Flexible Usage and Interconnectivity of Diverse Cell Death Pathways Protect against Intracellular Infection.

Programmed cell death contributes to host defense against pathogens. To investigate the relative importance of pyroptosis, necroptosis, and apoptosis during Salmonella infection, we infected mice and macrophages deficient for diverse combinations of caspases-1, -11, -12, and -8 and receptor interacting serine/threonine kinase 3 (RIPK3). Loss of pyroptosis, caspase-8-driven apoptosis, or necroptosis had minor impact on Salmonella control. However, combined deficiency of these cell death pathways caused loss of bacterial control in mice and their macrophages, demonstrating that host defense can employ varying components of several cell death pathways to limit intracellular infections. This flexible use of distinct cell death pathways involved extensive cross-talk between initiators and effectors of pyroptosis and apoptosis, where initiator caspases-1 and -8 also functioned as executioners when all known effectors of cell death were absent. These findings uncover a highly coordinated and flexible cell death system with in-built fail-safe processes that protect the host from intracellular infections.

MYB orchestrates T cell exhaustion and response to checkpoint inhibition.

CD8+ T cells that respond to chronic viral infections or cancer are characterized by the expression of inhibitory receptors such as programmed cell death protein 1 (PD-1) and by the impaired production of cytokines. This state of restrained functionality-which is referred to as T cell exhaustion1,2-is maintained by precursors of exhausted T (TPEX) cells that express the transcription factor T cell factor 1 (TCF1), self-renew and give rise to TCF1- exhausted effector T cells3-6. Here we show that the long-term proliferative potential, multipotency and repopulation capacity of exhausted T cells during chronic infection are selectively preserved in a small population of transcriptionally distinct CD62L+ TPEX cells. The transcription factor MYB is not only essential for the development of CD62L+ TPEX cells and maintenance of the antiviral CD8+ T cell response, but also induces functional exhaustion and thereby prevents lethal immunopathology. Furthermore, the proliferative burst in response to PD-1 checkpoint inhibition originates exclusively from CD62L+ TPEX cells and depends on MYB. Our findings identify CD62L+ TPEX cells as a stem-like population that is central to the maintenance of long-term antiviral immunity and responsiveness to immunotherapy. Moreover, they show that MYB is a transcriptional orchestrator of two fundamental aspects of exhausted T cell responses: the downregulation of effector function and the long-term preservation of self-renewal capacity.

ING4 and ING5 are essential for histone H3 lysine 14 acetylation and epicardial cell lineage development.

Inhibitor of growth 4 and 5 (ING4, ING5) are structurally similar chromatin-binding proteins in the KAT6A, KAT6B and KAT7 histone acetyltransferase protein complexes. Heterozygous mutations in the KAT6A or KAT6B gene cause human disorders with cardiac defects, but the contribution of their chromatin-adaptor proteins to development is unknown. We found that Ing5-/- mice had isolated cardiac ventricular septal defects. Ing4-/-Ing5-/- embryos failed to undergo chorioallantoic fusion and arrested in development at embryonic day 8.5, displaying loss of histone H3 lysine 14 acetylation, reduction in H3 lysine 23 acetylation levels and reduced developmental gene expression. Embryonic day 12.5 Ing4+/-Ing5-/- hearts showed a paucity of epicardial cells and epicardium-derived cells, failure of myocardium compaction, and coronary vasculature defects, accompanied by reduced expression of epicardium genes. Cell adhesion gene expression and proepicardium outgrowth were defective in the ING4- and ING5-deficient state. Our findings suggest that ING4 and ING5 are essential for heart development and promote epicardium and epicardium-derived cell fates and imply mutation of the human ING5 gene as a possible cause of isolated ventricular septal defects.

Loss of NF-κB1 Causes Gastric Cancer with Aberrant Inflammation and Expression of Immune Checkpoint Regulators in a STAT-1-Dependent Manner.

