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Supplementary Figures S1-S8, with legends

Supplementary Table 1

Mutations in the TP53 tumor suppressor gene are common in many cancer types, including the acute myeloid leukemia (AML) subtype known as complex karyotype AML (CK-AML). Here, we identify a gain-of-function (GOF) Trp53 mutation that accelerates CK-AML initiation beyond p53 loss and, surprisingly, is required for disease maintenance. The Trp53R172H mutation (TP53R175H in humans) exhibits a neomorphic function by promoting aberrant self-renewal in leukemic cells, a phenotype that is present in hematopoietic stem and progenitor cells (HSPC) even prior to their transformation. We identify FOXH1 as a critical mediator of mutant p53 function that binds to and regulates stem cell–associated genes and transcriptional programs. Our results identify a context where mutant p53 acts as a bona fide oncogene that contributes to the pathogenesis of CK-AML and suggests a common biological theme for TP53 GOF in cancer.Significance:Our study demonstrates how a GOF p53 mutant can hijack an embryonic transcription factor to promote aberrant self-renewal. In this context, mutant Trp53 functions as an oncogene to both initiate and sustain myeloid leukemia and suggests a potential convergent activity of mutant Trp53 across cancer types.This article is highlighted in the In This Issue feature, p. 813

SUMMARYSomatic chromosomal deletions are prevalent in cancer, yet their functional contributions remain ill-defined. Among the most prominent of these events are deletions of chromosome 9p21.3, which disable a cell intrinsic barrier to tumorigenesis by eliminating the CDKN2A/B tumor suppressor genes. However, half of 9p21.3 deletions encompass a cluster of 16 type I interferons (IFNs) whose co-deletions have not been functionally characterized. To dissect how 9p21.3 and other genomic deletions impact cancer, we developed MACHETE (Molecular Alteration of Chromosomes with Engineered Tandem Elements), a genome engineering strategy that enables flexible modeling of megabase-sized deletions. Generation of 9p21.3-syntenic deletions in a mouse model of pancreatic cancer revealed that concomitant loss of Cdkn2a/b and the IFN cluster led to immune evasion and metastasis compared to Cdkn2a/b-only deletions. Mechanistically, IFN co-deletion disrupted type I IFN signaling, altered antigen-presenting cells, and facilitated escape from CD8+ T cell surveillance in a cell extrinsic manner requiring loss of interferon epsilon (Ifne). Our results establish co-deletions of the IFN cluster as a pervasive route to tumor immune evasion and metastasis, revealing how deletions can disable physically linked cell intrinsic and extrinsic tumor suppression. Our study establishes a framework to dissect the functions of genomic deletions in cancer and beyond.

The most prominent homozygous deletions in cancer affect chromosome 9p21.3 and eliminate CDKN2A/B tumor suppressors, disabling a cell-intrinsic barrier to tumorigenesis. Half of 9p21.3 deletions, however, also encompass a type I interferon (IFN) gene cluster; the consequences of this co-deletion remain unexplored. To functionally dissect 9p21.3 and other large genomic deletions, we developed a flexible deletion engineering strategy, MACHETE (molecular alteration of chromosomes with engineered tandem elements). Applying MACHETE to a syngeneic mouse model of pancreatic cancer, we found that co-deletion of the IFN cluster promoted immune evasion, metastasis and immunotherapy resistance. Mechanistically, IFN co-deletion disrupted type I IFN signaling in the tumor microenvironment, leading to marked changes in infiltrating immune cells and escape from CD8+ T-cell surveillance, effects largely driven by the poorly understood interferon epsilon. These results reveal a chromosomal deletion that disables both cell-intrinsic and cell-extrinsic tumor suppression and provide a framework for interrogating large deletions in cancer and beyond.

