Mouse models to investigate in situ cell fate decisions induced by p53.

Investigating how transcription factors control complex cellular processes requires tools that enable responses to be visualised at the single-cell level and their cell fate to be followed over time. For example, the tumour suppressor p53 (also called TP53 in humans and TRP53 in mice) can initiate diverse cellular responses by transcriptional activation of its target genes: Puma to induce apoptotic cell death and p21 to induce cell cycle arrest/cell senescence. However, it is not known how these processes are regulated and initiated in different cell types. Also, the context-dependent interaction partners and binding loci of p53 remain largely elusive. To be able to examine these questions, we here developed knock-in mice expressing triple-FLAG-tagged p53 to facilitate p53 pull-down and two p53 response reporter mice, knocking tdTomato and GFP into the Puma/Bbc3 and p21 gene loci, respectively. By crossing these reporter mice into a p53-deficient background, we show that the new reporters reliably inform on p53-dependent and p53-independent initiation of both apoptotic or cell cycle arrest/senescence programs, respectively, in vitro and in vivo.

Mutant TRP53 exerts a target gene-selective dominant-negative effect to drive tumor development.

Mutations in Trp53, prevalent in human cancer, are reported to drive tumorigenesis through dominant-negative effects (DNEs) over wild-type TRP53 function as well as neomorphic gain-of-function (GOF) activity. We show that five TRP53 mutants do not accelerate lymphomagenesis on a TRP53-deficient background but strongly synergize with c-MYC overexpression in a manner that distinguishes the hot spot Trp53 mutations. RNA sequencing revealed that the mutant TRP53 DNE does not globally repress wild-type TRP53 function but disproportionately impacts a subset of wild-type TRP53 target genes. Accordingly, TRP53 mutant proteins impair pathways for DNA repair, proliferation, and metabolism in premalignant cells. This reveals that, in our studies of lymphomagenesis, mutant TRP53 drives tumorigenesis primarily through the DNE, which modulates wild-type TRP53 function in a manner advantageous for neoplastic transformation.

BCL-XL exerts a protective role against anemia caused by radiation-induced kidney damage.

Studies of gene-targeted mice identified the roles of the different pro-survival BCL-2 proteins during embryogenesis. However, little is known about the role(s) of these proteins in adults in response to cytotoxic stresses, such as treatment with anti-cancer agents. We investigated the role of BCL-XL in adult mice using a strategy where prior bone marrow transplantation allowed for loss of BCL-XL exclusively in non-hematopoietic tissues to prevent anemia caused by BCL-XL deficiency in erythroid cells. Unexpectedly, the combination of total body γ-irradiation (TBI) and genetic loss of Bcl-x caused secondary anemia resulting from chronic renal failure due to apoptosis of renal tubular epithelium with secondary obstructive nephropathy. These findings identify a critical protective role of BCL-XL in the adult kidney and inform on the use of BCL-XL inhibitors in combination with DNA damage-inducing drugs for cancer therapy. Encouragingly, the combination of DNA damage-inducing anti-cancer therapy plus a BCL-XL inhibitor could be tolerated in mice, at least when applied sequentially.

Genome-scale clustered regularly interspaced short palindromic repeats screen identifies nucleotide metabolism as an actionable therapeutic vulnerability in diffuse large B-cell lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is the most common malignancy that develops in patients with ataxia-telangiectasia, a cancer-predisposing inherited syndrome characterized by inactivating germline ATM mutations. ATM is also frequently mutated in sporadic DLBCL. To investigate lymphomagenic mechanisms and lymphoma-specific dependencies underlying defective ATM, we applied ribonucleic acid (RNA)-seq and genome-scale loss-offunction clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 screens to systematically interrogate B-cell lymphomas arising in a novel murine model (Atm-/-nu-/-) with constitutional Atm loss, thymic aplasia but residual T-cell populations. Atm-/-nu-/-lymphomas, which phenotypically resemble either activated B-cell-like or germinal center Bcell-like DLBCL, harbor a complex karyotype, and are characterized by MYC pathway activation. In Atm-/-nu-/-lymphomas, we discovered nucleotide biosynthesis as a MYCdependent cellular vulnerability that can be targeted through the synergistic nucleotidedepleting actions of mycophenolate mofetil (MMF) and the WEE1 inhibitor, adavosertib (AZD1775). The latter is mediated through a synthetically lethal interaction between RRM2 suppression and MYC dysregulation that results in replication stress overload in Atm-/-nu-/-lymphoma cells. Validation in cell line models of human DLBCL confirmed the broad applicability of nucleotide depletion as a therapeutic strategy for MYC-driven DLBCL independent of ATM mutation status. Our findings extend current understanding of lymphomagenic mechanisms underpinning ATM loss and highlight nucleotide metabolism as a targetable therapeutic vulnerability in MYC-driven DLBCL.

