Sir Peter MacCallum Department of Oncology - Research Publications

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    Tucatinib promotes immune activation and synergizes with programmed cell death-1 and programmed cell death-ligand 1 inhibition in HER2-positive breast cancer
    Li, R ; Sant, S ; Brown, E ; Caramia, F ; Nikolic, B ; Clarke, K ; Byrne, A ; Gonzalez, LEL ; Savas, P ; Luen, SJ ; Teo, ZL ; Virassamy, B ; Neeson, PJ ; Darcy, PK ; Loi, S (OXFORD UNIV PRESS INC, 2023-07-06)
    BACKGROUND: Programmed cell death-1 (PD-1) and programmed cell death-ligand 1 (PD-L1) inhibitors have poor efficacy in patients with trastuzumab-resistant advanced HER2-positive breast cancer. Tucatinib is a potent, selective anti-HER2 tyrosine kinase inhibitor with proven clinical benefit in the advanced setting in patients with trastuzumab resistance. We investigated if tucatinib can alter the tumor microenvironment and if this could be harnessed for therapeutic efficacy. METHODS: We investigated the antitumor efficacy and contribution of the immune response of tucatinib using 2 immunocompetent, HER2-positive murine breast cancer models (trastuzumab-sensitive H2N113; trastuzumab-resistant Fo5) and the efficacy of tucatinib with trastuzumab and PD-1 or PD-L1 checkpoint inhibitors. RESULTS: In both models, tucatinib statistically significantly inhibited tumor growth and demonstrated dose-dependent efficacy. Ex vivo analysis by flow cytometry of tumor-infiltrating lymphocytes in mice treated with tucatinib showed increased frequency, higher proliferation, and enhanced effector function of CD8+ effector memory T cells. Tucatinib treatment also increased frequency of CD8+PD-1+ and CD8+TIM3+ T cells, CD49+ natural killer cells, monocytes, and major histocompatibility complex II expression on dendritic cells and macrophages and a decrease in myeloid-derived suppressor cells. Gene expression analysis revealed statistically significant enrichment in pathways associated with immune activation, type I and II interferon response, adaptive immune response, and antigen receptor signaling. In vivo, tucatinib and α-PD-L1 or α-PD-1 demonstrated statistically significantly increased efficacy and improved survival of mice compared with tucatinib alone. CONCLUSION: Tucatinib modulates the immune microenvironment favorably, and combination treatment with α-PD-L1 or α-PD-1 demonstrated increased efficacy in preclinical HER2-positive tumor models. These findings provide a rationale for investigation of tucatinib and immune checkpoint inhibition in the clinic.
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    A2AR eGFP reporter mouse enables elucidation of A2AR expression dynamics during anti-tumor immune responses
    Todd, KL ; Lai, J ; Sek, K ; Huang, Y-K ; Newman, DM ; Derrick, EB ; Koay, H-F ; Nguyen, D ; Hoang, TX ; Petley, EV ; Chan, CW ; Munoz, I ; House, IG ; Lee, JN ; Kim, JS ; Li, J ; Tong, J ; N. de Menezes, M ; Scheffler, CM ; Yap, KM ; Chen, AXY ; Dunbar, PA ; Haugen, B ; Parish, IA ; Johnstone, RW ; Darcy, PK ; Beavis, PA (NATURE PORTFOLIO, 2023-11-01)
    There is significant clinical interest in targeting adenosine-mediated immunosuppression, with several small molecule inhibitors having been developed for targeting the A2AR receptor. Understanding of the mechanism by which A2AR is regulated has been hindered by difficulty in identifying the cell types that express A2AR due to a lack of robust antibodies for these receptors. To overcome this limitation, here an A2AR eGFP reporter mouse is developed, enabling the expression of A2AR during ongoing anti-tumor immune responses to be assessed. This reveals that A2AR is highly expressed on all tumor-infiltrating lymphocyte subsets including Natural Killer (NK) cells, NKT cells, γδ T cells, conventional CD4+ and CD8+ T lymphocytes and on a MHCIIhiCD86hi subset of type 2 conventional dendritic cells. In response to PD-L1 blockade, the emergence of PD-1+A2AR- cells correlates with successful therapeutic responses, whilst IL-18 is identified as a cytokine that potently upregulates A2AR and synergizes with A2AR deficiency to improve anti-tumor immunity. These studies provide insight into the biology of A2AR in the context of anti-tumor immunity and reveals potential combination immunotherapy approaches.
