CTCs in the bloodstream preclude longitudinal, in-depth studies of these cells using existing liquid biopsy techniques. Here, we present an optofluidic system that continuously collects fluorescently labeled CTCs from a genetically engineered mouse model (GEMM) for several hours per day over multiple days or weeks. The system is based on a microfluidic cell sorting chip connected serially to an unanesthetized mouse via an implanted arteriovenous shunt. Pneumatically controlled microfluidic valves capture CTCs as they flow through the device, and CTC-depleted blood is returned back to the mouse via the shunt. To demonstrate the utility of our system, we profile CTCs isolated longitudinally from animals over 4 days of treatment with the BET inhibitor JQ1 using single-cell RNA sequencing (scRNA-Seq) and show that our approach eliminates potential biases driven by intermouse heterogeneity that can occur when CTCs are collected across different mice. The CTC isolation and sorting technology presented here provides a research tool to help reveal details of how CTCs evolve over time, allowing studies to credential changes in CTCs as biomarkers of drug response and facilitating future studies to understand the role of CTCs in metastasis.

Prognostically relevant RNA expression states exist in pancreatic ductal adenocarcinoma (PDAC), but our understanding of their drivers, stability, and relationship to therapeutic response is limited. To examine these attributes systematically, we profiled metastatic biopsies and matched organoid models at single-cell resolution. In vivo, we identify a new intermediate PDAC transcriptional cell state and uncover distinct site- and state-specific tumor microenvironments (TMEs). Benchmarking models against this reference map, we reveal strong culture-specific biases in cancer cell transcriptional state representation driven by altered TME signals. We restore expression state heterogeneity by adding back in vivo-relevant factors and show plasticity in culture models. Further, we prove that non-genetic modulation of cell state can strongly influence drug responses, uncovering state-specific vulnerabilities. This work provides a broadly applicable framework for aligning cell states across in vivo and ex vivo settings, identifying drivers of transcriptional plasticity and manipulating cell state to target associated vulnerabilities.

Resistance to second-generation androgen receptor (AR) antagonists and CYP17 inhibitors in patients with castration-resistant prostate cancer (CRPC) develops rapidly through reactivation of the androgen signaling axis and has been attributed to AR overexpression, production of constitutively active AR splice variants, or the selection for AR mutants with altered ligand-binding specificity. It has been established that androgens induce cell-cycle progression, in part, through upregulation of cyclin D1 (CCND1) expression and subsequent activation of cyclin-dependent kinases 4 and 6 (CDK4/6). Thus, the efficacy of the newly described CDK4/6 inhibitors (G1T28 and G1T38), docetaxel and enzalutamide, was evaluated as single agents in clinically relevant in vitro and in vivo models of hormone-sensitive and treatment-resistant prostate cancer. CDK4/6 inhibition (CDK4/6i) was as effective as docetaxel in animal models of treatment-resistant CRPC but exhibited significantly less toxicity. The in vivo effects were durable and importantly were observed in prostate cancer cells expressing wild-type AR, AR mutants, and those that have lost AR expression. CDK4/6i was also effective in prostate tumor models expressing the AR-V7 variant or the AR F876L mutation, both of which are associated with treatment resistance. Furthermore, CDK4/6i was effective in prostate cancer models where AR expression was lost. It is concluded that CDK4/6 inhibitors are a viable alternative to taxanes as therapeutic interventions in endocrine therapy–refractory CRPC. Implications: The preclinical efficacy of CDK4/6 monotherapy observed here suggests the need for near-term clinical studies of these agents in advanced prostate cancer. Mol Cancer Res; 15(6); 660–9. ©2017 AACR.

Oncogenic KRAS can activate numerous effector pathways to drive malignant progression. However, the relationships between specific effectors and oncogenic phenotypes, and the extent to which these relationships vary across heterogeneous tumors, are incompletely understood. Recently in Cell Reports, a team of scientists described an innovative, combinatorial siRNA-based approach to functionally link KRAS effectors and phenotypes in a large panel of cancer cell lines. Central to this work was the identification of two major subtypes of KRAS-mutant cancers with distinct effector landscapes and tractable therapeutic vulnerabilities.

Background: Pancreatic adenocarcinoma (PDAC) is a rapidly progressive disease, with few molecular markers that stratify patients for survival time or treatment response. Bulk transcriptional analyses of PDAC have identified two major transcriptional subtypes, classical and basal, the latter of which is associated with worse overall survival and limited sensitivity to chemotherapy. However, spatial localization and classical-basal heterogeneity are incompletely understood at the individual cell level within a bulk tumor. Design: We built and validated a multiplex immunofluorescence assay that employs three classical and two basal subtype markers to measure subtype composition at spatially resolved, single cell resolution in a multi-institutional cohort of formalin-fixed paraffin-embedded primary pancreatic cancer resection specimens. Using digital image analysis, supervised machine learning, and Cox proportional hazards regression, we analyzed protein-level classical-basal subtype landscape, cell localization, and outcome associations. Results: Subtyping data were successfully generated for 1.2 million tumor cells across 290 resected tumor specimens (median 3579 cells/tumor). Tumor level analysis of cellular composition revealed that 88% of tumors are “mixed”, harboring any number of both classical and basal cells, whereas tumors composed only of classical or basal cells represented 11% and 1.4% of cases, respectively. In addition to pure classical and basal cells, we identified “hybrid cells” co-expressing basal and classical markers in 79% of cases, ranging in abundance from Citation Format: Hannah L. Williams, Jinming Zhang, Srivatsan Raghavan, Peter S. Winter, Kevin Kapner, Sara Vayrynen, Andressa Dias Costa, Chen Yuan, Mai Chan Lau, Vicente Morales-Oyarvide, Douglas Rubinson, Lauren Brais, Emma Reilly, Margaret Kozak, David Linehan, Richard Dunne, Daniel Chang, Albert Koong, Aram Hezel, William C. Hahn, Alek K. Shalek, Andrew J. Aguirre, Jonathan A. Nowak, Brian M Wolpin. Spatially resolved, single cell assessment of pancreatic ductal adenocarcinoma expression subtypes reveals mixed and hybrid basal-classical marker expression with prognostic significance and discrete spatial localization [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PR-006.

