Sadna Budhu

Inhibiting DNA Methylation Causes an Interferon Response in Cancer via dsRNA Including Endogenous Retroviruses.

We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.We show that DNA methyltransferase inhibitors (DNMTis) upregulate immune signaling in cancer through the viral defense pathway. In ovarian cancer (OC), DNMTis trigger cytosolic sensing of double-stranded RNA (dsRNA) causing a type I interferon response and apoptosis. Knocking down dsRNA sensors TLR3 and MAVS reduces this response 2-fold and blocking interferon beta or its receptor abrogates it. Upregulation of hypermethylated endogenous retrovirus (ERV) genes accompanies the response and ERV overexpression activates the response. Basal levels of ERV and viral defense gene expression significantly correlate in primary OC and the latter signature separates primary samples for multiple tumor types from The Cancer Genome Atlas into low versus high expression groups. In melanoma patients treated with an immune checkpoint therapy, high viral defense signature expression in tumors significantly associates with durable clinical response and DNMTi treatment sensitizes to anti-CTLA4 therapy in a pre-clinical melanoma model.

Overcoming resistance to checkpoint blockade therapy by targeting PI3Kγ in myeloid cells

Recent clinical trials using immunotherapy have demonstrated its potential to control cancer by disinhibiting the immune system. Immune checkpoint blocking (ICB) antibodies against cytotoxic-T-lymphocyte-associated protein 4 or programmed cell death protein 1/programmed death-ligand 1 have displayed durable clinical responses in various cancers. Although these new immunotherapies have had a notable effect on cancer treatment, multiple mechanisms of immune resistance exist in tumours. Among the key mechanisms, myeloid cells have a major role in limiting effective tumour immunity. Growing evidence suggests that high infiltration of immune-suppressive myeloid cells correlates with poor prognosis and ICB resistance. These observations suggest a need for a precision medicine approach in which the design of the immunotherapeutic combination is modified on the basis of the tumour immune landscape to overcome such resistance mechanisms. Here we employ a pre-clinical mouse model system and show that resistance to ICB is directly mediated by the suppressive activity of infiltrating myeloid cells in various tumours. Furthermore, selective pharmacologic targeting of the gamma isoform of phosphoinositide 3-kinase (PI3Kγ), highly expressed in myeloid cells, restores sensitivity to ICB. We demonstrate that targeting PI3Kγ with a selective inhibitor, currently being evaluated in a phase 1 clinical trial (NCT02637531), can reshape the tumour immune microenvironment and promote cytotoxic-T-cell-mediated tumour regression without targeting cancer cells directly. Our results introduce opportunities for new combination strategies using a selective small molecule PI3Kγ inhibitor, such as IPI-549, to overcome resistance to ICB in patients with high levels of suppressive myeloid cell infiltration in tumours.

Induction of tumoricidal function in CD4+ T cells is associated with concomitant memory and terminally differentiated phenotype

OX40 engagement induces a cytotoxic CD4+ T cell subpopulation to eradicate advance melanomas

GITR Pathway Activation Abrogates Tumor Immune Suppression through Loss of Regulatory T-cell Lineage Stability

