Sir Peter MacCallum Department of Oncology - Research Publications

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    A quantitative model to predict pathogenicity of missense variants in the TP53 gene
    Fortuno, C ; Cipponi, A ; Ballinger, ML ; Tavtigian, S ; Olivier, M ; Ruparel, V ; Haupt, Y ; Haupt, S ; Tucker, K ; Spurdle, AB ; Thomas, DM ; James, PA (WILEY, 2019-06)
    Germline pathogenic variants in the TP53 gene cause Li-Fraumeni syndrome, a condition that predisposes individuals to a wide range of cancer types. Identification of individuals carrying a TP53 pathogenic variant is linked to clinical management decisions, such as the avoidance of radiotherapy and use of high-intensity screening programs. The aim of this study was to develop an evidence-based quantitative model that integrates independent in silico data (Align-GVGD and BayesDel) and somatic to germline ratio (SGR), to assign pathogenicity to every possible missense variant in the TP53 gene. To do this, a likelihood ratio for pathogenicity (LR) was derived from each component calibrated using reference sets of assumed pathogenic and benign missense variants. A posterior probability of pathogenicity was generated by combining LRs, and algorithm outputs were validated using different approaches. A total of 730 TP53 missense variants could be assigned to a clinically interpretable class. The outputs of the model correlated well with existing clinical information, functional data, and ClinVar classifications. In conclusion, these quantitative outputs provide the basis for individualized assessment of cancer risk useful for clinical interpretation. In addition, we propose the value of the novel SGR approach for use within the ACMG/AMP guidelines for variant classification.
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    HIGH DOSE-RATE BRACHYTHERAPY OF LOCALIZED PROSTATE CANCER CONVERTS TUMORS FROM COLD TO HOT
    Keam, S ; Halse, H ; ThuNgoc, N ; Wang, M ; Losio, NVK ; Mitchell, C ; Caramia, F ; Byrne, D ; Haupt, S ; Ryland, G ; Darcy, P ; Sandhu, S ; Blombery, P ; Haupt, Y ; Williams, S ; Neeson, P (BMJ PUBLISHING GROUP, 2020-11)
    Background Prostate cancer is frequently cured with high dose-rate brachytherapy (HDRBT) radiation as a front-line treatment. Although considered to be an immune-excluded tissue, immune responses to radiation are implicated in driving tumour-eradication in prostate cancer.1 This has not been proven, and yet is used as the rationale for clinical trials combining radiation and immunotherapies.2 We hypothesise that there is a predictable relationship between radiation and the immune responses in prostate cancer that could be used to provide sound rationale for specific immune interventions in solid tumours that are made possible by radiation therapy. Methods We present here new results stemming from our recently published immunoprofiling study of world-unique pre- and post-radiation tissues from 24 prostate cancer patients (figure 1A), RadBank cohort).3 These samples were assessed using immune cell multiplex IHC, gene expression profiling, digital spatial profiling (DSP) and computational analysis of cell distribution. Results This study unequivocally revealed that high dose-rate radiation converts predominately ‘cold’ prostate tumour tissue to a more activated ‘hot’ state comprised of two sub-types (high and a less activated intermediate state). These changes were evident in increased tumour inflammation gene signatures and immune checkpoint expression, immune cell composition changes, and alterations in spatial interactions. However, as 20% of the patients did not respond, we also explored pre-treatment gene signatures of patient responses to radiation – identifying potential mechanisms that prime tissues to respond more favourably. Most recently, we have explored three other important facets of the immune response to HDRBT: (i) putative differential drivers of high and intermediate responses (figure 1B), (ii) TCR clonality changes (figure 1C), and (iii) the influence of clinical features (e.g. Gleason grade) and treatment (e.g. androgen deprivation) (figure 1D). Differential expression analysis has identified key molecules (e.g. CD40LG and Lck expression) which are associated with higher activation responses. TCR sequencing of pre- and post-HDRBT tissue and peripheral circulating cells is also suggestive of engagement of the adaptive immune system and the emergence of tumor-specific T cells. Finally, multivariate analysis has also revealed that higher grade tumours exhibit higher basal levels of activation and IC expression – making them less sensitive to immune activation by HDRBT. Abstract 580 Figure 