Ross A. Dickins

Senescence and tumour clearance is triggered by p53 restoration in murine liver carcinomas

Although cancer arises from a combination of mutations in oncogenes and tumour suppressor genes, the extent to which tumour suppressor gene loss is required for maintaining established tumours is poorly understood. p53 is an important tumour suppressor that acts to restrict proliferation in response to DNA damage or deregulation of mitogenic oncogenes, by leading to the induction of various cell cycle checkpoints, apoptosis or cellular senescence. Consequently, p53 mutations increase cell proliferation and survival, and in some settings promote genomic instability and resistance to certain chemotherapies. To determine the consequences of reactivating the p53 pathway in tumours, we used RNA interference (RNAi) to conditionally regulate endogenous p53 expression in a mosaic mouse model of liver carcinoma. We show that even brief reactivation of endogenous p53 in p53-deficient tumours can produce complete tumour regressions. The primary response to p53 was not apoptosis, but instead involved the induction of a cellular senescence program that was associated with differentiation and the upregulation of inflammatory cytokines. This program, although producing only cell cycle arrest in vitro, also triggered an innate immune response that targeted the tumour cells in vivo, thereby contributing to tumour clearance. Our study indicates that p53 loss can be required for the maintenance of aggressive carcinomas, and illustrates how the cellular senescence program can act together with the innate immune system to potently limit tumour growth.

Probing tumor phenotypes using stable and regulated synthetic microRNA precursors.

RNA interference is a powerful method for suppressing gene expression in mammalian cells. Stable knock-down can be achieved by continuous expression of synthetic short hairpin RNAs, typically from RNA polymerase III promoters. But primary microRNA transcripts, which are endogenous triggers of RNA interference, are normally synthesized by RNA polymerase II. Here we show that RNA polymerase II promoters expressing rationally designed primary microRNA–based short hairpin RNAs produce potent, stable and regulatable gene knock-down in cultured cells and in animals, even when present at a single copy in the genome. Most notably, by tightly regulating Trp53 knock-down using tetracycline-based systems, we show that cultured mouse fibroblasts can be switched between proliferative and senescent states and that tumors induced by Trp53 suppression and cooperating oncogenes regress upon re-expression of Trp53. In practice, this primary microRNA–based short hairpin RNA vector system is markedly similar to cDNA overexpression systems and is a powerful tool for studying gene function in cells and animals.

Role of genomic instability and p53 in AID-induced c-myc-Igh translocations

Chromosomal translocations involving the immunoglobulin switch region are a hallmark feature of B-cell malignancies. However, little is known about the molecular mechanism by which primary B cells acquire or guard against these lesions. Here we find that translocations between c-myc and the IgH locus (Igh) are induced in primary B cells within hours of expression of the catalytically active form of activation-induced cytidine deaminase (AID), an enzyme that deaminates cytosine to produce uracil in DNA. Translocation also requires uracil DNA glycosylase (UNG), which removes uracil from DNA to create abasic sites that are then processed to double-strand breaks. The pathway that mediates aberrant joining of c-myc and Igh differs from intrachromosomal repair during immunoglobulin class switch recombination in that it does not require histone H2AX, p53 binding protein 1 (53BP1) or the non-homologous end-joining protein Ku80. In addition, translocations are inhibited by the tumour suppressors ATM, Nbs1, p19 (Arf) and p53, which is consistent with activation of DNA damage- and oncogenic stress-induced checkpoints during physiological class switching. Finally, we demonstrate that accumulation of AID-dependent, IgH-associated chromosomal lesions is not sufficient to enhance c-myc–Igh translocations. Our findings reveal a pathway for surveillance and protection against AID-dependent DNA damage, leading to chromosomal translocations.

The genomic landscape of hypodiploid acute lymphoblastic leukemia

The genetic basis of hypodiploid acute lymphoblastic leukemia (ALL), a subtype of ALL characterized by aneuploidy and poor outcome, is unknown. Genomic profiling of 124 hypodiploid ALL cases, including whole-genome and exome sequencing of 40 cases, identified two subtypes that differ in the severity of aneuploidy, transcriptional profiles and submicroscopic genetic alterations. Near-haploid ALL with 24–31 chromosomes harbor alterations targeting receptor tyrosine kinase signaling and Ras signaling (71%) and the lymphoid transcription factor gene IKZF3 (encoding AIOLOS; 13%). In contrast, low-hypodiploid ALL with 32–39 chromosomes are characterized by alterations in TP53 (91.2%) that are commonly present in nontumor cells, IKZF2 (encoding HELIOS; 53%) and RB1 (41%). Both near-haploid and low-hypodiploid leukemic cells show activation of Ras-signaling and phosphoinositide 3-kinase (PI3K)-signaling pathways and are sensitive to PI3K inhibitors, indicating that these drugs should be explored as a new therapeutic strategy for this aggressive form of leukemia.

Dissecting the unique role of the retinoblastoma tumor suppressor during cellular senescence

The RB protein family (RB, p107, and p130) has overlapping and compensatory functions in cell-cycle control. However, cancer-associated mutations are almost exclusively found in RB, implying that RB has a nonredundant role in tumor suppression. We demonstrate that RB preferentially associates with E2F target genes involved in DNA replication and is uniquely required to repress these genes during senescence but not other growth states. Consequently, RB loss leads to inappropriate DNA synthesis following a senescence trigger and, together with disruption of a p21-mediated cell-cycle checkpoint, enables extensive proliferation and rampant genomic instability. Our results identify a nonredundant RB effector function that may contribute to tumor suppression and reveal how loss of RB and p53 cooperate to bypass senescence.

