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Defining tumor cell immune evasion Mouse models used to study cancer often lack a full immune system, allowing implantation of human tumors into the mice. By contrast, naturally evolving tumors must contend with a fully functional immune system and its destruction of some of the cells (see the Perspective by Ho and Wood). Two groups now report studies on mouse models with a fully intact immune system. Martin et al . started with preexisting murine tumor cell lines and examined their continued evolution in vivo, whereas Del Poggetto et al . examined the development of new pancreatic tumors in the context of inflammation, as is often seen in human patients. In each study, the authors found that the immune system exerted a selective pressure on cells that would give rise to tumors, promoting the survival of those that had lost expression of tumor suppressor genes or activated a specific oncogene. The findings suggest a major role for the immune system in driving tumor evolution across multiple types of cancer. —YN

Background & Aims Understanding the mechanisms by which tumors adapt to therapy is critical for developing effective combination therapeutic approaches to improve clinical outcomes for patients with cancer. Methods To identify promising and clinically actionable targets for managing colorectal cancer (CRC), we conducted a patient-centered functional genomics platform that includes approximately 200 genes and paired this with a high-throughput drug screen that includes 262 compounds in four patient-derived xenografts (PDXs) from patients with CRC. Results Both screening methods identified exportin 1 (XPO1) inhibitors as drivers of DNA damage-induced lethality in CRC. Molecular characterization of the cellular response to XPO1 inhibition uncovered an adaptive mechanism that limited the duration of response in TP53-mutated, but not in TP53-wild-type CRC models. Comprehensive proteomic and transcriptomic characterization revealed that the ATM/ATR-CHK1/2 axes were selectively engaged in TP53-mutant CRC cells upon XPO1 inhibitor treatment and that this response was required for adapting to therapy and escaping cell death. Administration of KPT-8602, an XPO1 inhibitor, followed by AZD-6738, an ATR inhibitor, resulted in dramatic antitumor effects and prolonged survival in TP53-mutant models of CRC. Conclusions Our findings anticipate tremendous therapeutic benefit and support the further evaluation of XPO1 inhibitors, especially in combination with DNA damage checkpoint inhibitors, to elicit an enduring clinical response in patients with CRC harboring TP53 mutations.

Alterations in chromatin remodeling genes have been increasingly implicated in human oncogenesis. Specifically, the biallelic inactivation of the SWI/SNF subunit SMARCB1 results in the emergence of extremely aggressive pediatric malignancies. Here, we developed embryonic mosaic mouse models of malignant rhabdoid tumors (MRT) that faithfully recapitulate the clinical-pathological features of the human disease. We demonstrated that SMARCB1-deficient malignancies exhibit dramatic activation of the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress response via genetically intact MYC-p19(ARF)-p53 axis. As a consequence, these tumors display an exquisite sensitivity to agents inducing proteotoxic stress and inhibition of the autophagic machinery. In conclusion, our findings provide rationale for drug repositioning trials investigating combinations of agents targeting the UPR and autophagy in SMARCB1-deficient MRT.

Tumor functional heterogeneity has been recognized for decades, and technological advancements are fueling renewed interest in uncovering the cell-intrinsic and extrinsic factors that influence tumor development and therapeutic response. Intratumoral heterogeneity is now arguably one of the most-studied topics in tumor biology, leading to the discovery of new paradigms and reinterpretation of old ones, as we aim to understand the profound implications that genomic, epigenomic, and functional heterogeneity hold with regard to clinical outcomes. In spite of our improved understanding of the biological complexity of cancer, characterization of tumor metabolic heterogeneity has lagged behind, lost in a century-old controversy debating whether glycolysis or mitochondrial respiration is more influential. But is tumor metabolism really so simple? Here, we review historical and current views of intratumoral heterogeneity, with an emphasis on summarizing the emerging data that begin to illuminate just how vast the spectrum of metabolic strategies a tumor can employ may be, and what this means for how we might interpret other tumor characteristics, such as mutational landscape, contribution of microenvironmental influences, and treatment resistance.