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.

BCL-2 protein family: attractive targets for cancer therapy.

Acquired resistance to cell death is a hallmark of cancer. The BCL-2 protein family members play important roles in controlling apoptotic cell death. Abnormal over-expression of pro-survival BCL-2 family members or abnormal reduction of pro-apoptotic BCL-2 family proteins, both resulting in the inhibition of apoptosis, are frequently detected in diverse malignancies. The critical role of the pro-survival and pro-apoptotic BCL-2 family proteins in the regulation of apoptosis makes them attractive targets for the development of agents for the treatment of cancer. This review describes the roles of the various pro-survival and pro-apoptotic members of the BCL-2 protein family in normal development and organismal function and how defects in the control of apoptosis promote the development and therapy resistance of cancer. Finally, we discuss the development of inhibitors of pro-survival BCL-2 proteins, termed BH3-mimetic drugs, as novel agents for cancer therapy.

Impaired ribosome biogenesis checkpoint activation induces p53-dependent MCL-1 degradation and MYC-driven lymphoma death.

MYC-driven B-cell lymphomas are addicted to increased levels of ribosome biogenesis (RiBi), offering the potential for therapeutic intervention. However, it is unclear whether inhibition of RiBi suppresses lymphomagenesis by decreasing translational capacity and/or by p53 activation mediated by the impaired RiBi checkpoint (IRBC). Here we generated Eµ-Myc lymphoma cells expressing inducible short hairpin RNAs to either ribosomal protein L7a (RPL7a) or RPL11, the latter an essential component of the IRBC. The loss of either protein reduced RiBi, protein synthesis, and cell proliferation to similar extents. However, only RPL7a depletion induced p53-mediated apoptosis through the selective proteasomal degradation of antiapoptotic MCL-1, indicating the critical role of the IRBC in this mechanism. Strikingly, low concentrations of the US Food and Drug Administration-approved anticancer RNA polymerase I inhibitor Actinomycin D (ActD) dramatically prolonged the survival of mice harboring Trp53+/+;Eµ-Myc but not Trp53-/-;Eµ-Myc lymphomas, which provides a rationale for treating MYC-driven B-cell lymphomas with ActD. Importantly, the molecular effects of ActD on Eµ-Myc cells were recapitulated in human B-cell lymphoma cell lines, highlighting the potential for ActD as a therapeutic avenue for p53 wild-type lymphoma.

Intact TP-53 function is essential for sustaining durable responses to BH3-mimetic drugs in leukemias.

Selective targeting of BCL-2 with the BH3-mimetic venetoclax has been a transformative treatment for patients with various leukemias. TP-53 controls apoptosis upstream of where BCL-2 and its prosurvival relatives, such as MCL-1, act. Therefore, targeting these prosurvival proteins could trigger apoptosis across diverse blood cancers, irrespective of TP53 mutation status. Indeed, targeting BCL-2 has produced clinically relevant responses in blood cancers with aberrant TP-53. However, in our study, TP53-mutated or -deficient myeloid and lymphoid leukemias outcompeted isogenic controls with intact TP-53, unless sufficient concentrations of BH3-mimetics targeting BCL-2 or MCL-1 were applied. Strikingly, tumor cells with TP-53 dysfunction escaped and thrived over time if inhibition of BCL-2 or MCL-1 was sublethal, in part because of an increased threshold for BAX/BAK activation in these cells. Our study revealed the key role of TP-53 in shaping long-term responses to BH3-mimetic drugs and reconciled the disparate pattern of initial clinical response to venetoclax, followed by subsequent treatment failure among patients with TP53-mutant chronic lymphocytic leukemia or acute myeloid leukemia. In contrast to BH3-mimetics targeting just BCL-2 or MCL-1 at doses that are individually sublethal, a combined BH3-mimetic approach targeting both prosurvival proteins enhanced lethality and durably suppressed the leukemia burden, regardless of TP53 mutation status. Our findings highlight the importance of using sufficiently lethal treatment strategies to maximize outcomes of patients with TP53-mutant disease. In addition, our findings caution against use of sublethal BH3-mimetic drug regimens that may enhance the risk of disease progression driven by emergent TP53-mutant clones.