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    Combined PARP and WEE1 inhibition triggers anti-tumor immune response in BRCA1/2 wildtype triple-negative breast cancer
    Teo, ZL ; O'Connor, MJ ; Versaci, S ; Clarke, KA ; Brown, ER ; Percy, LW ; Kuykhoven, K ; Mintoff, CP ; Savas, P ; Virassamy, B ; Luen, SJ ; Byrne, A ; Sant, S ; Lindeman, GJ ; Darcy, PK ; Loi, S (NATURE PORTFOLIO, 2023-08-15)
    Novel therapeutic strategies that can effectively combine with immunotherapies are needed in the treatment of triple-negative breast cancer (TNBC). We demonstrate that combined PARP and WEE1 inhibition are synergistic in controlling tumour growth in BRCA1/2 wild-type TNBC preclinical models. The PARP inhibitor (PARPi) olaparib combined with the WEE1 inhibitor (WEE1i) adavosertib triggered increases in anti-tumour immune responses, including STING pathway activation. Combinations with a STING agonist resulted in further improved durable tumour regression and significant improvements in survival outcomes in murine tumour models of BRCA1/2 wild-type TNBC. In addition, we have identified baseline tumour-infiltrating lymphocyte (TIL) levels as a potential predictive biomarker of response to PARPi, WEE1i and immunotherapies in BRCA1/2 wild-type TNBC.
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    Low-dose carboplatin modifies the tumor microenvironment to augment CAR T cell efficacy in human prostate cancer models
    Porter, LH ; Zhu, JJ ; Lister, NL ; Harrison, SG ; Keerthikumar, S ; Goode, DL ; Urban, RQ ; Byrne, DJ ; Azad, A ; Vela, I ; Hofman, MS ; Neeson, PJ ; Darcy, PK ; Trapani, JA ; Taylor, RA ; Risbridger, GP (NATURE PORTFOLIO, 2023-09-02)
    Chimeric antigen receptor (CAR) T cells have transformed the treatment landscape for hematological malignancies. However, CAR T cells are less efficient against solid tumors, largely due to poor infiltration resulting from the immunosuppressive nature of the tumor microenvironment (TME). Here, we assessed the efficacy of Lewis Y antigen (LeY)-specific CAR T cells in patient-derived xenograft (PDX) models of prostate cancer. In vitro, LeY CAR T cells directly killed organoids derived from androgen receptor (AR)-positive or AR-null PDXs. In vivo, although LeY CAR T cells alone did not reduce tumor growth, a single prior dose of carboplatin reduced tumor burden. Carboplatin had a pro-inflammatory effect on the TME that facilitated early and durable CAR T cell infiltration, including an altered cancer-associated fibroblast phenotype, enhanced extracellular matrix degradation and re-oriented M1 macrophage differentiation. In a PDX less sensitive to carboplatin, CAR T cell infiltration was dampened; however, a reduction in tumor burden was still observed with increased T cell activation. These findings indicate that carboplatin improves the efficacy of CAR T cell treatment, with the extent of the response dependent on changes induced within the TME.
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    CRISPR-Cas9 screening identifies an IRF1-SOCS1-mediated negative feedback loop that limits CXCL9 expression and antitumor immunity
    House, IG ; Derrick, EB ; Sek, K ; Chen, AXY ; Li, J ; Lai, J ; Todd, KL ; Munoz, I ; Michie, J ; Chan, CW ; Huang, Y-K ; Chan, JD ; Petley, E ; Tong, J ; Nguyen, D ; Engel, S ; Savas, P ; Hogg, SJ ; Vervoort, SJ ; Kearney, CJ ; Burr, ML ; Lam, EYN ; Gilan, O ; Bedoui, S ; Johnstone, RW ; Dawson, MA ; Loi, S ; Darcy, PK ; Beavis, PA (CELL PRESS, 2023-08-29)
    CXCL9 expression is a strong predictor of response to immune checkpoint blockade therapy. Accordingly, we sought to develop therapeutic strategies to enhance the expression of CXCL9 and augment antitumor immunity. To perform whole-genome CRISPR-Cas9 screening for regulators of CXCL9 expression, a CXCL9-GFP reporter line is generated using a CRISPR knockin strategy. This approach finds that IRF1 limits CXCL9 expression in both tumor cells and primary myeloid cells through induction of SOCS1, which subsequently limits STAT1 signaling. Thus, we identify a subset of STAT1-dependent genes that do not require IRF1 for their transcription, including CXCL9. Targeting of either IRF1 or SOCS1 potently enhances CXCL9 expression by intratumoral macrophages, which is further enhanced in the context of immune checkpoint blockade therapy. We hence show a non-canonical role for IRF1 in limiting the expression of a subset of STAT1-dependent genes through induction of SOCS1.