Polymorphisms in NFKB1 that diminish its expression have been linked to human inflammatory diseases and increased risk for epithelial cancers. The underlying mechanisms are unknown, and the link is perplexing given that NF-κB signaling reportedly typically exerts pro-tumorigenic activity. Here we have shown that NF-κB1 deficiency, even loss of a single allele, resulted in spontaneous invasive gastric cancer (GC) in mice that mirrored the histopathological progression of human intestinal-type gastric adenocarcinoma. Bone marrow chimeras revealed that NF-κB1 exerted tumor suppressive functions in both epithelial and hematopoietic cells. RNA-seq analysis showed that NF-κB1 deficiency resulted in aberrant JAK-STAT signaling, which dysregulated expression of effectors of inflammation, antigen presentation, and immune checkpoints. Concomitant loss of STAT1 prevented these immune abnormalities and GC development. These findings provide mechanistic insight into how polymorphisms that attenuate NFKB1 expression predispose humans to epithelial cancers, highlighting the pro-tumorigenic activity of STAT1 and identifying targetable vulnerabilities in GC.

SARS-CoV-2 mouse adaptation selects virulence mutations that cause TNF-driven age-dependent severe disease with human correlates.

The diversity of COVID-19 disease in otherwise healthy people, from seemingly asymptomatic infection to severe life-threatening disease, is not clearly understood. We passaged a naturally occurring near-ancestral SARS-CoV-2 variant, capable of infecting wild-type mice, and identified viral genomic mutations coinciding with the acquisition of severe disease in young adult mice and lethality in aged animals. Transcriptomic analysis of lung tissues from mice with severe disease elucidated a host antiviral response dominated mainly by interferon and IL-6 pathway activation in young mice, while in aged animals, a fatal outcome was dominated by TNF and TGF-ß signaling. Congruent with our pathway analysis, we showed that young TNF-deficient mice had mild disease compared to controls and aged TNF-deficient animals were more likely to survive infection. Emerging clinical correlates of disease are consistent with our preclinical studies, and our model may provide value in defining aberrant host responses that are causative of severe COVID-19.

A single-cell RNA expression atlas of normal, preneoplastic and tumorigenic states in the human breast.

To examine global changes in breast heterogeneity across different states, we determined the single-cell transcriptomes of > 340,000 cells encompassing normal breast, preneoplastic BRCA1+/- tissue, the major breast cancer subtypes, and pairs of tumors and involved lymph nodes. Elucidation of the normal breast microenvironment revealed striking changes in the stroma of post-menopausal women. Single-cell profiling of 34 treatment-naive primary tumors, including estrogen receptor (ER)+ , HER2+ , and triple-negative breast cancers, revealed comparable diversity among cancer cells and a discrete subset of cycling cells. The transcriptomes of preneoplastic BRCA1+/- tissue versus tumors highlighted global changes in the immune microenvironment. Within the tumor immune landscape, proliferative CD8+ T cells characterized triple-negative and HER2+ cancers but not ER+ tumors, while all subtypes comprised cycling tumor-associated macrophages, thus invoking potentially different immunotherapy targets. Copy number analysis of paired ER+ tumors and lymph nodes indicated seeding by genetically distinct clones or mass migration of primary tumor cells into axillary lymph nodes. This large-scale integration of patient samples provides a high-resolution map of cell diversity in normal and cancerous human breast.

A risk-reward examination of sample multiplexing reagents for single cell RNA-Seq.

Single-cell RNA sequencing (scRNA-Seq) has emerged as a powerful tool for understanding cellular heterogeneity and function. However the choice of sample multiplexing reagents can impact data quality and experimental outcomes. In this study, we compared various multiplexing reagents, including MULTI-Seq, Hashtag antibody, and CellPlex, across diverse sample types such as human peripheral blood mononuclear cells (PBMCs), mouse embryonic brain and patient-derived xenografts (PDXs). We found that all multiplexing reagents worked well in cell types robust to ex vivo manipulation but suffered from signal-to-noise issues in more delicate sample types. We compared multiple demultiplexing algorithms which differed in performance depending on data quality. We find that minor improvements to laboratory workflows such as titration and rapid processing are critical to optimal performance. We also compared the performance of fixed scRNA-Seq kits and highlight the advantages of the Parse Biosciences kit for fragile samples. Highly multiplexed scRNA-Seq experiments require more sequencing resources, therefore we evaluated CRISPR-based destruction of non-informative genes to enhance sequencing value. Our comprehensive analysis provides insights into the selection of appropriate sample multiplexing reagents and protocols for scRNA-Seq experiments, facilitating more accurate and cost-effective studies.