Young People's Experiences of Returning to Physical Leisure Activities after a Severe Acquired Brain Injury.

AIMS: To understand the experiences of young people returning to physical leisure activities following a severe acquired brain injury (ABI). METHODS: Seven young people (5 male; 14-19 years) participated. Semi-structured interviews were conducted with young people who sustained a severe ABI 1-3 years prior to the study. Data thematically analyzed using Braun and Clarke's six-phase approach. RESULTS: Three main themes were created: My changing sense of identity around physical activity after my brain injury (how important physical activity was to them, how participation changed following their ABI); Why I take part in physical leisure activities (fun, friendships, help with recovery and physical and emotional health); and I can't do it alone (need for trusted adults to practically and emotionally support them to try and activities and continue to participate). DISCUSSION: Returning to physical leisure activities after ABI was important to young people, especially if they were active prior to their injury. However, participating with changed abilities was practically and emotionally challenging. Services need a multidisciplinary approach to ensure young people are supported with psychological processes of loss, adjustment, identity and resilience in addition to the practical help necessary to enable meaningful participation in activities they consider fun.

The MCL1 inhibitor S63845 is tolerable and effective in diverse cancer models.

Avoidance of apoptosis is critical for the development and sustained growth of tumours. The pro-survival protein myeloid cell leukemia 1 (MCL1) is overexpressed in many cancers, but the development of small molecules targeting this protein that are amenable for clinical testing has been challenging. Here we describe S63845, a small molecule that specifically binds with high affinity to the BH3-binding groove of MCL1. Our mechanistic studies demonstrate that S63845 potently kills MCL1-dependent cancer cells, including multiple myeloma, leukaemia and lymphoma cells, by activating the BAX/BAK-dependent mitochondrial apoptotic pathway. In vivo, S63845 shows potent anti-tumour activity with an acceptable safety margin as a single agent in several cancers. Moreover, MCL1 inhibition, either alone or in combination with other anti-cancer drugs, proved effective against several solid cancer-derived cell lines. These results point towards MCL1 as a target for the treatment of a wide range of tumours.

A new model to guide identity-focused multidisciplinary rehabilitation for children and young people following acquired brain injury: I-FoRM.

A complexity of biological, psychological, environmental and systemic factors influences a child's adaption after acquired brain injury (ABI), all of which transform as the child matures. Multidisciplinary rehabilitation teams are challenged by balancing family system needs and the child's needs, whilst promoting the child's functional skills in difficult or unappealing tasks. This paper presents the conceptual basis for a model for use in childhood ABI neurorehabilitation to address these challenges. A non-systematic narrative review of literature pertinent to integrated neurorehabilitation of pediatric ABI was conducted. Contemporary models of adult and pediatric psychosocial adaptation involving identity following ABI were reviewed. Key findings were then synthesized with models of pediatric resilience and self-concept development. The resulting model describes a cyclical adaptation process whereby the child learns experientially about their self and their world after ABI. Processes of identity development play a central role - particularly emotive processes of self-evaluation - by influencing the child's motivation for participation, tolerance for challenge, self-regulation and emerging self-awareness. The model directs clinicians to use the psychosocial processes of identity development to enhance the child's willingness and capacity to engage in the daily challenges of rehabilitation. Further systematic development and evaluation of the model is needed.

Targeting of MCL-1 kills MYC-driven mouse and human lymphomas even when they bear mutations in p53.

The transcriptional regulator c-MYC is abnormally overexpressed in many human cancers. Evasion from apoptosis is critical for cancer development, particularly c-MYC-driven cancers. We explored which anti-apoptotic BCL-2 family member (expressed under endogenous regulation) is essential to sustain c-MYC-driven lymphoma growth to reveal which should be targeted for cancer therapy. Remarkably, inducible Cre-mediated deletion of even a single Mcl-1 allele substantially impaired the growth of c-MYC-driven mouse lymphomas. Mutations in p53 could diminish but not obviate the dependency of c-MYC-driven mouse lymphomas on MCL-1. Importantly, targeting of MCL-1 killed c-MYC-driven human Burkitt lymphoma cells, even those bearing mutations in p53. Given that loss of one allele of Mcl-1 is well tolerated in healthy tissues, our results suggest that therapeutic targeting of MCL-1 would be an attractive therapeutic strategy for MYC-driven cancers.