Metastatic pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal malignancy with few therapeutic options. Tumor transcriptional state is a strong predictor of clinical outcome in PDAC, with two primary cell states, basal-like and classical, identified by bulk transcriptional profiling. Basal-like tumors carry a worse prognosis, but the mechanisms underlying this survival difference, the degree of cellular heterogeneity within a given tumor, and the subtype-specific contributions from the local immune microenvironment are not well understood. In addition, there are ongoing efforts to use patient-derived organoid models as functional surrogates for an individual patient’s disease, but the degree to which patient transcriptional phenotypes are preserved in their matched organoid models remains unclear. Here, we describe a pipeline that enables both direct characterization of the liver metastatic niche via single-cell RNA-sequencing and functional assessment of PDAC tumor biology in patient-matched organoid models. Starting from core needle biopsies of metastatic PDAC lesions, we applied this approach to profile 22 patient samples and their matched organoid models using single-cell RNA-sequencing with Seq-Well. We demonstrate significant heterogeneity at the single-cell level across the basal-like to classical transcriptional spectrum. Basal-like cells expressed more mesenchymal and stem-like features, while classical cells expressed features of epithelial and pancreatic progenitor transcriptional programs. A population of “hybrid” malignant cells co-expressed markers of both basal-like and classical states, suggesting that these phenotypes lie on a continuum rather than as discrete entities. Microenvironmental composition also differed by subtype across T/NK and macrophage populations. Specifically, basal-like tumors exhibited tumor cell crosstalk with specific macrophage subsets, while classical tumors harbored greater immune infiltration and a relatively pro-angiogenic microenvironment, raising important considerations for subtype-specific microenvironmental directed therapy. Finally, we found that matched organoids exhibited transcriptional drift along the basal-like to classical axis relative to their parent tumors, with evidence for selection against basal-like phenotypes in vitro. However, tumor cells in organoid culture exhibited remarkable plasticity and could recover in vivo basal-like phenotypes in response to changes in their growth conditions. Taken together, our work provides a framework for the analysis of human cancers and their matched models using single-cell methods to dissect tumor-intrinsic and extrinsic contributions, and reveals novel insights into the transcriptional heterogeneity and plasticity of PDAC. Citation Format: Srivatsan Raghavan, Peter S. Winter, Andrew W. Navia, Hannah L. Williams, Alan DenAdel, Radha L. Kalekar, Jennyfer Galvez-Reyes, Kristen E. Lowder, Nolawit Mulugeta, Manisha S. Raghavan, Ashir A. Borah, Sara A. Vayrynen, Andressa Dias Costa, Junning Wang, Emma Reilly, Dorisanne Y. Ragon, Lauren K. Brais, Alex M. Jaeger, James M. Cleary, Lorin Crawford, Jonathan A. Nowak, Brian M. Wolpin, William C. Hahn, Andrew J. Aguirre, Alex K. Shalek. Transcriptional subtype-specific microenvironmental crosstalk and tumor cell plasticity in metastatic pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-058.

Targeted inhibitors of essential oncogenic kinases induce high rates of clinical response but cure few patients due to the persistence of minimal residual disease (MRD). BCR-ABL mutant leukemias are a classic example of this paradigm where patients usually achieve deep remissions followed by near inevitable relapses. Multiple factors have been shown to influence how an individual patient’s leukemic cells will navigate treatment including differentiation state, mutational background, and communication with the microenvironment. Here, we use BCR-ABL-rearranged acute lymphoblastic leukemia (BCR-ABL ALL) to interrogate cell-autonomous features leading to therapeutic resistance using low-input single-cell assays. Specifically, we use a combination of primary samples and PDX models to dissect aberrant developmental hierarchies and monitor leukemic cell transcriptional and biophysical phenotype at pretreatment, MRD, and relapse. Using machine learning, we relate malignant B cells to normal development, allowing us to define leukemic developmental programs and demonstrate that these have consequences for the time to progression as well as the genetic alterations seen at relapse. Further, we determine that there are unique biophysical features tied to leukemic developmental states and that these integrative properties co-evolve with transcriptional state over the course of treatment. Finally, we demonstrate in PDX studies that it may be possible to intercept relapse by targeting specific features of MRD cells. Together, these data suggest that significant developmental hierarchies exist in ALL, tumor subpopulations can be identified directly within MRD, and their phenotypic and molecular characterization can be exploited to therapeutic effect. Citation Format: Peter S. Winter, Andrew Navia, Haley Strouf, Mahnoor Mirza, Jennyfer Galvez-Reyes, Nolawit Mulugeta, Laura Bilal, Nezha Senhaji, Peter Dennis, Catharine S. Leahy, Kay Shigemori, Foster Powers, Alejandro Gupta, Nicholas Calistri, Alex Van Scoyk, Kristen Jones, Huiyun Liu, Kristen E. Stevenson, Robert Kimmerling, Mark Stevens, David M. Weinstock, Scott R. Manalis, Mark A. Murakami, Alex K. Shalek. Aberrant leukemic developmental hierarchies and MRD-specific targeting informed by single-cell biophysical and molecular profiling [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr IA20.