The histone lysine acetyltransferase HBO1 (KAT7) regulates hematopoietic stem cell quiescence and self-renewal.

The histone acetyltransferase HBO1 (MYST2, KAT7) is indispensable for postgastrulation development, histone H3 lysine 14 acetylation (H3K14Ac), and the expression of embryonic patterning genes. In this study, we report the role of HBO1 in regulating hematopoietic stem cell function in adult hematopoiesis. We used 2 complementary cre-recombinase transgenes to conditionally delete Hbo1 (Mx1-Cre and Rosa26-CreERT2). Hbo1-null mice became moribund due to hematopoietic failure with pancytopenia in the blood and bone marrow 2 to 6 weeks after Hbo1 deletion. Hbo1-deleted bone marrow cells failed to repopulate hemoablated recipients in competitive transplantation experiments. Hbo1 deletion caused a rapid loss of hematopoietic progenitors. The numbers of lineage-restricted progenitors for the erythroid, myeloid, B-, and T-cell lineages were reduced. Loss of HBO1 resulted in an abnormally high rate of recruitment of quiescent hematopoietic stem cells (HSCs) into the cell cycle. Cycling HSCs produced progenitors at the expense of self-renewal, which led to the exhaustion of the HSC pool. Mechanistically, genes important for HSC functions were downregulated in HSC-enriched cell populations after Hbo1 deletion, including genes essential for HSC quiescence and self-renewal, such as Mpl, Tek(Tie-2), Gfi1b, Egr1, Tal1(Scl), Gata2, Erg, Pbx1, Meis1, and Hox9, as well as genes important for multipotent progenitor cells and lineage-specific progenitor cells, such as Gata1. HBO1 was required for H3K14Ac through the genome and particularly at gene loci required for HSC quiescence and self-renewal. Our data indicate that HBO1 promotes the expression of a transcription factor network essential for HSC maintenance and self-renewal in adult hematopoiesis.

Inhibitors of histone acetyltransferases KAT6A/B induce senescence and arrest tumour growth.

Acetylation of histones by lysine acetyltransferases (KATs) is essential for chromatin organization and function1. Among the genes coding for the MYST family of KATs (KAT5-KAT8) are the oncogenes KAT6A (also known as MOZ) and KAT6B (also known as MORF and QKF)2,3. KAT6A has essential roles in normal haematopoietic stem cells4-6 and is the target of recurrent chromosomal translocations, causing acute myeloid leukaemia7,8. Similarly, chromosomal translocations in KAT6B have been identified in diverse cancers8. KAT6A suppresses cellular senescence through the regulation of suppressors of the CDKN2A locus9,10, a function that requires its KAT activity10. Loss of one allele of KAT6A extends the median survival of mice with MYC-induced lymphoma from 105 to 413 days11. These findings suggest that inhibition of KAT6A and KAT6B may provide a therapeutic benefit in cancer. Here we present highly potent, selective inhibitors of KAT6A and KAT6B, denoted WM-8014 and WM-1119. Biochemical and structural studies demonstrate that these compounds are reversible competitors of acetyl coenzyme A and inhibit MYST-catalysed histone acetylation. WM-8014 and WM-1119 induce cell cycle exit and cellular senescence without causing DNA damage. Senescence is INK4A/ARF-dependent and is accompanied by changes in gene expression that are typical of loss of KAT6A function. WM-8014 potentiates oncogene-induced senescence in vitro and in a zebrafish model of hepatocellular carcinoma. WM-1119, which has increased bioavailability, arrests the progression of lymphoma in mice. We anticipate that this class of inhibitors will help to accelerate the development of therapeutics that target gene transcription regulated by histone acetylation.