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    Estrogen-induced immune changes within the normal mammary gland
    Tower, H ; Dall, G ; Davey, A ; Stewart, M ; Lanteri, P ; Ruppert, M ; Lambouras, M ; Nasir, I ; Yeow, S ; Darcy, PK ; Ingman, W ; Parker, B ; Haynes, NM ; Britt, KL (NATURE PORTFOLIO, 2022-11-08)
    Breast cancer (BCa) incidence increases following aberrant hormone exposure, which has been linked to direct effects on estrogen receptor (ER)+ mammary epithelium. While estrogen exposure during mammary involution has been shown to drive tumour growth via neutrophils, the potential for the ER + immune microenvironment to mediate part (in addition to mammary epithelial cells) of hormonally controlled BCa risk during normal development has not been assessed. We collected mammary tissue, lymph nodes and blood from tumour naïve mice treated with, oophorectomy, estrogen (17β estradiol) or Fulvestrant. Flow cytometry was used to examine the impact on the frequency of innate and adaptive immune cells. Oophorectomy and fulvestrant decreased the proportion of macrophages, particularly pro-tumour polarized M2 macrophages and neutrophils. Conversely, dendritic cells were increased by these therapies, as were eosinophils. Estrogen increased the proportion of M2 macrophages and to a lesser extent CD4-CD8- double negative and FoxP3+ regulatory T cells but decreased CD8 + T cells and B cells. Excluding eosinophils, these changes were restricted to the mammary tissue. This suggests that inhibiting estrogen action lowers the immune suppressive myeloid cells, increases in antigen presentation and eosinophil-mediated direct or indirect cytotoxic effects. In contrast, estrogen exposure, which drives BCa risk, increases the suppressive myeloid cells and reduces anti-tumour cytotoxic T cells. The impact of hormonal exposure on BCa risk, may in part be linked to its immune modulatory activity.
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    Targeting Protein Tyrosine Phosphatase 22 Does Not Enhance the Efficacy of Chimeric Antigen Receptor T Cells in Solid Tumors
    Du, X ; Darcy, PK ; Wiede, F ; Tiganis, T (AMER SOC MICROBIOLOGY, 2022-03-17)
    Adoptive cell therapy with chimeric antigen receptor (CAR) T cells has revolutionized the treatment of certain B cell malignancies but has been in ineffective against solid tumors. Recent studies have highlighted the potential of targeting negative regulators of T cell signaling to enhance the efficacy and extend the utility of CAR T cells to solid tumors. Autoimmunity-linked protein tyrosine phosphatase N22 (PTPN22) has been proposed as a target for cancer immunotherapy. Here, we have used CRISPR/Cas9 gene editing to generate PTPN22-deficient (Ptpn22Δ/Δ) mice (C57BL/6) and assessed the impact of PTPN22 deficiency on the cytotoxicity and efficacy of CAR T cells in vitro and in vivo. As reported previously, PTPN22 deficiency was accompanied by the promotion of effector T cell responses ex vivo and the repression of syngeneic tumor growth in vivo. However, PTPN22 deficiency did not enhance the cytotoxic activity of murine CAR T cells targeting the extracellular domain of the human oncoprotein HER2 in vitro. Moreover, PTPN22-deficient α-HER2 CAR T cells or ovalbumin-specific OT-I CD8+ T cells adoptively transferred into mice bearing HER2+ mammary tumors or ovalbumin-expressing mammary or colorectal tumors, respectively, were no more effective than their wild-type counterparts in suppressing tumor growth. The deletion of PTPN22 using CRISPR/Cas9 gene editing also did not affect the cytotoxic activity of human CAR T cells targeting the Lewis Y antigen that is expressed by many human solid tumors. Therefore, PTPN22 deficiency does not enhance the antitumor activity of CAR T cells in solid organ malignancies.
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    TSTEM-like CAR-T cells exhibit improved persistence and tumor control compared with conventional CAR-T cells in preclinical models
    Meyran, D ; Zhu, JJ ; Butler, J ; Tantalo, D ; MacDonald, S ; Nguyen, TN ; Wang, M ; Thio, N ; D'Souza, C ; Qin, VM ; Slaney, C ; Harrison, A ; Sek, K ; Petrone, P ; Thia, K ; Giuffrida, L ; Scott, AM ; Terry, RL ; Tran, B ; Desai, J ; Prince, HM ; Harrison, SJ ; Beavis, PA ; Kershaw, MH ; Solomon, B ; Ekert, PG ; Trapani, JA ; Darcy, PK ; Neeson, PJ (AMER ASSOC ADVANCEMENT SCIENCE, 2023-04-05)
    Patients who receive chimeric antigen receptor (CAR)-T cells that are enriched in memory T cells exhibit better disease control as a result of increased expansion and persistence of the CAR-T cells. Human memory T cells include stem-like CD8+ memory T cell progenitors that can become either functional stem-like T (TSTEM) cells or dysfunctional T progenitor exhausted (TPEX) cells. To that end, we demonstrated that TSTEM cells were less abundant in infused CAR-T cell products in a phase 1 clinical trial testing Lewis Y-CAR-T cells (NCT03851146), and the infused CAR-T cells displayed poor persistence in patients. To address this issue, we developed a production protocol to generate TSTEM-like CAR-T cells enriched for expression of genes in cell replication pathways. Compared with conventional CAR-T cells, TSTEM-like CAR-T cells had enhanced proliferative capacity and increased cytokine secretion after CAR stimulation, including after chronic CAR stimulation in vitro. These responses were dependent on the presence of CD4+ T cells during TSTEM-like CAR-T cell production. Adoptive transfer of TSTEM-like CAR-T cells induced better control of established tumors and resistance to tumor rechallenge in preclinical models. These more favorable outcomes were associated with increased persistence of TSTEM-like CAR-T cells and an increased memory T cell pool. Last, TSTEM-like CAR-T cells and anti-programmed cell death protein 1 (PD-1) treatment eradicated established tumors, and this was associated with increased tumor-infiltrating CD8+CAR+ T cells producing interferon-γ. In conclusion, our CAR-T cell protocol generated TSTEM-like CAR-T cells with enhanced therapeutic efficacy, resulting in increased proliferative capacity and persistence in vivo.