Schaer and colleagues show that GITR ligation by agonist antibody DTA-1 inhibits intratumor immune suppression by inducing tumor-dependent loss of Foxp3 and altered expression of transcription factors and cytokines important for Treg function, resulting in impaired Treg lineage stability and enhanced killing of tumor cells by T effectors. These results will inform the effective use of GITR therapy in humans. Ligation of GITR (glucocorticoid-induced TNF receptor-related gene, or TNFRSF18) by agonist antibody has recently entered into early-phase clinical trials for the treatment of advanced malignancies. Although the ability of GITR modulation to induce tumor regression is well documented in preclinical studies, the underlying mechanisms of action, particularly its effects on CD4+Foxp3+ regulatory T cells (Treg), have not been fully elucidated. We have previously shown that GITR ligation in vivo by agonist antibody DTA-1 causes more than 50% reduction of intratumor Tregs with down modulation of Foxp3 expression. Here, we show that the loss of Foxp3 is tumor dependent. Adoptively transferred Foxp3+ Tregs from tumor-bearing animals lose Foxp3 expression in the host when treated with DTA-1, whereas Tregs from naïve mice maintain Foxp3 expression. GITR ligation also alters the expression of various transcription factors and cytokines important for Treg function. Complete Foxp3 loss in intratumor Tregs correlates with a dramatic decrease in Helios expression and is associated with the upregulation of transcription factors, T-Bet and Eomes. Changes in Helios correspond with a reduction in interleukin (IL)-10 and an increase in IFN-γ expression in DTA-1–treated Tregs. Together, these data show that GITR agonist antibody alters Treg lineage stability inducing an inflammatory effector T-cell phenotype. The resultant loss of lineage stability causes Tregs to lose their intratumor immune-suppressive function, making the tumor susceptible to killing by tumor-specific effector CD8+ T cells. Cancer Immunol Res; 1(5); 320–31. ©2013 AACR.

The importance of animal models in tumor immunity and immunotherapy.

The clinical success and US FDA approval of two immunotherapies (sipuleucel-T and ipilimumab) have brought tumor immunology to the forefront of cancer research. It has been long recognized that the immune system can infiltrate and survey the tumor microenvironment. The field of tumor immunology has been actively examining this phenomenon since the 1890s when William Coley first treated patients with live pathogenic bacteria and observed occasional regressions leading to long term survival. Recent progress in understanding mechanisms of immune activation and tolerance has led to the development of novel therapies that aim to either overcome inhibitory pathways (i.e. checkpoint blockade such as anti-CTLA-4 and anti-PD-1) or stimulate immune cell activation (i.e. co-stimulation such as anti-GITR and anti-OX40). A major part of the success of immunotherapy has been the development of appropriate mouse models. This review will outline the history and the major findings leading to the accomplishments of modern day immunology with specific attention to the usefulness of animal models.

The New Era of Cancer Immunotherapy: Manipulating T-Cell Activity to Overcome Malignancy.

Using the immune system to control cancer has been investigated for over a century. Yet it is only over the last several years that therapeutic agents acting directly on the immune system have demonstrated improved overall survival for cancer patients in phase III clinical trials. Furthermore, it appears that some patients treated with such agents have been cured of metastatic cancer. This has led to increased interest and acceleration in the rate of progress in cancer immunotherapy. Most of the current immunotherapeutic success in cancer treatment is based on the use of immune-modulating antibodies targeting critical checkpoints (CTLA-4 and PD-1/PD-L1). Several other immune-modulating molecules targeting inhibitory or stimulatory pathways are being developed. The combined use of these medicines is the subject of intense investigation and holds important promise. Combination regimens include those that incorporate targeted therapies that act on growth signaling pathways, as well as standard chemotherapy and radiation therapy. In fact, these standard therapies have intrinsic immune-modulating properties that can support antitumor immunity. In the years ahead, adoptive T-cell therapy will also be an important part of treatment for some cancer patients. Other areas which are regaining interest are the use of oncolytic viruses that immunize patients against their own tumors and the use of vaccines against tumor antigens. Immunotherapy has demonstrated unprecedented durability in controlling multiple types of cancer and we expect its use to continue expanding rapidly.