Human and viral membrane-associated E3 ubiquitin ligases MARCH1 and MIR2 recognize different features of CD86 to downregulate surface expression.

Immune-stimulatory ligands, such as major histocompatibility complex molecules and the T-cell costimulatory ligand CD86, are central to productive immunity. Endogenous mammalian membrane-associated RING-CHs (MARCH) act on these and other targets to regulate antigen presentation and activation of adaptive immunity, whereas virus-encoded homologs target the same molecules to evade immune responses. Substrate specificity is encoded in or near the membrane-embedded domains of MARCHs and the proteins they regulate, but the exact sequences that distinguish substrates from nonsubstrates are poorly understood. Here, we examined the requirements for recognition of the costimulatory ligand CD86 by two different MARCH-family proteins, human MARCH1 and Kaposi's sarcoma herpesvirus modulator of immune recognition 2 (MIR2), using deep mutational scanning. We identified a highly specific recognition surface in the hydrophobic core of the CD86 transmembrane (TM) domain (TMD) that is required for recognition by MARCH1 and prominently features a proline at position 254. In contrast, MIR2 requires no specific sequences in the CD86 TMD but relies primarily on an aspartic acid at position 244 in the CD86 extracellular juxtamembrane region. Surprisingly, MIR2 recognized CD86 with a TMD composed entirely of valine, whereas many different single amino acid substitutions in the context of the native TM sequence conferred MIR2 resistance. These results show that the human and viral proteins evolved completely different recognition modes for the same substrate. That some TM sequences are incompatible with MIR2 activity, even when no specific recognition motif is required, suggests a more complicated mechanism of immune modulation via CD86 than was previously appreciated.

MOZ directs the distal-less homeobox gene expression program during craniofacial development.

Oral clefts are common birth defects. Individuals with oral clefts who have identical genetic mutations regularly present with variable penetrance and severity. Epigenetic or chromatin-mediated mechanisms are commonly invoked to explain variable penetrance. However, specific examples of these are rare. Two functional copies of the MOZ (KAT6A, MYST3) gene, encoding a MYST family lysine acetyltransferase chromatin regulator, are essential for human craniofacial development, but the molecular role of MOZ in this context is unclear. Using genetic interaction and genomic studies, we have investigated the effects of loss of MOZ on the gene expression program during mouse development. Among the more than 500 genes differentially expressed after loss of MOZ, 19 genes had previously been associated with cleft palates. These included four distal-less homeobox (DLX) transcription factor-encoding genes, Dlx1, Dlx2, Dlx3 and Dlx5 and DLX target genes (including Barx1, Gbx2, Osr2 and Sim2). MOZ occupied the Dlx5 locus and was required for normal levels of histone H3 lysine 9 acetylation. MOZ affected Dlx gene expression cell-autonomously within neural crest cells. Our study identifies a specific program by which the chromatin modifier MOZ regulates craniofacial development.

Novel drivers and modifiers of MPL-dependent oncogenic transformation identified by deep mutational scanning.

The single transmembrane domain (TMD) of the human thrombopoietin receptor (TpoR/myeloproliferative leukemia [MPL] protein), encoded by exon 10 of the MPL gene, is a hotspot for somatic mutations associated with myeloproliferative neoplasms (MPNs). Approximately 6% and 14% of JAK2 V617F- essential thrombocythemia and primary myelofibrosis patients, respectively, have "canonical" MPL exon 10 driver mutations W515L/K/R/A or S505N, which generate constitutively active receptors and consequent loss of Tpo dependence. Other "noncanonical" MPL exon 10 mutations have also been identified in patients, both alone and in combination with canonical mutations, but, in almost all cases, their functional consequences and relevance to disease are unknown. Here, we used a deep mutational scanning approach to evaluate all possible single amino acid substitutions in the human TpoR TMD for their ability to confer cytokine-independent growth in Ba/F3 cells. We identified all currently recognized driver mutations and 7 novel mutations that cause constitutive TpoR activation, and a much larger number of second-site mutations that enhance S505N-driven activation. We found examples of both of these categories in published and previously unpublished MPL exon 10 sequencing data from MPN patients, demonstrating that some, if not all, of the new mutations reported here represent likely drivers or modifiers of myeloproliferative disease.