1The effect of prostate brachytherapy on immune contexts(A) Study of immune response in 24 patients treated with HDRBT at Peter MacCallum Cancer Center ((DOI:10.1136/jitc2020-000792). Examples of new insights including (B) molecules associated with higher activation levels (e.g. Lck and CD40LG/CD154), (C) changes in T cell receptor dominance and diversity in tissue and peripheral circulation, and (D) effects of clinical attributes on immune modulators (e.g. TGFbeta) and TIS activation states. Conclusions We have begun to resolve clear patient and clinical classifiers based on immune responses to radiation, and identified patient groups likely to benefit from immune therapy alongside radiation. Importantly, these classifications are associated with baseline gene expression profiles that may be used for pre-clinical stratification and more sophisticated treatment paradigms. Ethics Approval All participants provided consent covering tissue research as part of a prospective tissue collection study for prostate radiobiology research, approved by the Human Research Ethics Committee at the Peter MacCallum Cancer Centre (PMCC; HREC approvals 10/68, 13/167, 18/204). Consent Written informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal. References Dudzinski SO, et al., Combination immunotherapy and radiotherapy causes an abscopal treatment response in a mouse model of castration resistant prostate cancer. J Immunother Cancer 2019. 7(1): p. 218. Kwon E.D., et al., Ipilimumab versus placebo after radiotherapy in patients with metastatic castration-resistant prostate cancer that had progressed after docetaxel chemotherapy (CA184-043): a multicentre, randomised, double-blind, phase 3 trial. Lancet Oncol 2014;15(7): p. 700–12. Keam SP, et al., High dose-rate brachytherapy of localized prostate cancer converts tumors from cold to hot. J Immunother Cancer 2020;8(1).
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    Identification of cancer sex-disparity in the functional integrity of p53 and its X chromosome network
    Haupt, S ; Caramia, F ; Herschtal, A ; Soussi, T ; Lozano, G ; Chen, H ; Liang, H ; Speed, TP ; Haupt, Y (NATURE PUBLISHING GROUP, 2019-11-26)
    The disproportionately high prevalence of male cancer is poorly understood. We tested for sex-disparity in the functional integrity of the major tumor suppressor p53 in sporadic cancers. Our bioinformatics analyses expose three novel levels of p53 impact on sex-disparity in 12 non-reproductive cancer types. First, TP53 mutation is more frequent in these cancers among US males than females, with poorest survival correlating with its mutation. Second, numerous X-linked genes are associated with p53, including vital genomic regulators. Males are at unique risk from alterations of their single copies of these genes. High expression of X-linked negative regulators of p53 in wild-type TP53 cancers corresponds with reduced survival. Third, females exhibit an exceptional incidence of non-expressed mutations among p53-associated X-linked genes. Our data indicate that poor survival in males is contributed by high frequencies of TP53 mutations and an inability to shield against deregulated X-linked genes that engage in p53 networks.
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    E6AP Promotes a Metastatic Phenotype in Prostate Cancer
    Gamell, C ; Bandilovska, I ; Gulati, T ; Kogan, A ; Lim, SC ; Kovacevic, Z ; Takano, EA ; Timpone, C ; Agupitan, AD ; Litchfield, C ; Blandino, G ; Horvath, LG ; Fox, SB ; Williams, SG ; Russo, A ; Gallo, E ; Paul, PJ ; Mitchell, C ; Sandhu, S ; Keam, SP ; Haupt, S ; Richardson, DR ; Haupt, Y (CELL PRESS, 2019-12-20)
    Although primary prostate cancer is largely curable, progression to metastatic disease is associated with very poor prognosis. E6AP is an E3 ubiquitin ligase and a transcriptional co-factor involved in normal prostate development. E6AP drives prostate cancer when overexpressed. Our study exposed a role for E6AP in the promotion of metastatic phenotype in prostate cells. We revealed that elevated levels of E6AP in primary prostate cancer correlate with regional metastasis and demonstrated that E6AP promotes acquisition of mesenchymal features, migration potential, and ability for anchorage-independent growth. We identified the metastasis suppressor NDRG1 as a target of E6AP and showed it is key in E6AP induction of mesenchymal phenotype. We showed that treatment of prostate cancer cells with pharmacological agents upregulated NDRG1 expression suppressed E6AP-induced cell migration. We propose that the E6AP-NDRG1 axis is an attractive therapeutic target for the treatment of E6AP-driven metastatic prostate cancer.
