A hallmark of prostate cancer progression is dysregulation of lipid metabolism via overexpression of fatty acid synthase (FASN), a key enzyme in de novo fatty acid synthesis. Metastatic ...castration-resistant prostate cancer (mCRPC) develops resistance to inhibitors of androgen receptor (AR) signaling through a variety of mechanisms, including the emergence of the constitutively active AR variant V7 (AR-V7). Here, we developed an FASN inhibitor (IPI-9119) and demonstrated that selective FASN inhibition antagonizes CRPC growth through metabolic reprogramming and results in reduced protein expression and transcriptional activity of both full-length AR (AR-FL) and AR-V7. Activation of the reticulum endoplasmic stress response resulting in reduced protein synthesis was involved in IPI-9119–mediated inhibition of the AR pathway. In vivo, IPI-9119 reduced growth of AR-V7–driven CRPC xenografts and human mCRPC-derived organoids and enhanced the efficacy of enzalutamide in CRPC cells. In human mCRPC, both FASN and AR-FL were detected in 87% of metastases. AR-V7 was found in 39% of bone metastases and consistently coexpressed with FASN. In patients treated with enzalutamide and/or abiraterone FASN/AR-V7 double-positive metastases were found in 77% of cases. These findings provide a compelling rationale for the use of FASN inhibitors in mCRPCs, including those overexpressing AR-V7.
Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its ...potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.
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.
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.
Few therapies target the loss of tumor suppressor genes in cancer. We examine CRISPR-SpCas9 and RNA-interference loss-of-function screens to identify new therapeutic targets associated with genomic ...loss of tumor suppressor genes. The endosomal sorting complexes required for transport (ESCRT) ATPases VPS4A and VPS4B score as strong synthetic lethal dependencies. VPS4A is essential in cancers harboring loss of VPS4B adjacent to SMAD4 on chromosome 18q and VPS4B is required in tumors with co-deletion of VPS4A and CDH1 (E-cadherin) on chromosome 16q. We demonstrate that more than 30% of cancers selectively require VPS4A or VPS4B. VPS4A suppression in VPS4B-deficient cells selectively leads to ESCRT-III filament accumulation, cytokinesis defects, nuclear deformation, G2/M arrest, apoptosis, and potent tumor regression. CRISPR-SpCas9 screening and integrative genomic analysis reveal other ESCRT members, regulators of abscission, and interferon signaling as modifiers of VPS4A dependency. We describe a compendium of synthetic lethal vulnerabilities and nominate VPS4A and VPS4B as high-priority therapeutic targets for cancers with 18q or 16q loss.
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•Analysis of synthetic lethal interactions with tumor suppressor gene loss in cancer•VPS4A and VPS4B form a synthetic lethal pair in SMAD4- or CDH1-deleted cancers•VPS4A ablation causes abscission defects, nuclear deformation, and apoptosis•Dependency on VPS4A is modulated by other ESCRT proteins and interferon signaling
Neggers et al. identify the ATPases VPS4A and VPS4B as selective vulnerabilities and potential therapeutic targets in cancers harboring loss of chromosome 18q or 16q. In VPS4B-deficient cancers, VPS4A suppression leads to ESCRT-III dysfunction, nuclear deformation, and abscission defects. Moreover, ESCRT proteins and interferons can modulate dependency on VPS4A.
JC virus is a member of the Polyomavirus family of DNA tumor viruses and the causative agent of progressive multifocal leukoencephalopathy (PML). PML is a disease that occurs primarily in people who ...are immunocompromised and is usually fatal. As with other Polyomavirus family members, the replication of JC virus (JCV) DNA is dependent upon the virally encoded protein T-antigen. To further our understanding of JCV replication, we have determined the crystal structure of the origin-binding domain (OBD) of JCV T-antigen. This structure provides the first molecular understanding of JCV T-ag replication functions; for example, it suggests how the JCV T-ag OBD site-specifically binds to the major groove of GAGGC sequences in the origin. Furthermore, these studies suggest how the JCV OBDs interact during subsequent oligomerization events. We also report that the OBD contains a novel "pocket"; which sequesters the A1 & B2 loops of neighboring molecules. Mutagenesis of a residue in the pocket associated with the JCV T-ag OBD interfered with viral replication. Finally, we report that relative to the SV40 OBD, the surface of the JCV OBD contains one hemisphere that is highly conserved and one that is highly variable.
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.
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•scRNA-seq of metastatic pancreatic cancer and matched organoid models•Ex vivo to in vivo comparisons reveal loss of cell state heterogeneity in models•Cell state is shaped by the microenvironment in vivo and can be controlled ex vivo•Cell state drives drug response
Systematic profiling of metastatic pancreatic cancer biopsies and matched organoid models provides a view of cellular states, their regulation by the tumor microenvironment, and the ability to modulate these states to impact drug responses.
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 intra-tumoral 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 intra-tumoral heterogeneity identified through this clinically applicable mIF pipeline may inform prognosis and treatment selection for patients with PDAC.