Adaptive drug-resistance mechanisms allow human tumors to evade treatment through selection and expansion of treatment-resistant clones. Modeling the functional heterogeneity of tumors can unmask critical contributions of distinct tumor cell sub-populations toward identifying rational drug combinations. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro adherent cultures and in vivo tumors are maintained by a common set of long term self-renewing tumorigenic cells that can be used to establish clonal replica tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of clonal dynamics and adaptive responses to therapeutic challenge over time, we uncovered that the tumorigenic compartment of pancreatic tumors maintains a multitude of functionally heterogeneous subpopulations of cells with differential degrees of sensitivity to therapeutics. High-throughput isolation and deep characterization of unique clonal lineages showed genetic and transcriptomic diversity underlying the functionally diverse subpopulations, positioning the origins of tumor heterogeneity within the long-term self-renewing compartment. Molecular annotation of gemcitabine-naïve clonal lineages with distinct responses to treatment in the context of CRTs generated signatures that can predict the response to chemotherapy and exposed pre-existing functional mechanisms of clonal resistance, primarily associated to DNA damage tolerance and mitochondrial respiration (OXPHOS). Further transcriptomic and metabolic characterization of residual tumor cells in patient derived xenograft models as well as in patients after chemoradiation showed that resistant cells that contribute to tumor relapse are metabolically rewired to upregulate OXPHOS. Combining a novel inhibitor of oxidative phosphorylation (IACS-10759) developed at the MD Anderson Institute for Applied Cancer Science, and currently in phase I clinical trial in acute myeloid leukemia and solid tumors, with standard of care drugs drastically reduces tumor clonal complexity, underscoring the promise of inhibiting mitochondrial respiration as a new therapeutic strategy to prolong patient survival by eradicating resistant clones that survive chemoradiation. Our study, correlating genomic and transcriptomic traits with specific functional phenotypes, uncovered new mechanisms that underlie intra-tumor sub-clonal heterogeneity, influence treatment response to drugs and sustain tumor relapse. Citation Format: Sahil Seth, Chieh-Yuan Li, Sara Loponte, I-Lin Ho, Denise Corti, Luigi Sapio, Edoardo Del Poggetto, Michael Peoples, Tatiana Karpinets, Frederick S. Robinson, Shan Jiang, Prasanta Dutta, Joseph Marszalek, Maria E. Di Francesco, Timothy P. Heffernan, Virginia Giuliani, Pratip K. Bhattacharya, Giannicola Genovese, Andrew Futreal, Giulio Draetta, Andrea Viale, Alessandro Carugo. Dissection of clonal heterogeneity unmasks pre-existing chemoresistance and new metabolic vulnerabilities in pancreatic cancer [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 2900.

A major barrier to achieving durable remission and definitive cure in oncology patients is the emergence of tumor resistance, a common outcome of different disease types independent from the therapeutic approach undertaken. Patients with pancreatic ductal adenocarcinoma (PDAC) continue to have a poor prognosis despite concerted efforts to advance new drugs to the clinic. One reason for this, in PDAC and other tumors, is that tumors are constantly adapting and evolving in response to external perturbations. To better investigate tumor evolution in response to therapy we developed a new clonal tracking platform that enables the in vivo study of long term self-renewing compartments and the generation of cohorts of patient-derived xenografts in which tumors are virtually identical and maintained by the same clones (clonal replica tumors), representing a unique tool to address fundamental questions about clonal dynamics in response to pharmacological treatment. Using this novel approach we demonstrate that standard of care in pancreatic cancer, despite inducing tumor regression, has minimal effect on the clonal composition of tumors that eventually relapse. Transcriptomic and metabolic characterization of residual tumor cells in patient derived xenograft models as well as in patients after chemoradiation shows that resistant cells that contribute to tumor relapse are metabolically rewired to upregulate mitochondrial respiration (OXPHOS). Combining a novel inhibitor of oxidative phosphorylation (IACS-010759), developed at the MD Anderson Institute for Applied Cancer Science and currently in phase I clinical trial in relapsed/refractory acute myelogenous leukemia and advanced solid tumors, with standard of care drugs drastically reduces tumor clonal complexity, underscoring the promise of inhibiting mitochondrial respiration as a new therapeutic strategy to prolong patient survival by eradicating resistant clones that survive chemoradiation. Citation Format: Sara Loponte, Denise Corti, Sahil Seth, Edoardo Del Poggetto, I-Lin Ho, Chieh-Yuan Li, Shan Jiang, Joseph R. Marszalek, Maria Emilia Di Francesco, Giannicola Genovese, Giulio Draetta, Alessandro Carugo, Andrea Viale. Metabolic targeting of chemoresistance perturbs clonal complexity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4953.

