Putting the STING back into BH3-mimetic drugs for TP53-mutant blood cancers.

TP53-mutant blood cancers remain a clinical challenge. BH3-mimetic drugs inhibit BCL-2 pro-survival proteins, inducing cancer cell apoptosis. Despite acting downstream of p53, functional p53 is required for maximal cancer cell killing by BH3-mimetics through an unknown mechanism. Here, we report p53 is activated following BH3-mimetic induced mitochondrial outer membrane permeabilization, leading to BH3-only protein induction and thereby potentiating the pro-apoptotic signal. TP53-deficient lymphomas lack this feedforward loop, providing opportunities for survival and disease relapse after BH3-mimetic treatment. The therapeutic barrier imposed by defects in TP53 can be overcome by direct activation of the cGAS/STING pathway, which promotes apoptosis of blood cancer cells through p53-independent BH3-only protein upregulation. Combining clinically relevant STING agonists with BH3-mimetic drugs efficiently kills TRP53/TP53-mutant mouse B lymphoma, human NK/T lymphoma, and acute myeloid leukemia cells. This represents a promising therapy regime that can be fast-tracked to tackle TP53-mutant blood cancers in the clinic.

Loss-of-Function but Not Gain-of-Function Properties of Mutant TP53 Are Critical for the Proliferation, Survival, and Metastasis of a Broad Range of Cancer Cells.

Mutations in the tumor suppressor TP53 cause cancer and impart poor chemotherapeutic responses, reportedly through loss-of-function, dominant-negative effects and gain-of-function (GOF) activities. The relative contributions of these attributes is unknown. We found that removal of 12 different TP53 mutants with reported GOFs by CRISPR/Cas9 did not impact proliferation and response to chemotherapeutics of 15 human cancer cell lines and colon cancer-derived organoids in culture. Moreover, removal of mutant TP53/TRP53 did not impair growth or metastasis of human cancers in immune-deficient mice or growth of murine cancers in immune-competent mice. DepMap mining revealed that removal of 158 different TP53 mutants had no impact on the growth of 391 human cancer cell lines. In contrast, CRISPR-mediated restoration of wild-type TP53 extinguished the growth of human cancer cells in vitro. These findings demonstrate that LOF but not GOF effects of mutant TP53/TRP53 are critical to sustain expansion of many tumor types. SIGNIFICANCE: This study provides evidence that removal of mutant TP53, thereby deleting its reported GOF activities, does not impact the survival, proliferation, metastasis, or chemotherapy responses of cancer cells. Thus, approaches that abrogate expression of mutant TP53 or target its reported GOF activities are unlikely to exert therapeutic impact in cancer. See related commentary by Lane, p. 211 . This article is featured in Selected Articles from This Issue, p. 201.

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.

BCL-W makes only minor contributions to MYC-driven lymphoma development.

The BH3-mimetic drug Venetoclax, a specific inhibitor of anti-apoptotic BCL-2, has had clinical success for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia. Attention has now shifted towards related pro-survival BCL-2 family members, hypothesising that new BH3-mimetic drugs targeting these proteins may emulate the success of Venetoclax. BH3-mimetics targeting pro-survival MCL-1 or BCL-XL have entered clinical trials, but managing on-target toxicities is challenging. While increasing evidence suggests BFL-1/A1 is a resistance factor for diverse chemotherapeutic agents and BH3-mimetic drugs in haematological malignancies, few studies have explored the role of BCL-W in the development, expansion, and therapeutic responses of cancer. Previously, we found that BCL-W was not required for the ongoing survival and growth of various established human Burkitt lymphoma and diffuse large B cell lymphoma cell lines. However, questions remained about whether BCL-W impacts lymphoma development. Here, we show that BCL-W appears dispensable for MYC-driven lymphomagenesis, and such tumours arising in the absence of BCL-W show no compensatory changes to BCL-2 family member expression, nor altered sensitivity to BH3-mimetic drugs. These results demonstrate that BCL-W does not play a major role in the development of MYC-driven lymphoma or the responses of these tumours to anti-cancer agents.

A Drosophila chemical screen reveals synergistic effect of MEK and DGKα inhibition in Ras-driven cancer.

Elevated Ras signalling is highly prevalent in human cancer; however, targeting Ras-driven cancers with Ras pathway inhibitors often leads to undesirable side effects and to drug resistance. Thus, identifying compounds that synergise with Ras pathway inhibitors would enable lower doses of the Ras pathway inhibitors to be used and also decrease the acquisition of drug resistance. Here, in a specialised chemical screen using a Drosophila model of Ras-driven cancer, we have identified compounds that reduce tumour size by synergising with sub-therapeutic doses of the Ras pathway inhibitor trametinib, which targets MEK, the mitogen-activated protein kinase kinase, in this pathway. Analysis of one of the hits, ritanserin, and related compounds revealed that diacyl glycerol kinase α (DGKα, Dgk in Drosophila) was the critical target required for synergism with trametinib. Human epithelial cells harbouring the H-RAS oncogene and knockdown of the cell polarity gene SCRIB were also sensitive to treatment with trametinib and DGKα inhibitors. Mechanistically, DGKα inhibition synergises with trametinib by increasing the P38 stress-response signalling pathway in H-RASG12V SCRIBRNAi cells, which could lead to cell quiescence. Our results reveal that targeting Ras-driven human cancers with Ras pathway and DGKα inhibitors should be an effective combination drug therapy.