Humanized Mcl-1 mice enable accurate preclinical evaluation of MCL-1 inhibitors destined for clinical use.

Myeloid cell leukemia-1 (MCL-1) is a prosurvival B-cell lymphoma 2 (BCL-2) family member required for the sustained growth of many cancers. Recently, a highly specific MCL-1 inhibitor, S63845, showing sixfold higher affinity to human compared with mouse MCL-1, has been described. To accurately test efficacy and tolerability of this BH3-mimetic (BH3-only protein mimetic) drug in preclinical cancer models, we developed a humanized Mcl-1 (huMcl-1) mouse strain in which MCL-1 was replaced with its human homolog. huMcl-1 mice are phenotypically indistinguishable from wild-type mice but are more sensitive to the MCL-1 inhibitor S63845. Importantly, nontransformed cells and lymphomas from huMcl-1;Eµ-Myc mice are more sensitive to S63845 in vitro than their control counterparts. When huMcl-1;Eµ-Myc lymphoma cells were transplanted into huMcl-1 mice, treatment with S63845 alone or alongside cyclophosphamide led to long-term remission in ∼60% or almost 100% of mice, respectively. These results demonstrate the potential of our huMcl-1 mouse model for testing MCL-1 inhibitors, allowing precise predictions of efficacy and tolerability for clinical translation.

Using child- and family-centred goal setting as an outcome measure in residential rehabilitation for children and youth with acquired brain injuries: The challenge of predicting expected levels of achievement.

PURPOSE: Collaborative, child- and family-centred goal setting is essential in paediatric, acquired brain injury (ABI) rehabilitation. This study aims to understand which goals children and families prioritize and how accurately therapists predict expected levels of achievement for these goals. METHODS: Routinely collected Goal Attainment Scale-Light data from 122 children with severe ABI receiving residential rehabilitation were retrospectively analysed. Goals were mapped onto the International Classification of Functioning, Disability and Health. Descriptive analysis of accuracy of therapists' prediction of goal achievement was conducted. RESULTS: Eight-hundred sixty goals were set: 82% in activities and participation domains, most commonly mobility, self-care, and communication chapters. Forty-six per cent of therapist-set expected levels of achievement for these goals were met at the expected level, and 24% were exceeded. Chapters with the highest prediction accuracy included two environmental chapters and one body structure and function. Accurate prediction of activity and participation goals varied (35% in general tasks and demands to 58.8% in major life areas). CONCLUSIONS: Children and families prioritize mobility, self-care, and communication during ABI residential rehabilitation. Setting expected outcomes for these goals is challenging, as demonstrated by the variety in accurate prediction rates between and within chapters. Families need to be aware of this uncertainty during goal-setting discussions.

Paediatric Rehabilitation Ingredients Measure: a new tool for identifying paediatric neurorehabilitation content.

AIM: To develop an instrument (Paediatric Rehabilitation Ingredients Measure [PRISM]) for quantitative estimation of contents of interdisciplinary neurorehabilitation for use in studies of relationships between rehabilitation treatment delivered and severity-adjusted outcomes after acquired brain injury (ABI). METHOD: The measure was developed using an ingredients-mediators-outcomes model consistent with the International Classification of Functioning, Disability and Health, a literature review, and other current initiatives in the development of rehabilitation treatment taxonomies, with item codevelopment in workshops with rehabilitation professionals. Interrater reliability was assessed in inpatient and residential paediatric rehabilitation settings. RESULTS: Although sometimes an initially unfamiliar perspective on rehabilitation practice, PRISM's acceptability amongst professionals was excellent. Internal consistency of scores was sometimes an issue for users unfamiliar with the tool; however, this improved with practice and interrater reliability (assessed by Kendall's W) was good. The tool was felt to have particular value in facilitating interdisciplinary communication and working. Modifications to the design of the tool have improved internal consistency. INTERPRETATION: PRISM supports identification of the 'active ingredients' of an interdisciplinary rehabilitation package and facilitates interdisciplinary communication. It also has potential as a research tool examining relationships between rehabilitation delivered and severity-adjusted outcomes observed after paediatric ABI. WHAT THIS PAPER ADDS: Identifying contribution of rehabilitation to outcomes after acquired brain injury requires quantification of rehabilitation 'dose' and 'content'. Previous approaches to 'parsing' of rehabilitation dose and content may have overemphasized one-to-one sessions with therapists. We present a novel, holistic tool for identification of ingredients of an interdisciplinary rehabilitation package. It supports interdisciplinary communication and has potential as a research tool.