Background Transforming growth factor (TGF)-β is a pluripotent cytokine that displays several tissue-specific biological activities. In the liver, TGF-β is considered a fundamental molecule, controlling organ size and growth by limiting hepatocyte proliferation. It is involved in fibrogenesis and, therefore, in worsening liver damage, as well as in triggering the development of hepatocellular carcinoma (HCC). TGF-β is known to act as an oncosuppressor and also as a tumour promoter in HCC, but its role is still unclear. Design In this review, we discuss the potential role of TGF-β in regulating the tumoural progression of HCC, and therefore the rationale for targeting this molecule in patients with HCC. Results A considerable amount of experimental preclinical evidence suggests that TGF-β is a promising druggable target in patients with HCC. To support this hypothesis, a phase II clinical trial is currently ongoing using a TGF-β pathway inhibitor, and results will soon be available. Conclusions The identification of new TGF-β related biomarkers will help to select those patients most likely to benefit from therapy aimed at inhibiting the TGF-β pathway. New formulations that may provide a more controlled and sustained delivery of the drug will improve the therapeutic success of such treatments.

The majority of patients with pancreatic ductal adenocarcinoma (PDAC) present with metastatic disease at diagnosis and have median survival times of less than 12 months. Recent studies have demonstrated that PDAC tumors with distinct transcriptional signatures are associated with different clinical outcomes, and that the tumor microenvironment may contribute to PDAC pathogenesis. In parallel, there are ongoing efforts to understand if patient-derived organoid models can be used as functional surrogates for an individual patient’s disease. However, it remains unclear if patient transcriptional phenotypes are preserved in their matched organoid models. Here, we describe a pipeline that permits both direct characterization of the PDAC liver metastatic niche via single-cell RNA-sequencing and functional assessment of PDAC tumor biology in patient-matched organoid models. Starting from core needle biopsies of metastatic PDAC lesions containing 50-100k viable cells, we simultaneously perform (1) low-input single-cell RNA-sequencing using Seq-Well and (2) three-dimensional organoid culture generation. We have applied this approach to profile 21 patients and their matched early passage organoid models. Our pipeline yields high-quality single-cell measurements across diverse cell types—both tumor and nontumor stromal—enabling a principled dissection of tumor intrinsic and extrinsic factors. Evaluation of clinically relevant transcriptional signatures (e.g., basal-like vs. classical) revealed extensive heterogeneity at the single-cell level and identified new, hybrid expression states. We also observed evidence of significant subtype-specific crosstalk between immune populations and tumor cells—specifically between T cells and tumor cells originating from basal-like tumors. Serial sampling at different stages of treatment revealed transcriptional shifts in tumor cells suggestive of significant plasticity. We similarly found that organoids derived from basal-like tumors exhibited considerable plasticity in vitro and had decreased fitness in standard organoid culture conditions, suggesting the need for distinct environments to support specific transcriptional subtypes. Overall, our approach provides actionable insights into the heterogeneity and plasticity of human PDAC, as well as a pipeline and framework for the analysis of PDAC and other cancers. This abstract is also being presented as Poster A50. Citation Format: Peter S. Winter, Srivatsan Raghavan, Andrew W. Navia, Hannah Williams, Jennyfer Galvez-Reyes, Radha Kalekar, Ashir Borah, Alan DenAdel, Manisha Raghavan, Kristen Lowder, Nolawit Mulugeta, Junning Wang, Emma Reilly, Lauren Brais, Lorin Crawford, James McFarland, James M. Cleary, Jonathan Nowak, Brian M. Wolpin, Andrew J. Aguirre, William C. Hahn, Alex K. Shalek. Matched metastatic pancreatic ductal adenocarcinoma biopsies and organoid models reveal tumor cell transcriptional plasticity and subtype-specific microenvironmental crosstalk [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr PR02.

Patients with metastatic pancreatic ductal adenocarcinoma (PDAC) have few treatment options and continue to have dismal prognoses due to the rapid development of resistance to both standard-of-care and experimental therapies. Several recent studies have demonstrated that patients with distinct PDAC transcriptional subtypes have differing clinical courses, and that the tumor microenvironment can also contribute to patient outcome. However, deep cellular characterization of metastatic PDAC tumors and their stromal microenvironments has been challenging due to limited tissue availability from metastatic liver biopsies. Here, we present a focused assessment of the PDAC liver metastatic niche—encompassing tumor, immune, and stromal cells—via low-input single-cell transcriptional profiling of patient specimens with the goal of developing a deeper understanding of tumor heterogeneity and the tumor microenvironment. Our pipeline accesses core needle biopsies from liver metastases, splitting each core for 1) single-cell RNA sequencing using Seq-Well and 2) organoid generation. Using this pipeline, we have successfully profiled liver metastases from 15 patients along with matched early-passage organoid models. Assessment of clinically relevant transcriptional signatures reveals extensive heterogeneity at the single-cell level and identifies new, hybrid transcriptional states occupied by these metastases. In addition, we observe evidence of significant crosstalk between stromal and immune populations and tumor cells. Serial samples at different stages of therapy show transcriptional shifts in tumor cells suggestive of significant plasticity that likely contributes to therapeutic resistance. Initial analysis of matched organoids at successive passages demonstrates a skew in their clonal composition, as well as evolution of their transcriptional state as compared to their in vivo phenotypes. Overall, our work provides an important window into the biology of metastatic PDAC, as well as some of the first direct comparisons of clonality and transcriptional phenotypes across in vivo specimens and their in vitro organoid counterparts. This abstract is also being presented as Poster C43. Citation Format: Srivatsan Raghavan, Peter S. Winter, Andrew Navia, Radha Kalekar, Jennyfer Galvez-Reyes, Sanjay Prakadan, Junning Wang, Emma Reilly, Lauren Brais, James M. Cleary, Jonathan Nowak, Brian M. Wolpin, Alex K. Shalek, Andrew J. Aguirre, William C. Hahn. Assessment of tumor heterogeneity, clonal evolution, and the stromal microenvironment in metastatic pancreatic ductal adenocarcinoma and matched patient-derived organoids [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 PR15.