Abstract
Background: Discovery of new biomarker-linked cancer therapeutic targets may enable novel drug development and ultimately lead to advances in clinical care. Somatic copy number alterations ...(CNAs) leading to loss of tumor suppressor gene function constitute important driver events in tumorigenesis. Unfortunately, there are few existing therapeutic options to target the oncogenic processes evoked by tumor suppressor inactivation. However, developing drugs that target tractable synthetic lethal interactions with common somatic CNAs represents a promising approach to attain cancer-selective therapeutics. Synthetic lethality refers to the observation that for certain gene pairs, inactivation of either gene is tolerated but combined loss-of-function of both genes results in decreased cell viability. Synthetic lethal relationships in cancer have been defined in several different contexts, including among paralog genes for which dependency on one paralog is conferred by loss of a second functionally redundant paralog gene. Since targeting synthetic lethal relationships in cancer may yield a wide therapeutic window of efficacy between tumor and normal cells, identification of pharmacologically tractable synthetic lethal targets remains a priority for oncology drug development programs. Results and Discussion: To systematically define synthetic lethal vulnerabilities associated with genomic loss of established tumor suppressor genes, we analyzed genome-scale CRISPR-SpCas9 and RNA interference loss-of-function screening data from over 600 cancer cell lines. We identified and prioritized 193 synthetic lethal interactions with genomic loss of one or more of 51 common tumor suppressor genes. In particular, we discovered that the paralog genes encoding vacuolar protein sorting 4 homolog A and B (VPS4A and VPS4B) are selective genetic vulnerabilities for tumors harboring genomic copy loss of SMAD4 or CDH1 due to co-deletion of VPS4B or VPS4A, respectively. VPS4B is located on the long arm (q) of chromosome 18, 12.3 Mb away from SMAD4, while VPS4A is located 0.476 Mb downstream of CDH1 (encoding E-cadherin) on chromosome 16q. Thus, cancer cells with genomic loss of VPS4B selectively depend on expression of VPS4A for survival, and tumors with loss of VPS4A depend on VPS4B expression. Co-deletion of SMAD4 and VPS4B is commonly observed in approximately 33% of human cancer, with particularly high rates of loss in pancreatic cancers (68%), colorectal (71%) and renal cell carcinomas (17%) and to a lesser extent in cancers of the bile duct, lung, prostate, esophagus, uterus, cervix and ovary. Meanwhile, loss of CDH1 and VPS4A occurs frequently in cancers of the stomach, breast, skin, colon and prostate. VPS4A and B function as AAA ATPases which are critical for the regulation of endosomal sorting complex required for transport (ESCRT), a multimeric protein complex essential for inverse membrane remodeling. The ESCRT machinery is involved in a range of cellular processes, including cytokinesis, membrane repair, autophagy and endosomal processing. VPS4A/B are believed to form asymmetric hexameric complexes that are recruited to ESCRT-III filaments to drive ESCRT-mediated membrane fission and sealing. Here, we demonstrate that suppression of VPS4A in cancer cells with reduced copy number of VPS4B leads to accumulation of CHMP4B-containing ESCRT-III filaments, cytokinesis defects, nuclear membrane abnormalities and micronucleation, ultimately resulting in G2/M cell cycle arrest and apoptosis. We also observed that VPS4 suppression leads to defects in endosomal and endoplasmic reticulum structure. Furthermore, upon VPS4A suppression, we observed potent in vivo tumor regressions, which led to markedly prolonged survival in mouse xenograft models of pancreatic cancer and rhabdomyosarcoma harboring genomic loss of VPS4B. To understand regulators of VPS4A dependency, we performed a CRISPR-SpCas9 genome-scale screen in a pancreatic cancer cell line in the context of VPS4A suppression. We identified multiple genes that promote or suppress VPS4A dependency. Cancer cell sensitivity to VPS4A suppression was potently enhanced by disruption of regulators of the abscission checkpoint, including genes encoding the ULK3 kinase and the ESCRT-III proteins CHMP1A and CHMP1B. The abscission checkpoint is a genome protection mechanism that relies on Aurora B kinase (AURKB) and ESCRT-III subunits to delay abscission in response to chromosome mis-segregation to avoid DNA damage and aneuploidy. These findings suggest that inhibition of the ESCRT pathway and blockade of the abscission checkpoint could provide strategies to further enhance sensitivity of cancer cells to VPS4A suppression. Moreover, through CRISPR-SpCas9 screening and integrative transcriptomic and proteomic analysis, we also identified a strong correlation between baseline interferon response gene expression and VPS4A dependency. Indeed, when we treated VPS4B-deficient cells with interferon-β and interferon-γ to induce interferon signaling, we observed a pronounced sensitization of these cells to VPS4A depletion, thus suggesting that immune signals from the tumor microenvironment may influence VPS4 dependency. These data collectively suggest potential future therapeutic strategies for combination with VPS4A inhibition. Finally, we demonstrate through mutant rescue experiments that the ATPase domain is critical for the function of VPS4A in mediating survival of cells with partial copy loss of VPS4B. Furthermore, we provide data that elucidate the degree to which VPS4A and VPS4B cooperate and form functional complexes in human cancer cells. Although VPS4A and B demonstrate 80.5% homology, the development of small molecules that differentially target VPS4A in cells with VPS4B loss or VPS4B in cells with VPS4A loss remains a tractable possibility due to small structural differences near the ATP-binding pocket. Moreover, combined inhibition of VPS4A and VPS4B may also prove effective and clinically tolerable given a potential therapeutic window arising from gene dosage alterations and differences in total VPS4A/B levels in tumor versus normal cells.
Citation Format: Jasper E. Neggers, Brenton Paolella, Adhana Asfaw, Michael V. Rothberg, Tom A. Skipper, Radha Kalekar, Michael Burger, Neekesh Dharia, Guillaume Kugener, Jeremie Kalfon, Nancy Dumont, Yvonne Li, Liam Spurr, Annan Yang, Wenbo Wu, AndrewAdam Durbin, Brian M. Wolpin, David E. Root, Jesse Boehm, Andrew D. Cherniack, Aviad Tsherniak, Andrew L. Hong, William C. Hahn, Kimberly Stegmaier, Todd Golub, Francisca Vazquez, Andrew J. Aguirre. Synthetic lethal interaction between the ESCRT paralog enzymes VPS4A and VPS4B in cancers harboring loss of chromosome 18q or 16q abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr NG01.
Abstract
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.
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