Gross Motor Function Measure-66 trajectories in children recovering after severe acquired brain injury.

AIM: To explore the appropriateness of using the interval-scale version of the Gross Motor Function Measure (GMFM-66) in paediatric acquired brain injury (ABI), and to characterize GMFM-66 recovery trajectories and factors that affect them. METHOD: An observational study of gross motor recovery trajectories during rehabilitation at a single specialist paediatric in-patient rehabilitation centre using repeated GMFM-66 observations. The cohort comprised children rehabilitating after severe ABI of various causes. RESULTS: A total of 287 GMFM observations were made on 74 children (45 males, 29 females; age-at-injury range 0.3-17.3y, median age 11.3y, interquartile range 6.6-15.0y). Differences in item-difficulty estimates between this sample and the cerebral palsy population in which the GMFM-66 was initially developed are not detectable at this sample size. Changes in GMFM over time show lag-exponential forms. Children sustaining hypoxic-ischaemic injuries made the slowest and least complete recoveries. Older children made faster gross motor recoveries after controlling for aetiology. The time at which gross motor ability began to rise coincided approximately with admission to the rehabilitation facility. INTERPRETATION: Aetiology is strongly associated with gross motor recovery after ABI. Younger age at injury was associated with slower recovery. Comparable item-difficulty scores in this sample and in the cerebral palsy population suggest comparable sequences of gross motor ability reacquisition.

Different patterns of Epstein-Barr virus latency in endemic Burkitt lymphoma (BL) lead to distinct variants within the BL-associated gene expression signature.

Epstein-Barr virus (EBV) is present in all cases of endemic Burkitt lymphoma (BL) but in few European/North American sporadic BLs. Gene expression arrays of sporadic tumors have defined a consensus BL profile within which tumors are classifiable as "molecular BL" (mBL). Where endemic BLs fall relative to this profile remains unclear, since they not only carry EBV but also display one of two different forms of virus latency. Here, we use early-passage BL cell lines from different tumors, and BL subclones from a single tumor, to compare EBV-negative cells with EBV-positive cells displaying either classical latency I EBV infection (where EBNA1 is the only EBV antigen expressed from the wild-type EBV genome) or Wp-restricted latency (where an EBNA2 gene-deleted virus genome broadens antigen expression to include the EBNA3A, -3B, and -3C proteins and BHRF1). Expression arrays show that both types of endemic BL fall within the mBL classification. However, while EBV-negative and latency I BLs show overlapping profiles, Wp-restricted BLs form a distinct subgroup, characterized by a detectable downregulation of the germinal center (GC)-associated marker Bcl6 and upregulation of genes marking early plasmacytoid differentiation, notably IRF4 and BLIMP1. Importantly, these same changes can be induced in EBV-negative or latency I BL cells by infection with an EBNA2-knockout virus. Thus, we infer that the distinct gene profile of Wp-restricted BLs does not reflect differences in the identity of the tumor progenitor cell per se but differences imposed on a common progenitor by broadened EBV gene expression.

Don't fear the reaper: The role of regulated cell death in tumorigenesis and BH3-mimetics for cancer therapy.

From its earliest characterization, it has been recognized that there is a role for regulated (programmed) cell death in cancer. As our understanding of the different types of programmed cell death processes and their molecular control has advanced, so have the technologies that allow us to manipulate these processes to, for example, fight against cancer. In this review, we describe the roles of the different forms of regulated cell death in the development of cancer as well as their potential therapeutic exploitation. In that vein, we explore the development and use of BH3-mimetics, a unique class of drugs that can directly activate the apoptotic cell death machinery to treat cancer. Finally, we address key challenges that face the field to improve the use of these therapeutics and the efforts that are being undertaken to do so.

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.