ARF functions as a melanoma tumor suppressor by inducing p53-independent senescence.

Inactivation of the p53 pathway represents the most common molecular defect of human cancer. But in the setting of melanoma, a highly aggressive and invariably fatal malignancy in its advanced disseminated form, mutation/deletion of p53 is relatively rare, whereas its positive regulator ARF is often lost. Here, we show that genetic deficiency in Arf but not p53 facilitates rapid development of melanoma in a genetically engineered mouse model. This difference is accounted for, at least in part, by the unanticipated observation that, unlike fibroblasts, senescence control in melanocytes is strongly regulated by Arf and not p53. Moreover, oncogenic NRAS collaborates with deficiency in Arf, but not p53, to fully transform melanocytes. Our data demonstrate that ARF and p53, although linked in a common pathway, suppress tumorigenesis through distinct, lineage-dependent mechanisms and suggest that ARF helps restrict melanoma progression by executing the oncogene-induced senescence program in benign nevi. Thus, therapeutics designed to restore wild-type p53 function may be insufficient to counter melanoma and other malignancies in which ARF holds p53-independent tumor suppressor activity.

MLL3 Is a Haploinsufficient 7q Tumor Suppressor in Acute Myeloid Leukemia

Recurring deletions of chromosome 7 and 7q [-7/del(7q)] occur in myelodysplastic syndromes and acute myeloid leukemia (AML) and are associated with poor prognosis. However, the identity of functionally relevant tumor suppressors on 7q remains unclear. Using RNAi and CRISPR/Cas9 approaches, we show that an ∼50% reduction in gene dosage of the mixed lineage leukemia 3 (MLL3) gene, located on 7q36.1, cooperates with other events occurring in -7/del(7q) AMLs to promote leukemogenesis. Mll3 suppression impairs the differentiation of HSPC. Interestingly, Mll3-suppressed leukemias, like human -7/del(7q) AMLs, are refractory to conventional chemotherapy but sensitive to the BET inhibitor JQ1. Thus, our mouse model functionally validates MLL3 as a haploinsufficient 7q tumor suppressor and suggests a therapeutic option for this aggressive disease.

A pipeline for the generation of shRNA transgenic mice

RNA interference (RNAi) is an extremely effective tool for studying gene function in almost all metazoan and eukaryotic model systems. RNAi in mice, through the expression of short hairpin RNAs (shRNAs), offers something not easily achieved with traditional genetic approaches—inducible and reversible gene silencing. However, technical variability associated with the production of shRNA transgenic strains has so far limited their widespread use. Here we describe a pipeline for the generation of miR30-based shRNA transgenic mice that enables efficient and consistent targeting of doxycycline-regulated, fluorescence-linked shRNAs to the Col1a1 locus. Notably, the protocol details crucial steps in the design and testing of miR30-based shRNAs to maximize the potential for developing effective transgenic strains. In all, this 14-week procedure provides a fast and cost-effective way for any laboratory to investigate gene function in vivo in the mouse.

The Ubiquitin Ligase Component Siah1a Is Required for Completion of Meiosis I in Male Mice

ABSTRACT The mammalian Siah genes encode highly conserved proteins containing a RING domain. As components of E3 ubiquitin ligase complexes, Siah proteins facilitate the ubiquitination and degradation of diverse protein partners including β-catenin, N-CoR, and DCC. We used gene targeting in mice to analyze the function of Siah1a during mammalian development and reveal novel roles in growth, viability, and fertility. Mutant animals have normal weights at term but are postnatally growth retarded, despite normal levels of pituitary growth hormone. Embryonic fibroblasts isolated from mutant animals grow normally. Most animals die before weaning, and few survive beyond 3 months. Serum gonadotropin levels are normal in Siah1a mutant mice; however, females are subfertile and males are sterile due to a block in spermatogenesis. Although spermatocytes in mutant mice display normal meiotic prophase and meiosis I spindle formation, they accumulate at metaphase to telophase of meiosis I and subsequently undergo apoptosis. The requirement of Siah1a for normal progression beyond metaphase I suggests that Siah1a may be part of a novel E3 complex acting late in the first meiotic division.

Generation and analysis of Siah2 mutant mice.

ABSTRACT Siah proteins function as E3 ubiquitin ligase enzymes to target the degradation of diverse protein substrates. To characterize the physiological roles of Siah2, we have generated and analyzed Siah2 mutant mice. In contrast to Siah1a knockout mice, which are growth retarded and exhibit defects in spermatogenesis, Siah2 mutant mice are fertile and largely phenotypically normal. While previous studies implicate Siah2 in the regulation of TRAF2, Vav1, OBF-1, and DCC, we find that a variety of responses mediated by these proteins are unaffected by loss of Siah2. However, we have identified an expansion of myeloid progenitor cells in the bone marrow of Siah2 mutant mice. Consistent with this, we show that Siah2 mutant bone marrow produces more osteoclasts in vitro than wild-type bone marrow. The observation that combined Siah2 and Siah1a mutation causes embryonic and neonatal lethality demonstrates that the highly homologous Siah proteins have partially overlapping functions in vivo.