While inhibitors of BCL-2 family proteins (BH3 mimetics) have shown promise as anti-cancer agents, the various dependencies or co-dependencies of diverse cancers on BCL-2 genes remain poorly understood. Here we develop a drug screening approach to define the sensitivity of cancer cells from ten tissue types to all possible combinations of selective BCL-2, BCL-XL, and MCL-1 inhibitors and discover that most cell lines depend on at least one combination for survival. We demonstrate that expression levels of BCL-2 genes predict single mimetic sensitivity, whereas EMT status predicts synergistic dependence on BCL-XL+MCL-1. Lastly, we use a CRISPR/Cas9 screen to discover that BFL-1 and BCL-w promote resistance to all tested combinations of BCL-2, BCL-XL, and MCL-1 inhibitors. Together, these results provide a roadmap for rationally targeting BCL-2 family dependencies in diverse human cancers and motivate the development of selective BFL-1 and BCL-w inhibitors to overcome intrinsic resistance to BH3 mimetics., Dependency of diverse cancers on specific BCL-2 family members and their combinations is unknown. Here they perform drug screening and find most cell lines to be dependent on at least one combination of BCL-2 family members, and using a CRISPR screen find BCL-w and BFL-1 to mediate resistance to BH3 mimetics

The entry of malignant hepatocytes into blood vessels is a key step in the dissemination and metastasis of hepatocellular carcinoma (HCC). The identification of molecular mechanisms involved in the transmigration of malignant hepatocytes through the endothelial barrier is of high relevance for therapeutic intervention and metastasis prevention. In this study, we employed a model of hepatocellular transmigration that mimics vascular invasion using hepatic sinusoidal endothelial cells and malignant hepatocytes evincing a mesenchymal-like, invasive phenotype by transforming growth factor (TGF)-β. Labelling of respective cell populations with various stable isotopes and subsequent mass spectrometry analyses allowed the “real-time” detection of molecular changes in both transmigrating hepatocytes and endothelial cells. Interestingly, the proteome profiling revealed 36 and 559 regulated proteins in hepatocytes and endothelial cells, respectively, indicating significant changes during active transmigration that mostly depends on cell–cell interaction rather than on TGF-β alone. Importantly, matching these in vitro findings with HCC patient data revealed a panel of common molecular alterations including peroxiredoxin-3, epoxide hydrolase, transgelin-2 and collectin 12 that are clinically relevant for the patient’s survival. We conclude that hepatocellular plasticity induced by TGF-β is crucially involved in blood vessel invasion of HCC cells.

Combinatorial inhibition of effector and feedback pathways is a promising treatment strategy for KRAS mutant cancers. However, the particular pathways that should be targeted to optimize therapeutic responses are unclear. Using CRISPR/Cas9, we systematically mapped the pathways whose inhibition cooperates with drugs targeting the KRAS effectors MEK, ERK, and PI3K. By performing 70 screens in models of KRAS mutant colorectal, lung, ovarian, and pancreas cancers, we uncovered universal and tissue-specific sensitizing combinations involving inhibitors of cell cycle, metabolism, growth signaling, chromatin regulation, and transcription. Furthermore, these screens revealed secondary genetic modifiers of sensitivity, yielding a SRC inhibitor-based combination therapy for KRAS/PIK3CA double-mutant colorectal cancers (CRCs) with clinical potential. Surprisingly, acquired resistance to combinations of growth signaling pathway inhibitors develops rapidly following treatment, but by targeting signaling feedback or apoptotic priming, it is possible to construct three-drug combinations that greatly delay its emergence.

Therapies that efficiently induce apoptosis are likely to be required for durable clinical responses in patients with solid tumors. Using a pharmacological screening approach, we discovered that combined inhibition of B cell lymphoma–extra large (BCL-X L ) and the mammalian target of rapamycin (mTOR)/4E-BP axis results in selective and synergistic induction of apoptosis in cellular and animal models of PIK3CA mutant breast cancers, including triple-negative tumors. Mechanistically, inhibition of mTOR/4E-BP suppresses myeloid cell leukemia–1 (MCL-1) protein translation only in PIK3CA mutant tumors, creating a synthetic dependence on BCL-X L . This dual dependence on BCL-X L and MCL-1, but not on BCL-2, appears to be a fundamental property of diverse breast cancer cell lines, xenografts, and patient-derived tumors that is independent of the molecular subtype or PIK3CA mutational status. Furthermore, this dependence distinguishes breast cancers from normal breast epithelial cells, which are neither primed for apoptosis nor dependent on BCL-X L /MCL-1, suggesting a potential therapeutic window. By tilting the balance of pro- to antiapoptotic signals in the mitochondria, dual inhibition of MCL-1 and BCL-X L also sensitizes breast cancer cells to standard-of-care cytotoxic and targeted chemotherapies. Together, these results suggest that patients with PIK3CA mutant breast cancers may benefit from combined treatment with inhibitors of BCL-X L and the mTOR/4E-BP axis, whereas alternative methods of inhibiting MCL-1 and BCL-X L may be effective in tumors lacking PIK3CA mutations.