Mutations in genes encoding components of chromatin modifying and remodeling complexes are among the most frequently observed somatic events in human cancers. For example, missense and nonsense mutations targeting the mixed lineage leukemia family member 3 (MLL3/KMT2C) histone methyltransferase occur in a range of solid tumors and heterozygous deletions encompassing MLL3 occur in a subset of aggressive leukemias. Although MLL3 loss can promote tumorigenesis in mice, the molecular targets and biological processes by which MLL3 suppresses tumorigenesis remain poorly characterized. Here we combined genetic, epigenomic, and animal modeling approaches to demonstrate that one of the mechanisms by which MLL3 links chromatin remodeling to tumor suppression is by co-activating the Cdkn2a tumor suppressor locus. Disruption of Mll3 cooperates with Myc overexpression in the development of murine hepatocellular carcinoma (HCC), in which MLL3 binding to the Cdkn2a locus is blunted, resulting in reduced H3K4 methylation and low expression levels of the locus-encoded genes, Ink4a and Arf. Conversely, elevated MLL3 expression increases its binding to the CDKN2A locus and co-activates gene transcription. Endogenous Mll3 restoration reverses these chromatin and transcriptional effects and triggers Ink4a/Arf-dependent apoptosis. Underscoring the human relevance of this epistasis, we found that genomic alterations in MLL3 and CDKN2A display mutual exclusivity in human HCC samples. These results collectively point to a new mechanism for disrupting CDKN2A activity during cancer development and, in doing so, link MLL3 to an established tumor suppressor network.

Primary and metastatic tumors typically share the same driver gene mutations, but it is unclear if these mutations are functionally relevant across different anatomical sites. Among such mutations, inactivation of the tumor suppressor gene SMAD4 is a hallmark of pancreatic and other gastrointestinal cancers and has been associated with metastatic disease. Here, we develop a mouse model of pancreatic ductal adenocarcinoma that enables Smad4 depletion in the pre-malignant Kras-mutant pancreas and subsequent Smad4 reactivation in late-stage metastatic tumors. Whereas early Smad4 deficiency facilitated tumor formation, later Smad4 restoration had unexpected organ-specific outcomes: no effect on primary tumor growth, suppression of liver metastases, and promotion of lung metastases. Integrative multiomic analysis revealed a near-universal genomic deletion of the Cdkn2a/b locus and organ-specific changes in the tumor cells’ epigenomic state. In particular, the liver and lung differentially favored KLF vs. RUNX dominated chromatin programs, which were confirmed to underpin the divergent effects of Smad4 restoration in functional studies. Our results show how organ-dependent epigenomic changes lead to altered driver gene function in metastatic disease. This organ-specific gene-chromatin interplay may be a generalizable principle in cancer biology and invites a revised paradigm for precision oncology that considers anatomical site in the interpretation of tumor genetics. Citation Format: Kaloyan M. Tsanov, Francisco M. Barriga, Yu-Jui Ho, Direna Alonso-Curbelo, Geulah Livshits, Richard Koche, Timour Baslan, Alexandra N. Wuest, Janelle Simon, Sha Tian, Wei Luan, Scott W. Lowe. Organ-specific effects of Smad4 in metastatic pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3512.

The era of gene therapy has begun. In recent years, potentially breakthrough datasets and rapidly expanding company pipelines have begun to overshadow the unfulfilled promise characteristic of the gene therapy sector in decades prior. One barometer for progress in the space can be seen in stock markets, where NASDAQ-listed in vivo gene therapy companies we follow have increased from 4 companies with $1.9 billion in market capitalization on January 31, 2014, to 24 companies with $30.5 billion in market capitalization on October 31, 2018. For many in the financial community, a tangible signal for the emergence of the broader gene therapy space is the recent notable mergers and acquisitions activity, a signal that previously heralded the arrival of blockbuster biotechnologies like monoclonal antibodies. Notably, Novartis' $8.7 billion acquisition of in vivo adeno-associated virus 9–based gene therapy player, AveXis, earlier this year has focused many on looking for new investment opportunities in the space, thereby increasing interest in the valuation of gene therapy companies. This perspective discusses the theoretical underpinnings of company valuation and explains why traditional approaches have limitations when valuing in vivo gene therapy companies, which produce single treatments that may achieve durable or curative benefits. We use the AveXis case study to illustrate certain points on the valuation of breakthrough innovation that we think have broader applicability throughout the in vivo gene therapy space. This publication is the first in a three-part series. Future discussions in this series on in vivo gene therapy companies will explore real-world approaches and considerations that have already proven successful in mitigating the limitations of traditional valuation approaches as well as those that may soon emerge.