Intrinsic and adaptive drug-resistance mechanisms allow human tumors to evade treatment through the demonstrated expansion of treatment-resistant clones. Thus, tumors are complex, dynamic ecosystems wherein populations of cells harboring both founder clones and unique, subclonal mutations coexist and progressively evolve. Modeling this functional heterogeneity of tumors can uncover critical contributions of distinct tumor cell sub-populations toward identifying rational drug combinations. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro adherent cultures and in vivo tumors are maintained by a common set of long-term self-renewing cells that can be used to establish Clonal Replica Tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of clonal dynamics and adaptive responses to therapeutic challenge across different animals over time, we uncovered that the long term self-renewing compartment of pancreatic cancer is represented by a multitude of functionally heterogeneous subpopulations of cells with differential degrees of sensitivity to therapeutics. Consistent with the stem cell hypothesis, although tumors respond to treatments and undergo a transient regression, their clonal complexity at the time of relapse is only partially compromised, implying that many tumorigenic cells survive the treatment and sustain tumor relapse. Moreover, our ability to track the same cell populations in different animals enabled us to demonstrate that the clonal composition of relapsed pancreatic tumors varied across the different drug treatment groups (gemcitabine, MEK1 inhibitor and dual PI3K/mTOR inhibitor), suggesting that the compartment of long-term self-renewing tumorigenic cells is highly functionally diverse in mediating drug resistance to different therapies. Notably, high-throughput isolation and deep characterization of unique clonal lineages isolated through CRTs demonstrated that individual self-renewing populations display a remarkable genetic and molecular heterogeneity that can account for the differential functional responses and adaptation to perturbations. So, our findings portend a model in which the genomic and functional heterogeneity within human tumors is maintained, propagated and recapitulated entirely by distinct pools of long-term self-renewing cells. This concept has important implications for the efficacy of pharmacological combinations, which has historically been ascribed to their synergistic effects to abrogate the emergence of resistance, may instead be linked to the ability of mechanistically unrelated drugs to delay relapse by targeting multiple populations of tumorigenic cells simultaneously. Citation Format: Sahil Seth, Chieh-Yuan Li, I-Lin Ho, Denise Corti, Sara Loponte, Luigi Sapio, Edoardo Del Poggetto, Michael Peoples, Tatiana Karpinets, Giannicola Genovese, Andrew Futreal, Giulio Draetta, Alessandro Carugo, Andrea Viale. Clonal dissection of pancreatic tumors unmasks functional and genomic heterogeneous long-term self-renewing compartments at the origin of treatment resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1177.

Tumor evolution and adaptation, especially in response to therapy, are well-established concepts in clinical oncology and a major causes of treatment failure. The inability of current experimental models, including genetically engineered mouse models, to inform on the breadth of functional heterogeneity within human tumors has substantially limited our understanding of the evolution of tumor architecture under perturbations such as pharmacologic treatments with a profound negative impact on cancer drug discovery research, where drug efficacy continues to be measured in terms of tumor volume. To address this technologic gap, we developed a new approach based on clonal tracking to model human tumors in vivo supporting the dissection of functional heterogeneity at the clonal level as well as the study of tumor evolution and clonal dynamics in response to external perturbations in real time. Lentivirus-based systems have been extensively used as a tool to investigate the clonal dynamics and tumor cell heterogeneities of solid tumors, but they have been limited by a lack of sensitivity and the impossibility of tracking identical clones in different animals. Using a revised strategy to barcode patient-derived pancreatic cancer cells coupled with deep-sequencing analysis, we created PDX cohorts harboring Clonal Replica Tumors (hereafter CRTs), in which all mice bear human tumors maintained by the same clones. Because they are maintained by thousands of common clones and are virtually identical, CRTs enable the evaluation of single- and combined-therapy approaches as well as mechanistic studies where the evolution and dynamics of single clones can be tracked with extremely high precision to monitor the contribution of even low-represented clones to tumor growth and relapse. Since functional heterogeneity in tumors can only be fully appreciated upon different perturbations, the availability of large cohorts of animals bearing clonally identical tumors makes CRTs a critical instrument to investigate tumor complexity in vivo. Further, another advancement supported by the CRT platform is the isolation and expansion of any clone of interest identified in vivo through bioinformatics analysis. High-throughput isolation and functional characterization of virtually every clonal population of cells within a CRT provides an invaluable tool to identify exploitable vulnerabilities of resistant clones. In conclusion, CRTs represent an innovative approach to dissect the complexity of human tumors at an unprecedented level of resolution, enabling the investigation of mechanisms of tumor evolution and drug resistance. Understanding clonal dynamics, tumor composition, and adaptive mechanisms, and how these factors may influence treatment response, is essential to reach new horizons in cancer care. Citation Format: Sahil Seth, Chieh-Yuan Li, Denise Corti, Sara Loponte, I-Lin Ho, Edoardo Del Poggetto, Michael Peoples, Christopher Bristow, Joseph Marszalek, Timothy Heffernan, Giannicola Genovese, Giulio Draetta, Alessandro Carugo, Andrea Viale. Generation of clonal replica tumors to interrogate complexity of human cancer in vivo [abstract]. In: Proceedings of the AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; 2017 Sep 24-27; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(10 Suppl):Abstract nr A15.