Lymphoma cells lacking pro-apoptotic BAX are highly resistant to BH3-mimetics targeting pro-survival MCL-1 but retain sensitivity to conventional DNA-damaging drugs.

BH3-mimetic drugs are an anti-cancer therapy that can induce apoptosis in malignant cells by directly binding and inhibiting pro-survival proteins of the BCL-2 family. The BH3-mimetic drug venetoclax, which targets BCL-2, has been approved for the treatment of chronic lymphocytic leukaemia and acute myeloid leukaemia by regulatory authorities worldwide. However, while most patients initially respond well, resistance and relapse while on this drug is an emerging and critical issue in the clinic. Though some studies have begun uncovering the factors involved in resistance to BCL-2-targeting BH3-mimetic drugs, little focus has been applied to pre-emptively tackle resistance for the next generation of BH3-mimetic drugs targeting MCL-1, which are now in clinical trials for diverse blood cancers. Therefore, using pre-clinical mouse and human models of aggressive lymphoma, we sought to predict factors likely to contribute to the development of resistance in patients receiving MCL-1-targeting BH3-mimetic drugs. First, we performed multiple whole genome CRISPR/Cas9 KO screens and identified that loss of the pro-apoptotic effector protein BAX, but not its close relative BAK, could confer resistance to MCL-1-targeting BH3-mimetic drugs in both short-term and long-term treatment regimens, even in lymphoma cells lacking the tumour suppressor TRP53. Furthermore, we found that mouse Eµ-Myc lymphoma cells selected for loss of BAX, as well as upregulation of the untargeted pro-survival BCL-2 family proteins BCL-XL and A1, when made naturally resistant to MCL-1 inhibitors by culturing them in increasing doses of drug over time, a situation mimicking the clinical application of these drugs. Finally, we identified therapeutic approaches which could overcome these two methods of resistance: the use of chemotherapeutic drugs or combined BH3-mimetic treatment, respectively. Collectively, these results uncover some key factors likely to cause resistance to MCL-1 inhibition in the clinic and suggest rational therapeutic strategies to overcome resistance that should be investigated further.

The anti-cancer agent APR-246 can activate several programmed cell death processes to kill malignant cells.

Mutant TP53 proteins are thought to drive the development and sustained expansion of cancers at least in part through the loss of the wild-type (wt) TP53 tumour suppressive functions. Therefore, compounds that can restore wt TP53 functions in mutant TP53 proteins are expected to inhibit the expansion of tumours expressing mutant TP53. APR-246 has been reported to exert such effects in malignant cells and is currently undergoing clinical trials in several cancer types. However, there is evidence that APR-246 may also kill malignant cells that do not express mutant TP53. To support the clinical development of APR-246 it is important to understand its mechanism(s) of action. By establishing isogenic background tumour cell lines with different TP53/TRP53 states, we found that APR-246 can kill malignant cells irrespective of their TP53/TRP53 status. Accordingly, RNAseq analysis revealed that treatment with APR-246 induces expression of the same gene set in Eµ-Myc mouse lymphoma cells of all four possible TRP53 states, wt, wt alongside mutant, knockout and knockout alongside mutant. We found that depending on the type of cancer cell and the concentration of APR-246 used, this compound can kill malignant cells through induction of various programmed cell death pathways, including apoptosis, necroptosis and ferroptosis. The sensitivity of non-transformed cells to APR-246 also depended on the cell type. These findings reveal that the clinical testing of APR-246 should not be limited to cancers expressing mutant TP53 but expanded to cancers that express wt TP53 or are TP53-deficient.

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.

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.

Generation of a CRISPR activation mouse that enables modelling of aggressive lymphoma and interrogation of venetoclax resistance.

CRISPR technologies have advanced cancer modelling in mice, but CRISPR activation (CRISPRa) methods have not been exploited in this context. We establish a CRISPRa mouse (dCas9a-SAMKI) for inducing gene expression in vivo and in vitro. Using dCas9a-SAMKI primary lymphocytes, we induce B cell restricted genes in T cells and vice versa, demonstrating the power of this system. There are limited models of aggressive double hit lymphoma. Therefore, we transactivate pro-survival BCL-2 in Eµ-MycT/+;dCas9a-SAMKI/+ haematopoietic stem and progenitor cells. Mice transplanted with these cells rapidly develop lymphomas expressing high BCL-2 and MYC. Unlike standard Eµ-Myc lymphomas, BCL-2 expressing lymphomas are highly sensitive to the BCL-2 inhibitor venetoclax. We perform genome-wide activation screens in these lymphoma cells and find a dominant role for the BCL-2 protein A1 in venetoclax resistance. Here we show the potential of our CRISPRa model for mimicking disease and providing insights into resistance mechanisms towards targeted therapies.