Tissue damage increases the risk of cancer through poorly understood mechanisms1. In mouse models of pancreatic cancer, pancreatitis associated with tissue injury collaborates with activating mutations in the Kras oncogene to markedly accelerate the formation of early neoplastic lesions and, ultimately, adenocarcinoma2,3. Here, by integrating genomics, single-cell chromatin assays and spatiotemporally controlled functional perturbations in autochthonous mouse models, we show that the combination of Kras mutation and tissue damage promotes a unique chromatin state in the pancreatic epithelium that distinguishes neoplastic transformation from normal regeneration and is selected for throughout malignant evolution. This cancer-associated epigenetic state emerges within 48 hours of pancreatic injury, and involves an ‘acinar-to-neoplasia’ chromatin switch that contributes to the early dysregulation of genes that define human pancreatic cancer. Among the factors that are most rapidly activated after tissue damage in the pre-malignant pancreatic epithelium is the alarmin cytokine interleukin 33, which recapitulates the effects of injury in cooperating with mutant Kras to unleash the epigenetic remodelling program of early neoplasia and neoplastic transformation. Collectively, our study demonstrates how gene–environment interactions can rapidly produce gene-regulatory programs that dictate early neoplastic commitment, and provides a molecular framework for understanding the interplay between genetic and environmental cues in the initiation of cancer. In mouse models of pancreatic cancer, a cooperative interaction between tissue damage and Kras gene mutation rapidly induces cancer-associated chromatin states in pre-malignant tissue, leading to gene dysregulation and neoplastic transformation.

Mutations in the TP53 tumor suppressor gene are common in many cancer types, including the acute myeloid leukemia (AML) subtype known as complex karyotype AML (CK-AML). Here, we identify a gain-of-function (GOF) Trp53 mutation that accelerates CK-AML initiation beyond p53 loss and, surprisingly, is required for disease maintenance. The Trp53R172H mutation (TP53R175H in humans) exhibits a neomorphic function by promoting aberrant self-renewal in leukemic cells, a phenotype that is present in hematopoietic stem and progenitor cells (HSPC) even prior to their transformation. We identify FOXH1 as a critical mediator of mutant p53 function that binds to and regulates stem cell–associated genes and transcriptional programs. Our results identify a context where mutant p53 acts as a bona fide oncogene that contributes to the pathogenesis of CK-AML and suggests a common biological theme for TP53 GOF in cancer. Significance: Our study demonstrates how a GOF p53 mutant can hijack an embryonic transcription factor to promote aberrant self-renewal. In this context, mutant Trp53 functions as an oncogene to both initiate and sustain myeloid leukemia and suggests a potential convergent activity of mutant Trp53 across cancer types. This article is highlighted in the In This Issue feature, p. 813

The pancreas produces many different hormones, as well as several substances important for digestion. To perform these roles, the pancreas contains different types of cells; for example, acinar cells make digestive enzymes that help to break down food. But, like other cells in the body, pancreatic cells can accumulate mutations in their DNA that cause them to divide, acquire an altered identity and form a cancerous tumor. The DNA of cells is packed into a structure called chromatin. While the DNA sequence is essentially the same across all normal cells of a given individual, chromatin can be more or less compacted in the different cell types that comprise our body tissues. A collection of proteins called the SWI/SNF complex can reorganize the chromatin to change how tightly the DNA is packed. This determines which genes in the DNA are accessible and can be activated, and which ones cannot. Around 25% of pancreatic cancers contain mutations in genes that produce proteins of the SWI/SNF complex. These mutations normally occur with an additional mutation that over-activates the gene that produces a potentially cancer-causing protein called Kras. Livshits et al. have now genetically engineered mice to investigate how one such SWI/SNF complex protein, called Arid1a, affects how pancreatic cancer develops using a genetic approach that made possible to temporarily halt the production of Arid1a in acinar cells by feeding these mice an antibiotic. The gene that produces Kras was also over-activated in the pancreases of the mice, making them more likely to develop cancer. Within just two weeks of stopping the production of Arid1a, the acinar cells stopped producing digestive enzymes and started making other proteins that are typically found in cancerous cells, indicating that Arid1a is involved in maintaining the normal identity and activity of these cells. Restoring the ability of altered acinar cells to produce normal levels of Arid1a (by removing the mice from the antibiotic diet) did not reverse these changes. Biochemical experiments showed that acinar cells with reduced levels of Arid1a have altered chromatin. In particular, the genes that produce digestive enzymes, which are normally active in healthy pancreases, were less accessible in mice who had over-active Kras and reduced levels of Arid1a. The results presented by Livshits et al. provide the first evidence of how alterations to Arid1a can lead to irreversible changes in the identity and activity of pancreatic acinar cells. These results will need to be carefully considered by researchers who are developing treatments for cancer patients with mutations in Arid1a and other SWI/SNF proteins. In particular, methods that attempt to restore the functions of absent SWI/SNF proteins to cancer cells are unlikely to treat the cancer successfully.

