Sir Peter MacCallum Department of Oncology - Research Publications
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ItemJAK2 is dispensable for maintenance of JAK2 mutant B-cell acute lymphoblastic leukemias(COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT, 2018-06-01)Activating JAK2 point mutations are implicated in the pathogenesis of myeloid and lymphoid malignancies, including high-risk B-cell acute lymphoblastic leukemia (B-ALL). In preclinical studies, treatment of JAK2 mutant leukemias with type I JAK2 inhibitors (e.g., Food and Drug Administration [FDA]-approved ruxolitinib) provided limited single-agent responses, possibly due to paradoxical JAK2Y1007/1008 hyperphosphorylation induced by these agents. To determine the importance of mutant JAK2 in B-ALL initiation and maintenance, we developed unique genetically engineered mouse models of B-ALL driven by overexpressed Crlf2 and mutant Jak2, recapitulating the genetic aberrations found in human B-ALL. While expression of mutant Jak2 was necessary for leukemia induction, neither its continued expression nor enzymatic activity was required to maintain leukemia survival and rapid proliferation. CRLF2/JAK2 mutant B-ALLs with sustained depletion or pharmacological inhibition of JAK2 exhibited enhanced expression of c-Myc and prominent up-regulation of c-Myc target genes. Combined indirect targeting of c-Myc using the BET bromodomain inhibitor JQ1 and direct targeting of JAK2 with ruxolitinib potently killed JAK2 mutant B-ALLs.
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ItemHow do tumor cells respond to HDAC inhibition?(WILEY, 2016-11)It is now well recognized that mutations, deregulated expression, and aberrant recruitment of epigenetic readers, writers, and erasers are fundamentally important processes in the onset and maintenance of many human tumors. The molecular, biological, and biochemical characteristics of a particular class of epigenetic erasers, the histone deacetylases (HDACs), have been extensively studied and small-molecule HDAC inhibitors (HDACis) have now been clinically approved for the treatment of human hemopoietic malignancies. This review explores our current understanding of the biological and molecular effects on tumor cells following HDACi treatment. The predominant responses include induction of tumor cell death and inhibition of proliferation that in experimental models have been linked to therapeutic efficacy. However, tumor cell-intrinsic responses to HDACi, including modulating tumor immunogenicity have also been described and may have substantial roles in mediating the antitumor effects of HDACi. We posit that the field has failed to fully reconcile the biological consequences of exposure to HDACis with the molecular events that underpin these responses, however progress is being made. Understanding the pleiotrophic activities of HDACis on tumor cells will hopefully fast track the development of more potent and selective HDACi that may be used alone or in combination to improve patient outcomes.
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ItemLong term, continuous exposure to panobinostat induces terminal differentiation and long term survival in the TH-MYCN neuroblastoma mouse model(WILEY, 2016-07-01)Neuroblastoma is the most common extra-cranial malignancy in childhood and accounts for ∼15% of childhood cancer deaths. Amplification of MYCN in neuroblastoma is associated with aggressive disease and predicts for poor prognosis. Novel therapeutic approaches are therefore essential to improving patient outcomes in this setting. The histone deacetylases are known to interact with N-Myc and regulate numerous cellular processes via epigenetic modulation, including differentiation. In this study, we used the TH-MYCN mouse model of neuroblastoma to investigate the antitumor activity of the pan-HDAC inhibitor, panobinostat. In particular we sought to explore the impact of long term, continuous panobinostat exposure on the epigenetically driven differentiation process. Continuous treatment of tumor bearing TH-MYCN transgenic mice with panobinostat for nine weeks led to a significant improvement in survival as compared with mice treated with panobinostat for a three-week period. Panobinostat induced rapid tumor regression with no regrowth observed following a nine-week treatment period. Initial tumor response was associated with apoptosis mediated via upregulation of BMF and BIM. The process of terminal differentiation of neuroblastoma into benign ganglioneuroma, with a characteristic increase in S100 expression and reduction of N-Myc expression, occurred following prolonged exposure to the drug. RNA-sequencing analysis of tumors from treated animals confirmed significant upregulation of gene pathways associated with apoptosis and differentiation. Together our data demonstrate the potential of panobinostat as a novel therapeutic strategy for high-risk neuroblastoma patients.