Targeted inhibitors of oncogenic kinases have transformed cancer therapy by inducing high rates of clinical response across diverse tumor types, yet they cure few patients due to the persistence of minimal residual disease (MRD), which seeds relapse. Characterizing MRD biology would promote development of more effective therapies tailored to relapse-initiating cells. However, these efforts have been complicated by the technical challenges of isolating and profiling low-frequency tumor populations. We overcome these hurdles by measuring the mass and short-term growth rate of single cells ex vivo using a series of suspended microchannel resonators (SMR) followed by single-cell RNA-seq of the same cells. This platform enables measurement of low input samples to facilitate characterization of MRD subpopulations and functionally drug test MRD-specific vulnerabilities. PDX models of BCR-ABL-rearranged acute lymphoblastic leukemia (BCR-ABL ALL) treated with targeted kinase inhibitors provide a tractable experimental system in which to deploy this platform to interrogate in vivo tumor evolution, MRD, and acquired resistance. We conducted a statistically powered 4-arm randomized phase II-like trial combining ponatinib and the novel allosteric BCR-ABL inhibitor ABL001 in a panel of 13 PDX models of BCR-ABL ALL (total evaluable mice: n=10 vehicle, n=14 ABL001, n=30 ponatinib, n=33 combination). Six of 30 animals receiving ponatinib (20% [95% CI, 8-39%]) and a significantly higher proportion receiving combination (10 of 33, 30% [95% CI, 16-49%]) met the primary endpoint of progression-free survival at 120 days on treatment (p=0.030). No mice in the vehicle or ABL001 arms met this endpoint, and all mice eventually progressed or were found to harbor MRD when sacrificed after treatment courses of up to 248 days. These durable remissions prior to relapse allowed us to delineate in vivo tumor evolution, focusing on MRD, via serial bone marrow aspirates. Drug-persistent marrow-resident leukemia cells exhibit strong enrichment for cellular quiescence programs that extend through MRD and into relapse. These programs are driven by expression of genes known to govern quiescence in hematopoietic stem cells (e.g., EGR1, NR4A1), and their expression correlates with a rare subset of biophysically small cells (mass Citation Format: Mark A. Murakami, Peter S. Winter, Andrew W. Navia, Laura L. Bilal, Haley Strouf, Kay Shigemori, Alejandro Gupta, Mahnoor Mirza, Catharine S. Leahy, Nick Calistri, Kristen Jones, Alexandra Van Scoyk, Huiyun Liu, Foster Powers, Robert Kimmerling, Mark Stevens, Kristen E. Stevenson, Scott R. Manalis, David M. Weinstock, Alex K. Shalek. Single-cell profiling reveals programs mediating oncogenic kinase-independence and minimal residual disease in BCR-ABL-rearranged acute lymphoblastic leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2450.

Latent Epstein-Barr virus (EBV) infection is causally linked to several human cancers. EBV expresses viral oncogenes that promote cell growth and inhibit the apoptotic response to uncontrolled proliferation. The EBV oncoprotein LMP1 constitutively activates NFκB and is critical for survival of EBV-immortalized B cells. However, during early infection EBV induces rapid B cell proliferation with low levels of LMP1 and little apoptosis. Therefore, we sought to define the mechanism of survival in the absence of LMP1/NFκB early after infection. We used BH3 profiling to query mitochondrial regulation of apoptosis and defined a transition from uninfected B cells (BCL-2) to early-infected (MCL-1/BCL-2) and immortalized cells (BFL-1). This dynamic change in B cell survival mechanisms is unique to virus-infected cells and relies on regulation of MCL-1 mitochondrial localization and BFL-1 transcription by the viral EBNA3A protein. This study defines a new role for EBNA3A in the suppression of apoptosis with implications for EBV lymphomagenesis. DOI: http://dx.doi.org/10.7554/eLife.22509.001, eLife digest Over 90% of adults around the world are infected with the Epstein-Barr virus. Like other closely related viruses, such as those that cause chicken pox and cold sores, an infection lasts for the rest of the person’s life, although the virus generally remains in a latent or dormant state. However, under certain conditions the latent viruses can cause cancers to develop; in fact, it is estimated that such infections are responsible for nearly 2% of all cancer deaths worldwide. One way that healthy human cells prevent cancer is by triggering their own death in a process called apoptosis. The Epstein-Barr virus can block apoptosis, therefore making the cells more likely to become cancerous. Previous research identified one protein in the Epstein-Barr virus that promotes cancer by preventing infected cells from dying as normal. However, even in the absence of this protein, Epstein-Barr virus-infected cells remain resistant to apoptosis. This suggests that the virus has another way of blocking cell death. Price et al. have now used a technique that stresses living cells in a way that reveals which proteins prevent apoptosis to study human cells infected with the Epstein-Barr virus. This revealed that soon after infection, the virus could force the human cell to produce MCL-1, a protein that prevents cell death. Later, the Epstein-Barr virus enlisted a second human protein called BFL-1, which makes the infected cell further resistant to apoptosis. Price et al. discovered that a protein in the Epstein-Barr virus called EBNA3A controls the production of the MCL-1 and BFL-1 proteins. In the future, developing therapies that target these proteins may lead to new treatments for cancers caused by the Epstein-Barr virus. Such treatments would be likely to have fewer side effects for patients than traditional chemotherapies. DOI: http://dx.doi.org/10.7554/eLife.22509.002

ABT-199, a potent and selective small-molecule antagonist of BCL-2, is being clinically vetted as pharmacotherapy for the treatment of acute myeloid leukemia (AML). However, given that prolonged monotherapy tends to beget resistance, we sought to investigate the means by which resistance to ABT-199 might arise in AML and the extent to which those mechanisms might be preempted. Here we used a pathway-activating genetic screen to nominate MCL-1 and BCL-XL as potential nodes of resistance. We then characterized a panel of ABT-199-resistant myeloid leukemia cell lines derived through chronic exposure to ABT-199 and found that acquired drug resistance is indeed driven by the upregulation of MCL-1 and BCL-XL. By targeting MCL-1 and BCL-XL, resistant AML cell lines could be resensitized to ABT-199. Further, preemptively targeting MCL-1 and/or BCL-XL alongside administration of ABT-199 was capable of delaying or forestalling the acquisition of drug resistance. Collectively, these data suggest that in AML, (1) the selection of initial therapy dynamically templates the landscape of acquired resistance via modulation of MCL-1/BCL-XL and (2) appropriate selection of initial therapy may delay or altogether forestall the acquisition of resistance to ABT-199.