CRISPR-Cas9-based genome editing enables the rapid genetic manipulation of any genomic locus without the need for gene targeting by homologous recombination. Here we describe a conditional transgenic approach that allows temporal control of CRISPR-Cas9 activity for inducible genome editing in adult mice. We show that doxycycline-regulated Cas9 induction enables widespread gene disruption in multiple tissues and that limiting the duration of Cas9 expression or using a Cas9(D10A) (Cas9n) variant can regulate the frequency and size of target gene modifications, respectively. Further, we show that this inducible CRISPR (iCRISPR) system can be used effectively to create biallelic mutation in multiple target loci and, thus, provides a flexible and fast platform to study loss-of-function phenotypes in vivo.

Germinal centres are specialized structures wherein B lymphocytes undergo clonal expansion, class switch recombination, antibody gene diversification and affinity maturation. Three to four antigen-specific B cells colonize a follicle to establish a germinal centre and become rapidly dividing germinal-centre centroblasts that give rise to dark zones. Centroblasts produce non-proliferating centrocytes that are thought to migrate to the light zone of the germinal centre, which is rich in antigen-trapping follicular dendritic cells and CD4+ T cells. It has been proposed that centrocytes are selected in the light zone on the basis of their ability to bind cognate antigen. However, there have been no studies of germinal-centre dynamics or the migratory behaviour of germinal-centre cells in vivo. Here we report the direct visualization of B cells in lymph node germinal centres by two-photon laser-scanning microscopy in mice. Nearly all antigen-specific B cells participating in a germinal-centre reaction were motile and physically restricted to the germinal centre but migrated bi-directionally between dark and light zones. Notably, follicular B cells were frequent visitors to the germinal-centre compartment, suggesting that all B cells scan antigen trapped in germinal centres. Consistent with this observation, we found that high-affinity antigen-specific B cells can be recruited to an ongoing germinal-centre reaction. We conclude that the open structure of germinal centres enhances competition and ensures that rare high-affinity B cells can participate in antibody responses.

The recent discovery of the CRISPR/Cas system and repurposing of this technology to edit a variety of different genomes have revolutionized an array of scientific fields, from genetics and translational research, to agriculture and bioproduction. In particular, the prospect of rapid and precise genome editing in laboratory animals by CRISPR/Cas has generated an immense interest in the scientific community. Here we review current in vivo applications of CRISPR/Cas and how this technology can improve our knowledge of gene function and our understanding of biological processes in animal models.

Using ultrasound-guided in utero infections of fluorescently traceable lentiviruses carrying RNAi or Cre recombinase into mouse embryos, we have demonstrated noninvasive, highly efficient selective transduction of surface epithelium, in which progenitors stably incorporate and propagate the desired genetic alterations. We achieved epidermal-specific infection using small generic promoters of existing lentiviral short hairpin RNA libraries, thus enabling rapid assessment of gene function as well as complex genetic interactions in skin morphogenesis and disease in vivo. We adapted this technology to devise a new quantitative method for ascertaining whether a gene confers a growth advantage or disadvantage in skin tumorigenesis. Using alpha1-catenin as a model, we uncover new insights into its role as a widely expressed tumor suppressor and reveal physiological interactions between Ctnna1 and the Hras1-Mapk3 and Trp53 gene pathways in regulating skin cell proliferation and apoptosis. Our study illustrates the strategy and its broad applicability for investigations of tissue morphogenesis, lineage specification and cancers.