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ItemSerglycin determines secretory granule repertoire and regulates natural killer cell and cytotoxic T lymphocyte cytotoxicity(WILEY, 2016-03)The anionic proteoglycan serglycin is a major constituent of secretory granules in cytotoxic T lymphocyte (CTL)/natural killer (NK) cells, and is proposed to promote the safe storage of the mostly cationic granule toxins, granzymes and perforin. Despite the extensive defects of mast cell function reported in serglycin gene-disrupted mice, no comprehensive study of physiologically relevant CTL/NK cell populations has been reported. We show that the cytotoxicity of serglycin-deficient CTL and NK cells is severely compromised but can be partly compensated in both cell types when they become activated. Reduced intracellular granzyme B levels were noted, particularly in CD27(+) CD11b(+) mature NK cells, whereas serglycin(-/-) TCR-transgenic (OTI) CD8 T cells also had reduced perforin stores. Culture supernatants from serglycin(-/-) OTI T cells and interleukin-2-activated NK contained increased granzyme B, linking reduced storage with heightened export. By contrast, granzyme A was not significantly reduced in cells lacking serglycin, indicating differentially regulated trafficking and/or storage for the two granzymes. A quantitative analysis of different granule classes by transmission electronmicroscopy showed a selective loss of dense-core granules in serglycin(-/-) CD8(+) CTLs, although other granule types were maintained quantitatively. The findings of the present study show that serglycin plays a critical role in the maturation of dense-core cytotoxic granules in cytotoxic lymphocytes and the trafficking and storage of perforin and granzyme B, whereas granzyme A is unaffected. The skewed retention of cytotoxic effector molecules markedly reduces CTL/NK cell cytotoxicity, although this is partly compensated for as a result of activating the cells by physiological means.
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ItemHistone deacetylase inhibitors: potential targets responsible for their anti-cancer effect(SPRINGER, 2010-12)The histone deacetylase inhibitors (HDACi) have demonstrated anticancer efficacy across a range of malignancies, most impressively in the hematological cancers. It is uncertain whether this clinical efficacy is attributable predominantly to their ability to induce apoptosis and differentiation in the cancer cell, or to their ability to prime the cell to other pro-death stimuli such as those from the immune system. HDACi-induced apoptosis occurs through altered expression of genes encoding proteins in both intrinsic and extrinsic apoptotic pathways; through effects on the proteasome/aggresome systems; through the production of reactive oxygen species, possibly by directly inducing DNA damage; and through alterations in the tumor microenvironment. In addition HDACi increase the immunogenicity of tumor cells and modulate cytokine signaling and potentially T-cell polarization in ways that may contribute the anti-cancer effect in vivo. Here, we provide an overview of current thinking on the mechanisms of HDACi activity, with attention given to the hematological malignancies as well as scientific observations arising from the clinical trials. We also focus on the immune effects of these agents.
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ItemPreclinical screening of histone deacetylase inhibitors combined with ABT-737, rhTRAIL/MD5-1 or 5-azacytidine using syngeneic Vk*MYC multiple myeloma(NATURE PUBLISHING GROUP, 2013-09)Multiple myeloma (MM) is an incurable malignancy with an unmet need for innovative treatment options. Histone deacetylase inhibitors (HDACi) are a new class of anticancer agent that have demonstrated activity in hematological malignancies. Here, we investigated the efficacy and safety of HDACi (vorinostat, panobinostat, romidepsin) and novel combination therapies using in vitro human MM cell lines and in vivo preclinical screening utilizing syngeneic transplanted Vk*MYC MM. HDACi were combined with ABT-737, which targets the intrinsic apoptosis pathway, recombinant human tumour necrosis factor-related apoptosis-inducing ligand (rhTRAIL/MD5-1), that activates the extrinsic apoptosis pathway or the DNA methyl transferase inhibitor 5-azacytidine. We demonstrate that in vitro cell line-based studies provide some insight into drug activity and combination therapies that synergistically kill MM cells; however, they do not always predict in vivo preclinical efficacy or toxicity. Importantly, utilizing transplanted Vk*MYC MM, we report that panobinostat and 5-azacytidine synergize to prolong the survival of tumor-bearing mice. In contrast, combined HDACi/rhTRAIL-based strategies, while efficacious, demonstrated on-target dose-limiting toxicities that precluded prolonged treatment. Taken together, our studies provide evidence that the transplanted Vk*MYC model of MM is a useful screening tool for anti-MM drugs and should aid in the prioritization of novel drug testing in the clinic.