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    The long and the short of it: the MDM4 tail so far
    Haupt, S ; Mejia-Hernandez, JO ; Vijayakumaran, R ; Keam, SP ; Haupt, Y (OXFORD UNIV PRESS, 2019-03)
    The mouse double minute 4 (MDM4) is emerging from the shadow of its more famous relative MDM2 and is starting to steal the limelight, largely due to its therapeutic possibilities. MDM4 is a vital regulator of the tumor suppressor p53. It restricts p53 transcriptional activity and also, at least in development, facilitates MDM2's E3 ligase activity toward p53. These functions of MDM4 are critical for normal cell function and a proper response to stress. Their importance for proper cell maintenance and proliferation identifies them as a risk for deregulation associated with the uncontrolled growth of cancer. MDM4 tails are vital for its function, where its N-terminus transactivation domain engages p53 and its C-terminus RING domain binds to MDM2. In this review, we highlight recently identified cellular functions of MDM4 and survey emerging therapies directed to correcting its dysregulation in disease.
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    P53: A Guardian of Immunity Becomes Its Saboteur through Mutation
    Agupitan, AD ; Neeson, P ; Williams, S ; Howitt, J ; Haupt, S ; Haupt, Y (MDPI, 2020-05)
    Awareness of the importance of immunity in controlling cancer development triggered research into the impact of its key oncogenic drivers on the immune response, as well as their value as targets for immunotherapy. At the heart of tumour suppression is p53, which was discovered in the context of viral infection and now emerges as a significant player in normal and cancer immunity. Wild-type p53 (wt p53) plays fundamental roles in cancer immunity and inflammation. Mutations in p53 not only cripple wt p53 immune functions but also sinisterly subvert the immune function through its neomorphic gain-of-functions (GOFs). The prevalence of mutant p53 across different types of human cancers, which are associated with inflammatory and immune dysfunction, further implicates mutant p53 in modulating cancer immunity, thereby promoting tumorigenesis, metastasis and invasion. In this review, we discuss several mutant p53 immune GOFs in the context of the established roles of wt p53 in regulating and responding to tumour-associated inflammation, and regulating innate and adaptive immunity. We discuss the capacity of mutant p53 to alter the tumour milieu to support immune dysfunction, modulate toll-like receptor (TLR) signalling pathways to disrupt innate immunity and subvert cell-mediated immunity in favour of immune privilege and survival. Furthermore, we expose the potential and challenges associated with mutant p53 as a cancer immunotherapy target and underscore existing therapies that may benefit from inquiry into cancer p53 status.
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    MDM2 inhibition in combination with endocrine therapy and CDK4/6 inhibition for the treatment of ER-positive breast cancer
    Portman, N ; Milioli, HH ; Alexandrou, S ; Coulson, R ; Yong, A ; Fernandez, KJ ; Chia, KM ; Halilovic, E ; Segara, D ; Parker, A ; Haupt, S ; Haupt, Y ; Tilley, WD ; Swarbrick, A ; Caldon, CE ; Lim, E (BMC, 2020-08-12)
    BACKGROUND: Resistance to endocrine therapy is a major clinical challenge in the management of oestrogen receptor (ER)-positive breast cancer. In this setting, p53 is frequently wildtype and its activity may be suppressed via upregulation of its key regulator MDM2. This underlies our rationale to evaluate MDM2 inhibition as a therapeutic strategy in treatment-resistant ER-positive breast cancer. METHODS: We used the MDM2 inhibitor NVP-CGM097 to treat in vitro and in vivo models alone and in combination with fulvestrant or palbociclib. We perform cell viability, cell cycle, apoptosis and senescence assays to evaluate anti-tumour effects in p53 wildtype and p53 mutant ER-positive cell lines (MCF-7, ZR75-1, T-47D) and MCF-7 lines resistant to endocrine therapy and to CDK4/6 inhibition. We further assess the drug effects in patient-derived xenograft (PDX) models of endocrine-sensitive and endocrine-resistant ER-positive breast cancer. RESULTS: We demonstrate that MDM2 inhibition results in cell cycle arrest and increased apoptosis in p53-wildtype in vitro and in vivo breast cancer models, leading to potent anti-tumour activity. We find that endocrine therapy or CDK4/6 inhibition synergises with MDM2 inhibition but does not further enhance apoptosis. Instead, combination treatments result in profound regulation of cell cycle-related transcriptional programmes, with synergy achieved through increased antagonism of cell cycle progression. Combination therapy pushes cell lines resistant to fulvestrant or palbociclib to become senescent and significantly reduces tumour growth in a fulvestrant-resistant patient-derived xenograft model. CONCLUSIONS: We conclude that MDM2 inhibitors in combination with ER degraders or CDK4/6 inhibitors represent a rational strategy for treating advanced, endocrine-resistant ER-positive breast cancer, operating through synergistic activation of cell cycle co-regulatory programmes.