Cancer cells can activate diverse signaling pathways to evade the cytotoxic action of drugs. We created and screened a library of barcoded pathway-activating mutant complementary DNAs to identify those that enhanced the survival of cancer cells in the presence of 13 clinically relevant, targeted therapies. We found that activation of the RAS-MAPK (mitogen-activated protein kinase), Notch1, PI3K (phosphoinositide 3-kinase)-mTOR (mechanistic target of rapamycin), and ER (estrogen receptor) signaling pathways often conferred resistance to this selection of drugs. Activation of the Notch1 pathway promoted acquired resistance to tamoxifen (an ER-targeted therapy) in serially passaged breast cancer xenografts in mice, and treating mice with a γ-secretase inhibitor to inhibit Notch signaling restored tamoxifen sensitivity. Markers of Notch1 activity in tumor tissue correlated with resistance to tamoxifen in breast cancer patients. Similarly, activation of Notch1 signaling promoted acquired resistance to MAPK inhibitors in BRAF(V600E) melanoma cells in culture, and the abundance of Notch1 pathway markers was increased in tumors from a subset of melanoma patients. Thus, Notch1 signaling may be a therapeutic target in some drug-resistant breast cancers and melanomas. Additionally, multiple resistance pathways were activated in melanoma cell lines with intrinsic resistance to MAPK inhibitors, and simultaneous inhibition of these pathways synergistically induced drug sensitivity. These data illustrate the potential for systematic identification of the signaling pathways controlling drug resistance that could inform clinical strategies and drug development for multiple types of cancer. This approach may also be used to advance clinical options in other disease contexts.

Myeloproliferative neoplasms (MPNs) frequently have an activating mutation in the gene encoding Janus kinase 2 (JAK2). Thus, targeting the pathway mediated by JAK and its downstream substrate, signal transducer and activator of transcription (STAT), may yield clinical benefit for patients with MPNs containing the JAK2V617F mutation. Although JAK inhibitor therapy reduces splenomegaly and improves systemic symptoms in patients, this treatment does not appreciably reduce the number of neoplastic cells. To identify potential mechanisms underlying this inherent resistance phenomenon, we performed pathway-centric, gain-of-function screens in JAK2V617F hematopoietic cells and found that the activation of the guanosine triphosphatase (GTPase) RAS or its effector pathways (mediated by the kinases AKT and ERK) renders cells insensitive to JAK inhibition. Resistant MPN cells became sensitized to JAK inhibitors when also exposed to inhibitors of the AKT or ERK pathways. Mechanistically, in JAK2V617F cells a JAK2-mediated inactivating phosphorylation of the pro-apoptotic protein BAD [B-cell lymphoma 2 (BCL-2)-associated death promoter] promoted cell survival. In sensitive cells, exposure to a JAK inhibitor resulted in dephosphorylation of BAD, enabling BAD to bind and sequester the pro-survival protein BCL-XL (also known as BCL2-like 1), thereby triggering apoptosis. In resistant cells, RAS effector pathways maintained BAD phosphorylation in the presence of JAK inhibitors, yielding a specific dependence on BCL-XL for survival. BCL-XL inhibitors potently induced apoptosis in JAK inhibitor-resistant cells. In patients with MPNs, activating mutations in RAS co-occur with the JAK2V617F mutation in the malignant cells, suggesting that RAS effector pathways likely play an important role in clinically observed resistance.

Transforming growth factor-beta (TGF-β) signaling has gained extensive interest in hepatocellular carcinoma (HCC). The small molecule kinase inhibitor galunisertib, targeting the TGF-β receptor I (TGF-βRI), blocks HCC progression in preclinical models and shows promising effects in ongoing clinical trials. As the drug is not similarly effective in all patients, this study was aimed at identifying new companion diagnostics biomarkers for patient stratification. Next-generation sequencing-based massive analysis of cDNA ends was used to investigate the transcriptome of an invasive HCC cell line responses to TGF-β1 and galunisertib. These identified mRNA were validated in 78 frozen HCC samples and in 26 ex-vivo HCC tissues treated in culture with galunisertib. Respective protein levels in patients blood were measured by enzyme-linked immunosorbent assay. SKIL, PMEPA1 ANGPTL4, SNAI1, Il11 and c4orf26 were strongly upregulated by TGF-β1 and downregulated by galunisertib in different HCC cell lines. In the 78 HCC samples, only SKIL and PMEPA1 (Pβ1. In ex-vivo samples, SKIL and PMEPA1 were strongly downregulated (PPβ1. SKIL and PMEPA1 mRNA expression in tumor tissues was significantly increased compared with controls and not correlated with protein levels in the blood of paired HCC patients. SKIL and PMEPA1 mRNA levels were positively correlated with TGF-β1 mRNA concentrations in HCC tissues and strongly downregulated by galunisertib. The target genes identified here may serve as biomarkers for the stratification of HCC patients undergoing treatment with galunisertib.