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.

Of the many cellular responses activated by TP53, which ones are critical for tumour suppression?

The tumour suppressor TP53 is a master regulator of several cellular processes that collectively suppress tumorigenesis. The TP53 gene is mutated in ~50% of human cancers and these defects usually confer poor responses to therapy. The TP53 protein functions as a homo-tetrameric transcription factor, directly regulating the expression of ~500 target genes, some of them involved in cell death, cell cycling, cell senescence, DNA repair and metabolism. Originally, it was thought that the induction of apoptotic cell death was the principal mechanism by which TP53 prevents the development of tumours. However, gene targeted mice lacking the critical effectors of TP53-induced apoptosis (PUMA and NOXA) do not spontaneously develop tumours. Indeed, even mice lacking the critical mediators for TP53-induced apoptosis, G1/S cell cycle arrest and cell senescence, namely PUMA, NOXA and p21, do not spontaneously develop tumours. This suggests that TP53 must activate additional cellular responses to mediate tumour suppression. In this review, we will discuss the processes by which TP53 regulates cell death, cell cycling/cell senescence, DNA damage repair and metabolic adaptation, and place this in context of current understanding of TP53-mediated tumour suppression.

Should mutant TP53 be targeted for cancer therapy?

Mutations in the TP53 tumour suppressor gene are found in ~50% of human cancers [1-6]. TP53 functions as a transcription factor that directly regulates the expression of ~500 genes, some of them involved in cell cycle arrest/cell senescence, apoptotic cell death or DNA damage repair, i.e. the cellular responses that together prevent tumorigenesis [1-6]. Defects in TP53 function not only cause tumour development but also impair the response of malignant cells to anti-cancer drugs, particularly those that induce DNA damage [1-6]. Most mutations in TP53 in human cancers cause a single amino acid substitution, usually within the DNA binding domain of the TP53 protein. These mutant TP53 proteins are often expressed at high levels in the malignant cells. Three cancer causing attributes have been postulated for mutant TP53 proteins: the inability to activate target genes controlled by wt TP53 (loss-of-function, LOF) that are critical for tumour suppression, dominant negative effects (DNE), i.e. blocking the function of wt TP53 in cells during early stages of transformation when mutant and wt TP53 proteins are co-expressed, and gain-of-function (GOF) effects whereby mutant TP53 impacts diverse cellular pathways by interacting with proteins that are not normally engaged by wt TP53 [1-6]. The GOF effects of mutant TP53 were reported to be essential for the sustained proliferation and survival of malignant cells and it was therefore proposed that agents that can remove mutant TP53 protein would have substantial therapeutic impact [7-9]. In this review article we discuss evidence for and against the value of targeting mutant TP53 protein for cancer therapy.

Flow cytometric single cell-based assay to simultaneously detect cell death, cell cycling, DNA content and cell senescence.

Cell death, cell cycle arrest and cellular senescence are three distinct cellular responses that can be induced by oncogene activation and diverse anti-cancer agents, and this often requires the action of the tumour suppressor TP53. Within a cell population, or even within an individual cell, these processes are not necessarily mutually exclusive. It is therefore important to measure all these processes simultaneously. However, current assays generally visualise only one or at best two responses, often only detecting the dominant one. Here, we present a novel flow cytometric assay that allows simultaneous assessment of cell viability and cell cycling through measurement of DNA content and DNA synthesis, and markers of cell senescence at the single cell level. We demonstrate that this assay can be performed on both human and murine cells, that are either cancerous or non-transformed, and can help to dissect complex cell fate decisions. We believe that this experimental tool will be useful for the study of diverse biological processes.

Demonstration of functional rehabilitation treatment effects in children and young people after severe acquired brain injury.

PURPOSE: To examine relationships between functional outcomes after pediatric acquired brain injury (ABI) and measures of rehabilitation dose. METHODS: An observational study of children receiving residential neurorehabilitation after severe ABI. RESULTS: Basic total rehabilitation dose shows a paradoxical inverse relationship to global outcome. This is due to confounding by both initial injury severity and length of stay, and variation in treatment content for a given total rehabilitation dose. Content-aware rehabilitation dose measures show robust positive correlations between fractions of rehabilitation treatment received and plausibly related aspects of outcome: specifically, between rates of recovery of gross motor function and the fraction of rehabilitation effort directed to active practice and motor learning. This relationship was robust to adjustment for therapists' expectations of recovery. CONCLUSION: Content-aware measures of rehabilitation dose are robustly causally related to pertinent aspects of outcome. These findings are step toward a goal of comparative effectiveness research in pediatric neurorehabilitation.