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    Intratumoral CD8+T cells with a tissue-resident memory phenotype mediate local immunity and immune checkpoint responses in breast cancer
    Virassamy, B ; Caramia, F ; Savas, P ; Sant, S ; Wang, J ; Christo, SN ; Byrne, A ; Clarke, K ; Brown, E ; Teo, ZL ; von Scheidt, B ; Freestone, D ; Gandolfo, LC ; Weber, K ; Teply-Szymanski, J ; Li, R ; Luen, SJ ; Denkert, C ; Loibl, S ; Lucas, O ; Swanton, C ; Speed, TP ; Darcy, PK ; Neeson, PJ ; Mackay, LK ; Loi, S (CELL PRESS, 2023-03-13)
    CD8+ tumor-infiltrating lymphocytes with a tissue-resident memory T (TRM) cell phenotype are associated with favorable prognosis in patients with triple-negative breast cancer (TNBC). However, the relative contribution of CD8+ TRM cells to anti-tumor immunity and immune checkpoint blockade efficacy in breast cancer remains unknown. Here, we show that intratumoral CD8+ T cells in murine mammary tumors transcriptionally resemble those from TNBC patients. Phenotypic and transcriptional studies established two intratumoral sub-populations: one more enriched in markers of terminal exhaustion (TEX-like) and the other with a bona fide resident phenotype (TRM-like). Treatment with anti-PD-1 and anti-CTLA-4 therapy resulted in expansion of these intratumoral populations, with the TRM-like subset displaying significantly enhanced cytotoxic capacity. TRM-like CD8+ T cells could also provide local immune protection against tumor rechallenge and a TRM gene signature extracted from tumor-free tissue was significantly associated with improved clinical outcomes in TNBC patients treated with checkpoint inhibitors.
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    IL-15 Preconditioning Augments CAR T Cell Responses to Checkpoint Blockade for Improved Treatment of Solid Tumors
    Giuffrida, L ; Sek, K ; Henderson, MA ; House, IG ; Lai, J ; Chen, AXY ; Todd, KL ; Petley, E ; Mardiana, S ; Todorovski, I ; Gruber, E ; Kelly, MJ ; Solomon, BJ ; Vervoort, SJ ; Johnstone, RW ; Parish, IA ; Neeson, PJ ; Kats, LM ; Darcy, PK ; Beavis, PA (CELL PRESS, 2020-11-04)
    Chimeric antigen receptor (CAR) T cell therapy has been highly successful in hematological malignancies leading to their US Food and Drug Administration (FDA) approval. However, the efficacy of CAR T cells in solid tumors is limited by tumor-induced immunosuppression, leading to the development of combination approaches, such as adjuvant programmed cell death 1 (PD-1) blockade. Current FDA-approved methods for generating CAR T cells utilize either anti-CD3 and interleukin (IL)-2 or anti-CD3/CD28 beads, which can generate a T cell product with an effector/exhausted phenotype. Whereas different cytokine preconditioning milieu, such as IL-7/IL-15, have been shown to promote T cell engraftment, the impact of this approach on CAR T cell responses to adjuvant immune-checkpoint blockade has not been assessed. In the current study, we reveal that the preconditioning of CAR T cells with IL-7/IL-15 increased CAR T cell responses to anti-PD-1 adjuvant therapy. This was associated with the emergence of an intratumoral CD8+CD62L+TCF7+IRF4- population that was highly responsive to anti-PD-1 therapy and mediated the vast majority of transcriptional and epigenetic changes in vivo following PD-1 blockade. Our data indicate that preservation of CAR T cells in a TCF7+ phenotype is crucial for their responsiveness to adjuvant immunotherapy approaches and should be a key consideration when designing clinical protocols.