Activation of CD4+ T cells results in rapid proliferation and differentiation into effector and regulatory subsets. CD4+ effector T cell (Teff) (Th1 and Th17) and Treg subsets are metabolically distinct, yet the specific metabolic differences that modify T cell populations are uncertain. Here, we evaluated CD4+ T cell populations in murine models and determined that inflammatory Teffs maintain high expression of glycolytic genes and rely on high glycolytic rates, while Tregs are oxidative and require mitochondrial electron transport to proliferate, differentiate, and survive. Metabolic profiling revealed that pyruvate dehydrogenase (PDH) is a key bifurcation point between T cell glycolytic and oxidative metabolism. PDH function is inhibited by PDH kinases (PDHKs). PDHK1 was expressed in Th17 cells, but not Th1 cells, and at low levels in Tregs, and inhibition or knockdown of PDHK1 selectively suppressed Th17 cells and increased Tregs. This alteration in the CD4+ T cell populations was mediated in part through ROS, as N-acetyl cysteine (NAC) treatment restored Th17 cell generation. Moreover, inhibition of PDHK1 modulated immunity and protected animals against experimental autoimmune encephalomyelitis, decreasing Th17 cells and increasing Tregs. Together, these data show that CD4+ subsets utilize and require distinct metabolic programs that can be targeted to control specific T cell populations in autoimmune and inflammatory diseases.

Vascular plants possess multiple mechanisms for defending themselves against pathogens. One well-characterized defense mechanism is systemic acquired resistance (SAR). In SAR, a plant detects the presence of a pathogen and transmits a signal throughout the plant, inducing changes in the expression of various pathogenesis-related (PR) genes. Once SAR is established, the plant is capable of mounting rapid responses to subsequent pathogen attacks. SAR has been characterized in numerous angiosperm and gymnosperm species; however, despite several pieces of evidence suggesting SAR may also exist in non-vascular plants6–8, its presence in non-vascular plants has not been conclusively demonstrated, in part due to the lack of an appropriate culture system. Here, we describe and use a novel culture system to demonstrate that the moss species Amblystegium serpens does initiate a SAR-like reaction upon inoculation with Pythium irregulare, a common soil-borne oomycete. Infection of A. serpens gametophores by P. irregulare is characterized by localized cytoplasmic shrinkage within 34 h and chlorosis and necrosis within 7 d of inoculation. Within 24 h of a primary inoculation (induction), moss gametophores grown in culture became highly resistant to infection following subsequent inoculation (challenge) by the same pathogen. This increased resistance was a response to the pathogen itself and not to physical wounding. Treatment with β-1,3 glucan, a structural component of oomycete cell walls, was equally effective at triggering SAR. Our results demonstrate, for the first time, that this important defense mechanism exists in a non-vascular plant, and, together with previous studies, suggest that SAR arose prior to the divergence of vascular and non-vascular plants. In addition, this novel moss – pathogen culture system will be valuable for future characterization of the mechanism of SAR in moss, which is necessary for a better understanding of the evolutionary history of SAR in plants.

Current models of CML blast crisis (BC) propose that expression of BCR-ABL results in genomic instability and the acquisition of genetic alterations that affect cell proliferation and survival, self-renewal and differentiation. To characterize the molecular events that underlie progression, we performed whole genome sequencing of paired samples of the same patient at CP and at BC (n = 12), as well as expression and methylation arrays of these samples and a larger validation cohort of unpaired CD34-selected samples (n = 38). Contrary to expectations, we found that the CML BC genome is relatively quiescent with regards to SNVs, indels and structural variations. In contrast, we observed widespread hyper-methylation in BC that was associated with distinct changes in expression and was independent of lineage/differentiation state. These findings suggest that in addition to genetic alterations, epigenomic events are likely to contribute substantively to BC progression. To understand the functional effects of the dysregulated transcriptome and epigenome in BC CML, we employed both pharmacologic and genetic methods to target candidate genes of interest identified in our earlier studies. To induce de-methylation of the BC genome, we treated primary samples with low doses of decitabine, a DNMT inhibitor. We found that decitabine impaired colony formation ability of BC CD34+ progenitors and concomitantly activated regulators of myeloid differentiation that were both hyper-methylated and down-regulated in BC CD34+ progenitors, such as MPO and KLF1. These results suggest that hyper-methylation does contribute to BC CD34+ progenitor function, and support the use of epigenetic therapies as a rational approach to targeting BC. The genetic approach we chose was a CRISPR-based in vitro pooled screen. We created a custom library targeting 200 genes, with an average of 5 sgRNAs per gene, and 50 non-targeting controls. We transduced K562 with the library and harvested samples at different time-points post-transduction/selection - Day 0, 7 and 21 - for deep sequencing. As expected, sgRNAs targeting essential genes such as MYC and MCM 2-7 were recurrently depleted in the population over time. More importantly, enriched sgRNAs targeted genes including TET2, which has been previously reported to be inactivated in myeloid malignancies, as well as novel candidates including RREB1, a transcription factor that binds to RAS-responsive elements (RREs) and may be involved in MAPK signaling. We will validate these targets by knocking them out individually and assessing their effect on the ability of CP cells to serially replate and/or engraft immune-deficient mice. Disclosures Chuah: Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Chiltern International: Honoraria. Takahashi:Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; BMS: Honoraria, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Masis: Consultancy; Otsuka: Membership on an entity's Board of Directors or advisory committees; Celgene: Speakers Bureau; Sysmex: Research Funding, Speakers Bureau; Astellas: Speakers Bureau. Valent:Novartis: Consultancy, Honoraria, Research Funding; Ariad: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Celgene: Honoraria.