For more than two decades, genetically engineered mouse models have been key to our mechanistic understanding of tumorigenesis and cancer progression. Recently, the massive quantity of data emerging from cancer genomics studies has demanded a corresponding increase in the efficiency and throughput of in vivo models for functional testing of putative cancer genes. Already a mainstay of cancer research, recent innovations in RNA interference (RNAi) technology have extended its utility for studying gene function and genetic interactions, enabling tissue-specific, inducible and reversible gene silencing in vivo. Concurrent advances in embryonic stem cell (ESC) culture and genome engineering have accelerated several steps of genetically engineered mouse model production and have facilitated the incorporation of RNAi technology into these models. Here, we review the current state of these technologies and examine how their integration has the potential to dramatically enhance the throughput and capabilities of animal models for cancer.

Cadherin/catenin-based adhesions coordinate cellular growth, survival, migration, and differentiation within a tissue by mechanically anchoring cells to their neighbors. They also intersect with diverse signaling pathways in development and cancer. Although the adhesive functions of adherens junction proteins are well characterized, their contribution to other signaling pathways is less well understood. Here, we show that ablation of α-catenin in the epidermis selectively induces apoptosis in suprabasal differentiating keratinocytes while sparing basal cell progenitors. This protection from death is coupled to elevated focal adhesion signaling, faster migration, and an altered distribution of growth factor receptors. We show that simultaneous depletion of α-catenin and focal adhesion kinase or p21-activated kinase eliminates basal cell protection as well as the elevated migration and proliferation of cells. The increased dependency of cells upon matrix interactions for their survival when cell–cell adhesions are destabilized has important implications for cancer progression and metastasis.

The cyclin-dependent kinases (CDKs) that drive the eukaryotic cell cycle must be phosphorylated within the activation segment (T-loop) by a CDK-activating kinase (CAK) to achieve full activity. Although a requirement for CDK-activating phosphorylation is conserved throughout eukaryotic evolution, CAK itself has diverged between metazoans and budding yeast, and fission yeast has two CAKs, raising the possibility that additional mammalian enzymes remain to be identified. We report here the characterization of PNQALRE (also known as CCRK or p42), a member of the mammalian CDK family most similar to the cell-cycle effectors Cdk1 and Cdk2 and to the CAK, Cdk7. Although PNQALRE/CCRK was recently proposed to activate Cdk2, we show that the monomeric protein has no intrinsic CAK activity. Depletion of PNQALRE by >80% due to RNA interference (RNAi) impairs cell proliferation, but fails to arrest the cell cycle at a discrete point. Instead, both the fraction of cells with a sub-G(1) DNA content and cleavage of poly(ADP-ribose) polymerase (PARP) increase. PNQALRE knockdown did not diminish Cdk2 T-loop phosphorylation in vivo or decrease CAK activity of a cell extract. In contrast, depletion of Cdk7 by RNAi causes a proportional decrease in the ability of an extract to activate recombinant Cdk2. Our data do not support the proposed function of PNQALRE/CCRK in activating CDKs, but instead reinforce the notion of Cdk7 as the major, and to date the only, CAK in mammalian cells.

Colorectal cancer (CRC) is a leading cause of death in the developed world, yet facile preclinical models that mimic the natural stages of CRC progression are lacking. Through the orthotopic engraftment of colon organoids we describe a broadly usable immunocompetent CRC model that recapitulates the entire adenoma-adenocarcinoma-metastasis axis in vivo. The engraftment procedure takes less than 5 minutes, shows efficient tumor engraftment in two-thirds of mice, and can be achieved using organoids derived from genetically engineered mouse models (GEMMs), wild-type organoids engineered ex vivo, or from patient-derived human CRC organoids. In this model, we describe the genotype and time-dependent progression of CRCs from adenocarcinoma (6 weeks), to local disseminated disease (11-12 weeks), and spontaneous metastasis (>20 weeks). Further, we use the system to show that loss of dysregulated Wnt signaling is critical for the progression of disseminated CRCs. Thus, our approach provides a fast and flexible means to produce tailored CRC mouse models for genetic studies and pre-clinical investigation.