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    High dose-rate brachytherapy of localized prostate cancer converts tumors from cold to hot
    Keam, SP ; Halse, H ; Nguyen, T ; Wang, M ; Van Kooten Losio, N ; Mitchell, C ; Caramia, F ; Byrne, DJ ; Haupt, S ; Ryland, G ; Darcy, PK ; Sandhu, S ; Blombery, P ; Haupt, Y ; Williams, SG ; Neeson, PJ (BMJ PUBLISHING GROUP, 2020)
    BACKGROUND: Prostate cancer (PCa) has a profoundly immunosuppressive microenvironment and is commonly immune excluded with few infiltrative lymphocytes and low levels of immune activation. High-dose radiation has been demonstrated to stimulate the immune system in various human solid tumors. We hypothesized that localized radiation therapy, in the form of high dose-rate brachytherapy (HDRBT), would overcome immune suppression in PCa. METHODS: To investigate whether HDRBT altered prostate immune context, we analyzed preradiation versus postradiation human tissue from a cohort of 24 patients with localized PCa that received HDRBT as primary treatment (RadBank cohort). We performed Nanostring immune gene expression profiling, digital spatial profiling, and high-throughput immune cell multiplex immunohistochemistry analysis. We also resolved tumor and nontumor zones in spatial and bioinformatic analyses to explore the immunological response. RESULTS: Nanostring immune profiling revealed numerous immune checkpoint molecules (eg, B7-H3, CTLA4, PDL1, and PDL2) and TGFβ levels were increased in response to HDRBT. We used a published 16-gene tumor inflammation signature (TIS) to divide tumors into distinct immune activation states (high:hot, intermediate and low:cold) and showed that most localized PCa are cold tumors pre-HDRBT. Crucially, HDRBT converted 80% of these 'cold'-phenotype tumors into an 'intermediate' or 'hot' class. We used digital spatial profiling to show these HDRBT-induced changes in prostate TIS scores were derived from the nontumor regions. Furthermore, these changes in TIS were also associated with pervasive changes in immune cell density and spatial relationships-in particular, between T cell subsets and antigen presenting cells. We identified an increased density of CD4+ FOXP3+ T cells, CD68+ macrophages and CD68+ CD11c+ dendritic cells in response to HDRBT. The only subset change specific to tumor zones was PDL1- macrophages. While these immune responses were heterogeneous, HDRBT induced significant changes in immune cell associations, including a gained T cell and HMWCK+ PDL1+ interaction in tumor zones. CONCLUSION: In conclusion, we showed HDRBT converted "cold" prostate tumors into more immunologically activated "hot" tissues, with accompanying spatially organized immune infiltrates and signaling changes. Understanding and potentially harnessing these changes will have widespread implications for the future treatment of localized PCa, including rational use of combination radio-immunotherapy.