Despite significant advances in the development of cancer treatment in the last 20 years the best available therapies have marginally improved the survival rate of treated pancreatic cancer patients (from 3% to 6% five-year survival), making it still one the deadliest malignancies. Single standard of care cytotoxic and targeted therapies demonstrate little to no effect on patient survival. Even though we now understand some of the main genetic factors that are mutatated in pancreatic tumors, their role in drug response remains poorly understood. To identify drug-sensitizing genetic targets in cancer cells we have developed a high-throughput screening platform. In this methodology, pooled lentiviral libraries containing hundreds of knockout constructs specific for cancer-related genes are used to infect cancer cells. By treating the infected populations with therapeutic agents we are able to identify genetic targets that when inhibited synergize with existing drugs to cause increased cell death. We piloted this type of screening platform in HCT-116 cells, a KRAS mutant, MEK inhibitor resistant colorectal cancer cell line, using the MEK-inhibitor AZD-2644 and a pooled shRNA library. This study identified 4-7 target genes that when knocked down sensitized the previously resistant HCT-116 cells to AZD-2644. To conduct a more robust and representative sensitization screen we developed a lentiviral CRISPR-Cas9 library. By performing screens in a panel of pancreatic cancer cell lines, we have identified both cell line dependencies and sensitizers to targeted and cytotoxic chemotherapies. The findings from these screens provide potential mechanistic insights into the key survival signaling programs in pancreatic cancer as well as new therapeutic strategies. Citation Format: Edgar Ferrer-Lorenzo, Daniel P. Nussbaum, Peter Winter, Kris Wood. Systematic interrogation of druggable pathways in pancreatic adenocarcinomas using pooled gene-knockdown lentiviral libraries. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5465. doi:10.1158/1538-7445.AM2015-5465

Understanding the mechanisms of resistance to anticancer therapies may improve personalized treatment regimens and responses to therapy. The mitochondrial apoptosis pathway is activated by most targeted and cytotoxic therapies and represents a node that may be modulated for broad therapy resistance. It has been shown that some cancers, especially those of hematopoietic origin, are primed for apoptosis (as measured by BH3 Profiling) and consequently undergo apoptosis readily in response to therapies while others are unprimed and resistant. However, despite the importance of apoptotic priming in governing response to anticancer therapies, the upstream molecular determinants of apoptotic priming are unknown. Prior work has shown that oncogenes, including c-Myc, can sensitize some cells to apoptosis. We therefore hypothesized that lineage programming and activation of oncogenes may modulate apoptotic priming in normal cells undergoing neoplastic transformation. Using numerous in vitro and in vivo ovarian, breast, kidney and liver tumorigenesis models we discovered that the strongest determinant of apoptotic priming and chemosensitivity in a cancer cell is the level of priming measured in the cell of origin. To illustrate, ovarian adenocarcinomas, which originate from primed ovarian or fallopian tube epithelia, are considerably more primed than hepatocellular carcinomas originating from unprimed precursors. Notably, we also found that activation of certain oncogenes can meaningfully increase apoptotic priming and chemosensitivity while others decrease it. For example, transformation of normal epithelial cells via forced expression of the c-Myc oncogene increased priming and chemosensitivity. In contrast, activation of Ras in isogenic cells dramatically decreased both priming and chemosensitivity. We have systematically characterized the effects of 27 oncogenes on apoptotic priming and chemosensitivity including PI3K, Notch, Src, BRAF, β-catenin, Akt, and Aurora B kinase. Certain subsets of oncogenes consistently reduced apoptotic priming in cells undergoing transformation and, in combination with cell lineage programs, established broad resistance to targeted and cytotoxic therapies. Importantly, our experimental findings are complemented by clinical correlates. Overall, this work may aid efforts to deploy personalized and efficacious cancer treatments and enhance our knowledge of how cancers evade cell death, which is a hallmark of cancer. Citation Format: Kristopher A. Sarosiek, Alison Karst, Peter Winter, Antonio Sorrentino, Sourav Bandyopadhyay, Andrei Goga, Kris C. Wood, Ronny Drapkin, Anthony Letai. Broad therapy resistance is induced by suppression of apoptotic priming by lineage programs and oncogenic activation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 970. doi:10.1158/1538-7445.AM2015-970

Background: MLN2480 is an orally administered, investigational small molecule pan-RAF kinase inhibitor currently in Phase 1 clinical development. Here, we built integrated PK/PD/efficacy models to understand the relationship between MAPK pathway inhibition and efficacy as well as the effect of dose schedule in two melanoma xenograft models, A375 and SKMEL-2. Materials and Methods: We fit a one-compartment PK model to MLN2480 plasma concentration time profiles following a single dose in mice. Tumor pERK levels in xenograft-bearing mice, measured by Western blotting following single (A375) or multiple (SKMEL-2) doses of MLN2480 were fit to a direct or indirect inhibitory Emax model, respectively, to describe the PK/PD relationship between the plasma concentration and %pERK inhibition. Activity in each xenograft was evaluated as the percent growth rate inhibition (%GRI), the percent change between treated and control exponential tumor volume growth rates over the treatment cycle. To connect PK, PD, and efficacy for each xenograft, we simulated the PK and PD profiles over the treatment cycle for each dose group, and estimated the averages of plasma concentration (Cavg) and %pERK inhibition, respectively, using non-compartmental analysis. Results: From a multiple linear regression, schedule-related parameters are not predictive for drug effect demonstrating schedule-independent activity. For a variety of dose schedules, the relationship between total dose and SKMEL-2 xenograft growth rate is approximately linear (R = 0.90, p < 0.0001). A sigmoidal PK/efficacy model captures the relationship between Cavg and %GRI for both xenografts. A375 exhibits greater sensitivity than SKMEL-2 to the same Cavg of MLN2480, with the models predicting tumor regression (Emax = 172% GRI) and stasis (Emax = 102% GRI) as the maximal effects, respectively. Both xenografts have steep, sigmoidal PD/efficacy relationships, which show the same pERK inhibition is associated with greater %GRI in A375, and %GRI saturates with residual pERK (EC90 = 38% in A375, 22% in SKMEL-2). Conclusions: Integrated PK/PD and PK/efficacy modeling for MLN2480 response in A375 and SKMEL-2 xenografts shows a strong relationship between pERK inhibition and preclinical activity, and that maximal effect is achieved without complete inhibition of the MAPK pathway. The findings of pERK inhibition-dependent but not schedule-dependent activity provide translational guidance to clinical dose and schedule selection.