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ItemHDAC inhibitors induce tumor-cell-selective pro-apoptotic transcriptional responses(SPRINGERNATURE, 2013-02)The identification of recurrent somatic mutations in genes encoding epigenetic enzymes has provided a strong rationale for the development of compounds that target the epigenome for the treatment of cancer. This notion is supported by biochemical studies demonstrating aberrant recruitment of epigenetic enzymes such as histone deacetylases (HDACs) and histone methyltransferases to promoter regions through association with oncogenic fusion proteins such as PML-RARα and AML1-ETO. HDAC inhibitors (HDACi) are potent inducers of tumor cell apoptosis; however, it remains unclear why tumor cells are more sensitive to HDACi-induced cell death than normal cells. Herein, we assessed the biological and molecular responses of isogenic normal and transformed cells to the FDA-approved HDACi vorinostat and romidepsin. Both HDACi selectively killed cells of diverse tissue origin that had been transformed through the serial introduction of different oncogenes. Time-course microarray expression profiling revealed that normal and transformed cells transcriptionally responded to vorinostat treatment. Over 4200 genes responded differently to vorinostat in normal and transformed cells and gene ontology and pathway analyses identified a tumor-cell-selective pro-apoptotic gene-expression signature that consisted of BCL2 family genes. In particular, HDACi induced tumor-cell-selective upregulation of the pro-apoptotic gene BMF and downregulation of the pro-survival gene BCL2A1 encoding BFL-1. Maintenance of BFL-1 levels in transformed cells through forced expression conferred vorinostat resistance, indicating that specific and selective engagement of the intrinsic apoptotic pathway underlies the tumor-cell-selective apoptotic activities of these agents. The ability of HDACi to affect the growth and survival of tumor cells whilst leaving normal cells relatively unharmed is fundamental to their successful clinical application. This study provides new insight into the transcriptional effects of HDACi in human donor-matched normal and transformed cells, and implicates specific molecules and pathways in the tumor-selective cytotoxic activity of these compounds.
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ItemEnhancing the antitumor effects of radiotherapy with combinations of immunostimulatory antibodies(LANDES BIOSCIENCE, 2012-12)The development and use of combination immunotherapy-based anticancer regimens is at an early but clearly exciting stage. We now demonstrate that the antibody-based co-targeting of multiple immunostimulatory and/or inhibitory pathways can be used safely and effectively in combination with single dose or fractionated radiotherapy to cure mice bearing established mammary tumors.
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ItemOverview of Histone Deacetylase Inhibitors in Haematological Malignancies(MDPI, 2010-08)Histone deacetylase inhibitors (HDACi) can induce hyperacetylation of both histone and non-histone target resulting in epigenetic reprogramming and altered activity, stability and localisation of non-histone proteins to ultimately mediate diverse biological effects on cancer cells and their microenvironment. Clinical trials have demonstrated single agent HDACi to have activity in hematological malignancies, in particular T-cell lymphoma and Hodgkin lymphoma. Combination strategies with standard therapies based on pre-clinical data are being employed with significant success due to their excellent side effect profile. Correlative studies will provide valuable information on the sub-groups of patients more likely to respond or be resistant to HDACi therapy, while long-term monitoring for toxicities is also needed.
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ItemThe combination of histone deacetylase inhibitors with immune-stimulating antibodies has potent anti-cancer effects(LANDES BIOSCIENCE, 2012-05-01)The use of immunotherapy to treat cancer is rapidly gaining momentum. Using pre-clinical mouse models, we have recently demonstrated potent and long lasting tumor regression can be elicited by immune-stimulating monoclonal antibodies (mAbs) when combined with histone deacetylase inhibitors (HDACi) and believe this therapy will have broad application in humans.