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    TP53 Status, Patient Sex, and the Immune Response as Determinants of Lung Cancer Patient Survival
    Freudenstein, D ; Litchfield, C ; Caramia, F ; Wright, G ; Solomon, BJ ; Ball, D ; Keam, SP ; Neeson, P ; Haupt, Y ; Haupt, S (MDPI, 2020-06)
    Lung cancer poses the greatest cancer-related death risk and males have poorer outcomes than females, for unknown reasons. Patient sex is not a biological variable considered in lung cancer standard of care. Correlating patient genetics with outcomes is predicted to open avenues for improved management. Using a bioinformatics approach across non-small cell lung cancer (NSCLC) subtypes, we identified where patient sex, mutation of the major tumor suppressor gene, Tumour protein P53 (TP53), and immune signatures stratified outcomes in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC), among datasets of The Cancer Genome Atlas (TCGA). We exposed sex and TP53 gene mutations as prognostic for LUAD survival. Longest survival in LUAD occurred among females with wild-type (wt) TP53 genes, high levels of immune infiltration and enrichment for pathway signatures of Interferon Gamma (INF-γ), Tumour Necrosis Factor (TNF) and macrophages-monocytes. In contrast, poor survival in men with LUAD and wt TP53 genes corresponded with enrichment of Transforming Growth Factor Beta 1 (TGFB1, hereafter TGF-β) and wound healing signatures. In LUAD with wt TP53 genes, elevated gene expression of immune checkpoint CD274 (hereafter: PD-L1) and also protein 53 (p53) negative-regulators of the Mouse Double Minute (MDM)-family predict novel avenues for combined immunotherapies. LUSC is dominated by male smokers with TP53 gene mutations, while a minor population of TCGA LC patients with wt TP53 genes unexpectedly had the poorest survival, suggestive of a separate etiology. We conclude that advanced approaches to LUAD and LUSC therapy lie in the consideration of patient sex, TP53 gene mutation status and immune signatures.
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    Regulation of PRMT5-MDM4 axis is critical in the response to CDK4/6 inhibitors in melanoma
    AbuHammad, S ; Cullinane, C ; Martin, C ; Bacolas, Z ; Ward, T ; Chen, H ; Slater, A ; Ardley, K ; Kirby, L ; Chan, KT ; Brajanovski, N ; Smith, LK ; Rao, AD ; Lelliott, EJ ; Kleinschmidt, M ; Vergara, IA ; Papenfuss, AT ; Lau, P ; Ghosh, P ; Haupt, S ; Haupt, Y ; Sanij, E ; Poortinga, G ; Pearson, RB ; Falk, H ; Curtis, DJ ; Stupple, P ; Devlin, M ; Street, I ; Davies, MA ; McArthur, GA ; Sheppard, KE (NATL ACAD SCIENCES, 2019-09-03)
    Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors are an established treatment in estrogen receptor-positive breast cancer and are currently in clinical development in melanoma, a tumor that exhibits high rates of CDK4 activation. We analyzed melanoma cells with acquired resistance to the CDK4/6 inhibitor palbociclib and demonstrate that the activity of PRMT5, a protein arginine methyltransferase and indirect target of CDK4, is essential for CDK4/6 inhibitor sensitivity. By indirectly suppressing PRMT5 activity, palbociclib alters the pre-mRNA splicing of MDM4, a negative regulator of p53, leading to decreased MDM4 protein expression and subsequent p53 activation. In turn, p53 induces p21, leading to inhibition of CDK2, the main kinase substituting for CDK4/6 and a key driver of resistance to palbociclib. Loss of the ability of palbociclib to regulate the PRMT5-MDM4 axis leads to resistance. Importantly, combining palbociclib with the PRMT5 inhibitor GSK3326595 enhances the efficacy of palbociclib in treating naive and resistant models and also delays the emergence of resistance. Our studies have uncovered a mechanism of action of CDK4/6 inhibitors in regulating the MDM4 oncogene and the tumor suppressor, p53. Furthermore, we have established that palbociclib inhibition of the PRMT5-MDM4 axis is essential for robust melanoma cell sensitivity and provide preclinical evidence that coinhibition of CDK4/6 and PRMT5 is an effective and well-tolerated therapeutic strategy. Overall, our data provide a strong rationale for further investigation of novel combinations of CDK4/6 and PRMT5 inhibitors, not only in melanoma but other tumor types, including breast, pancreatic, and esophageal carcinoma.