PHF6 regulates hematopoietic stem and progenitor cells and its loss synergizes with expression of TLX3 to cause leukemia.

Somatically acquired mutations in PHF6 (plant homeodomain finger 6) frequently occur in hematopoietic malignancies and often coincide with ectopic expression of TLX3. However, there is no functional evidence to demonstrate whether these mutations contribute to tumorigenesis. Similarly, the role of PHF6 in hematopoiesis is unknown. We report here that Phf6 deletion in mice resulted in a reduced number of hematopoietic stem cells (HSCs), an increased number of hematopoietic progenitor cells, and an increased proportion of cycling stem and progenitor cells. Loss of PHF6 caused increased and sustained hematopoietic reconstitution in serial transplantation experiments. Interferon-stimulated gene expression was upregulated in the absence of PHF6 in hematopoietic stem and progenitor cells. The numbers of hematopoietic progenitor cells and cycling hematopoietic stem and progenitor cells were restored to normal by combined loss of PHF6 and the interferon α and ß receptor subunit 1. Ectopic expression of TLX3 alone caused partially penetrant leukemia. TLX3 expression and loss of PHF6 combined caused fully penetrant early-onset leukemia. Our data suggest that PHF6 is a hematopoietic tumor suppressor and is important for fine-tuning hematopoietic stem and progenitor cell homeostasis.

A preliminary molecular phylogeny of shield-bearer moths (Lepidoptera: Adeloidea: Heliozelidae) highlights rich undescribed diversity.

Heliozelidae are a widespread, evolutionarily early diverging family of small, day-flying monotrysian moths, for which a comprehensive phylogeny is lacking. We generated the first molecular phylogeny of the family using DNA sequences of two mitochondrial genes (COI and COII) and two nuclear genes (H3 and 28S) from 130 Heliozelidae specimens, including eight of the twelve known genera: Antispila, Antispilina, Coptodisca, Heliozela, Holocacista, Hoplophanes, Pseliastis, and Tyriozela. Our results provide strong support for five major Heliozelidae clades: (i) a large widespread clade containing the leaf-mining genera Antispilina, Coptodisca and Holocacista and some species of Antispila, (ii) a clade containing most of the described Antispila, (iii) a clade containing the leaf-mining genus Heliozela and the monotypic genus Tyriozela, (iv) an Australian clade containing Pseliastis and (v) an Australian clade containing Hoplophanes. Each clade includes several new species and potentially new genera. Collectively, our data uncover a rich and undescribed diversity that appears to be especially prevalent in Australia. Our work highlights the need for a major taxonomic revision of the family and for generating a robust molecular phylogeny using multi-gene approaches in order to resolve the relationships among clades.

Development of amplicon deep sequencing markers and data analysis pipeline for genotyping multi-clonal malaria infections.

BACKGROUND: Amplicon deep sequencing permits sensitive detection of minority clones and improves discriminatory power for genotyping multi-clone Plasmodium falciparum infections. New amplicon sequencing and data analysis protocols are needed for genotyping in epidemiological studies and drug efficacy trials of P. falciparum. METHODS: Targeted sequencing of molecular marker csp and novel marker cpmp was conducted in duplicate on mixtures of parasite culture strains and 37 field samples. A protocol allowing to multiplex up to 384 samples in a single sequencing run was applied. Software "HaplotypR" was developed for data analysis. RESULTS: Cpmp was highly diverse (He = 0.96) in contrast to csp (He = 0.57). Minority clones were robustly detected if their frequency was >1%. False haplotype calls owing to sequencing errors were observed below that threshold. CONCLUSIONS: To reliably detect haplotypes at very low frequencies, experiments are best performed in duplicate and should aim for coverage of >10'000 reads/amplicon. When compared to length polymorphic marker msp2, highly multiplexed amplicon sequencing displayed greater sensitivity in detecting minority clones.