Histone deacetylases (HDACs) catalyze the removal of acetyl molecules from histone and non-histone substrates playing important roles in chromatin remodeling and control of gene expression. Class I HDAC1 is a critical regulator of cell cycle progression, cellular proliferation and differentiation during development; it is also regulated by many post-translational modifications (PTMs). Herein we characterize a new mitosis-specific phosphorylation of HDAC1 driven by Aurora kinases A and B. We show that this phosphorylation affects HDAC1 enzymatic activity and it is critical for the maintenance of a proper proliferative and developmental plan in a complex organism. Notably, we find that Aurora-dependent phosphorylation of HDAC1 regulates histone acetylation by modulating the expression of genes directly involved in the developing zebrafish central nervous system. Our data represent a step towards the comprehension of HDAC1 regulation by its PTM code, with important implications in unravelling its roles both in physiology and pathology.

Intrinsic and adaptive drug-resistance mechanisms allow human tumors to evade treatment through the demonstrated expansion of treatment-resistant clones. Thus, tumors are complex, dynamic ecosystems wherein populations of cells harboring both founder clones and unique, subclonal mutations coexist and progressively evolve. Modeling this functional heterogeneity of tumors can uncover critical contributions of distinct tumor cell sub-populations toward identifying rational drug combinations. Here, studying clonal evolution of tumor cells derived from human pancreatic tumors, we demonstrate that in vitro adherent cultures and in vivo tumors are maintained by a common set of long-term self-renewing cells that can be used to establish Clonal Replica Tumors (CRTs), large cohorts of animals bearing human tumors with identical clonal composition. Using CRTs to conduct quantitative assessments of clonal dynamics and adaptive responses to therapeutic challenge across different animals over time, we uncovered that the long term self-renewing compartment of pancreatic cancer is represented by a multitude of functionally heterogeneous subpopulations of cells with differential degrees of sensitivity to therapeutics. Consistent with the stem cell hypothesis, although tumors respond to treatments and undergo a transient regression, their clonal complexity at the time of relapse is only partially compromised, implying that many tumorigenic cells survive the treatment and sustain tumor relapse. Moreover, our ability to track the same cell populations in different animals enabled us to demonstrate that the clonal composition of relapsed pancreatic tumors varied across the different drug treatment groups (gemcitabine, MEK1 inhibitor and dual PI3K/mTOR inhibitor), suggesting that the compartment of long-term self-renewing tumorigenic cells is highly functionally diverse in mediating drug resistance to different therapies. Notably, high-throughput isolation and deep characterization of unique clonal lineages isolated through CRTs demonstrated that individual self-renewing populations display a remarkable genetic and molecular heterogeneity that can account for the differential functional responses and adaptation to perturbations. So, our findings portend a model in which the genomic and functional heterogeneity within human tumors is maintained, propagated and recapitulated entirely by distinct pools of long-term self-renewing cells. This concept has important implications for the efficacy of pharmacological combinations, which has historically been ascribed to their synergistic effects to abrogate the emergence of resistance, may instead be linked to the ability of mechanistically unrelated drugs to delay relapse by targeting multiple populations of tumorigenic cells simultaneously.