Mitochondrial DNA diversity of present-day Aboriginal Australians and implications for human evolution in Oceania.

Aboriginal Australians are one of the more poorly studied populations from the standpoint of human evolution and genetic diversity. Thus, to investigate their genetic diversity, the possible date of their ancestors' arrival and their relationships with neighboring populations, we analyzed mitochondrial DNA (mtDNA) diversity in a large sample of Aboriginal Australians. Selected mtDNA single-nucleotide polymorphisms and the hypervariable segment haplotypes were analyzed in 594 Aboriginal Australians drawn from locations across the continent, chiefly from regions not previously sampled. Most (~78%) samples could be assigned to mtDNA haplogroups indigenous to Australia. The indigenous haplogroups were all ancient (with estimated ages >40 000 years) and geographically widespread across the continent. The most common haplogroup was P (44%) followed by S (23%) and M42a (9%). There was some geographic structure at the haplotype level. The estimated ages of the indigenous haplogroups range from 39 000 to 55 000 years, dates that fit well with the estimated date of colonization of Australia based on archeological evidence (~47 000 years ago). The distribution of mtDNA haplogroups in Australia and New Guinea supports the hypothesis that the ancestors of Aboriginal Australians entered Sahul through at least two entry points. The mtDNA data give no support to the hypothesis of secondary gene flow into Australia during the Holocene, but instead suggest long-term isolation of the continent.

Antiquity and diversity of aboriginal Australian Y-chromosomes.

OBJECTIVE: Understanding the origins of Aboriginal Australians is crucial in reconstructing the evolution and spread of Homo sapiens as evidence suggests they represent the descendants of the earliest group to leave Africa. This study analyzed a large sample of Y-chromosomes to answer questions relating to the migration routes of their ancestors, the age of Y-haplogroups, date of colonization, as well as the extent of male-specific variation. METHODS: Knowledge of Y-chromosome variation among Aboriginal Australians is extremely limited. This study examined Y-SNP and Y-STR variation among 657 self-declared Aboriginal males from locations across the continent. 17 Y-STR loci and 47 Y-SNPs spanning the Y-chromosome phylogeny were typed in total. RESULTS: The proportion of non-indigenous Y-chromosomes of assumed Eurasian origin was high, at 56%. Y lineages of indigenous Sahul origin belonged to haplogroups C-M130*(xM8,M38,M217,M347) (1%), C-M347 (19%), K-M526*(xM147,P308,P79,P261,P256,M231,M175,M45,P202) (12%), S-P308 (12%), and M-M186 (0.9%). Haplogroups C-M347, K-M526*, and S-P308 are Aboriginal Australian-specific. Dating of C-M347, K-M526*, and S-P308 indicates that all are at least 40,000 years old, confirming their long-term presence in Australia. Haplogroup C-M347 comprised at least three sub-haplogroups: C-DYS390.1del, C-M210, and the unresolved paragroup C-M347*(xDYS390.1del,M210). CONCLUSIONS: There was some geographic structure to the Y-haplogroup variation, but most haplogroups were present throughout Australia. The age of the Australian-specific Y-haplogroups suggests New Guineans and Aboriginal Australians have been isolated for over 30,000 years, supporting findings based on mitochondrial DNA data. Our data support the hypothesis of more than one route (via New Guinea) for males entering Sahul some 50,000 years ago and give no support for colonization events during the Holocene, from either India or elsewhere.

Marked genetic diversity within Blastocystis in Australian wildlife revealed using a next generation sequencing-phylogenetic approach.

Blastocystis is a genus of intestinal stramenopiles that infect vertebrates, and may cause disease of the alimentary tract. Currently, at least 40 genotypes ("subtypes") of Blastocystis are recognised worldwide based on sequence data for the small subunit of the nuclear ribosomal RNA (SSU-rRNA) gene. Despite the numerous studies of Blastocystis worldwide, very few studies have explored Blastocystis in wild animals, particularly in Australia. Here, we used a PCR-based next generation sequencing (NGS)-phylogenetic approach to genetically characterise and classify Blastocystis variants from selected wildlife in the Australian state of Victoria. In total, 1658 faecal samples were collected from nine host species, including eastern grey kangaroo, swamp wallaby, common wombat, deer, European rabbit, canines and emu. Genomic DNA was extracted from these samples, a 500 bp region of the SSU-rRNA gene amplified by polymerase chain reaction (PCR) and, then, a subset of samples sequenced using Illumina technology. Primary PCR detected Blastocystis in 482 of the 1658 samples (29%), with the highest percentage in fallow deer (63%). Subsequent, Illumina-based sequencing of a subset of 356 samples revealed 55 distinct amplicon sequence variants (ASVs) representing seven currently-recognised subtypes (STs) [ST13 (prominent in marsupials), ST10, ST14, ST21, ST23, ST24 and ST25 (prominent in deer)] and two novel STs (ST45 and ST46) in marsupials. Mixed infections of different STs were observed in macropods, deer, emu and canids (fox, feral dog or dingo), but no infection was detected in rabbits or wombats. This study reveals marked genetic diversity within Blastocystis in a small number of species of wild animals in Australia, suggesting complexity in the genetic composition and transmission patterns of members of the genus Blastocystis in this country.

Combined absence of TRP53 target genes ZMAT3, PUMA and p21 cause a high incidence of cancer in mice.

Transcriptional activation of target genes is essential for TP53-mediated tumour suppression, though the roles of the diverse TP53-activated target genes in tumour suppression remains poorly understood. Knockdown of ZMAT3, an RNA-binding zinc-finger protein involved in regulating alternative splicing, in haematopoietic cells by shRNA caused leukaemia only with the concomitant absence of the PUMA and p21, the critical effectors of TRP53-mediated apoptosis and cell cycle arrest respectively. We were interested to further investigate the role of ZMAT3 in tumour suppression beyond the haematopoietic system. Therefore, we generated Zmat3 knockout and compound gene knockout mice, lacking Zmat3 and p21, Zmat3 and Puma or all three genes. Puma-/-p21-/-Zmat3-/- triple knockout mice developed tumours at a significantly higher frequency compared to wild-type, Puma-/-Zmat3-/- or p21-/-Zmat3-/-deficient mice. Interestingly, we observed that the triple knockout and Puma-/-Zmat3-/- double deficient animals succumbed to lymphoma, while p21-/-Zmat3-/- animals developed mainly solid cancers. This analysis suggests that in addition to ZMAT3 loss, additional TRP53-regulated processes must be disabled simultaneously for TRP53-mediated tumour suppression to fail. Our findings reveal that the absence of different TRP53 regulated tumour suppressive processes changes the tumour spectrum, indicating that different TRP53 tumour suppressive pathways are more critical in different tissues.

Genome-wide CRISPR screening identifies a role for ARRDC3 in TRP53-mediated responses.

Whole-genome screens using CRISPR technologies are powerful tools to identify novel tumour suppressors as well as factors that impact responses of malignant cells to anti-cancer agents. Applying this methodology to lymphoma cells, we conducted a genome-wide screen to identify novel inhibitors of tumour expansion that are induced by the tumour suppressor TRP53. We discovered that the absence of Arrestin domain containing 3 (ARRDC3) increases the survival and long-term competitiveness of MYC-driven lymphoma cells when treated with anti-cancer agents that activate TRP53. Deleting Arrdc3 in mice caused perinatal lethality due to various developmental abnormalities, including cardiac defects. Notably, the absence of ARRDC3 markedly accelerated MYC-driven lymphoma development. Thus, ARRDC3 is a new mediator of TRP53-mediated suppression of tumour expansion, and this discovery may open new avenues to harness this process for cancer therapy.