Kinase regulation of Human MHC Class I Molecule Expression on Cancer Cells

Kinome screens revealed EGFR and MEK as key to reduced MHCI expression on many tumors. FDA-approved inhibitors of these kinases increased surface MHC-I, providing a rationale for clinically testing similar kinase inhibitors with immunotherapies dependent on MHC-I. The major histocompatibility complex I (MHC-1) presents antigenic peptides to tumor-specific CD8+ T cells. The regulation of MHC-I by kinases is largely unstudied, even though many patients with cancer are receiving therapeutic kinase inhibitors. Regulators of cell-surface HLA amounts were discovered using a pooled human kinome shRNA interference–based approach. Hits scoring highly were subsequently validated by additional RNAi and pharmacologic inhibitors. MAP2K1 (MEK), EGFR, and RET were validated as negative regulators of MHC-I expression and antigen presentation machinery in multiple cancer types, acting through an ERK output–dependent mechanism; the pathways responsible for increased MHC-I upon kinase inhibition were mapped. Activated MAPK signaling in mouse tumors in vivo suppressed components of MHC-I and the antigen presentation machinery. Pharmacologic inhibition of MAPK signaling also led to improved peptide/MHC target recognition and killing by T cells and TCR-mimic antibodies. Druggable kinases may thus serve as immediately applicable targets for modulating immunotherapy for many diseases. Cancer Immunol Res; 4(11); 936–47. ©2016 AACR.

The New Era of Cancer Immunotherapy

Using the immune system to control cancer has been investigated for over a century. Yet it is only over the last several years that therapeutic agents acting directly on the immune system have demonstrated improved overall survival for cancer patients in phase III clinical trials. Furthermore, it appears that some patients treated with such agents have been cured of metastatic cancer. This has led to increased interest and acceleration in the rate of progress in cancer immunotherapy. Most of the current immunotherapeutic success in cancer treatment is based on the use of immune-modulating antibodies targeting critical checkpoints (CTLA-4 and PD-1/PD-L1). Several other immune-modulating molecules targeting inhibitory or stimulatory pathways are being developed. The combined use of these medicines is the subject of intense investigation and holds important promise. Combination regimens include those that incorporate targeted therapies that act on growth signaling pathways, as well as standard chemotherapy and radiation therapy. In fact, these standard therapies have intrinsic immune-modulating properties that can support antitumor immunity. In the years ahead, adoptive T-cell therapy will also be an important part of treatment for some cancer patients. Other areas which are regaining interest are the use of oncolytic viruses that immunize patients against their own tumors and the use of vaccines against tumor antigens. Immunotherapy has demonstrated unprecedented durability in controlling multiple types of cancer and we expect its use to continue expanding rapidly.

Blockade of surface-bound TGF-β on regulatory T cells abrogates suppression of effector T cell function in the tumor microenvironment

Targeting a cytokine on the surface of regulatory T cells inhibits their suppression of tumor cell killing by effector T cells. Blocking immunosuppression The antitumor effects of CD8+ T cells can be blocked in the tumor microenvironment, including through the suppressive function of regulatory T cells (Tregs). Standard in vitro systems fail to recapitulate the conditions that immune cells are exposed to in vivo. Budhu et al. used a three-dimensional, collagen-fibrin gel system to investigate the effects of CD8+ T cells on cocultured melanoma cells excised from mouse tumors. The antitumor activity of the CD8+ T cells was inhibited by the presence of tumor-derived Tregs, which depended on cell-cell contact or close proximity, required the cytokine TGF-β on the Treg cell surface, and resulted in the increased cell surface expression of the immune checkpoint receptor PD-1 on the CD8+ T cells. A blocking antibody against TGF-β prevented immunosuppression, suggesting a therapeutic strategy to inhibit Treg activity in tumors. Regulatory T cells (Tregs) suppress antitumor immunity by inhibiting the killing of tumor cells by antigen-specific CD8+ T cells. To better understand the mechanisms involved, we used ex vivo three-dimensional collagen-fibrin gel cultures of dissociated B16 melanoma tumors. This system recapitulated the in vivo suppression of antimelanoma immunity, rendering the dissociated tumor cells resistant to killing by cocultured activated, antigen-specific T cells. Immunosuppression was not observed when tumors excised from Treg-depleted mice were cultured in this system. Experiments with neutralizing antibodies showed that blocking transforming growth factor–β (TGF-β) also prevented immunosuppression. Immunosuppression depended on cell-cell contact or cellular proximity because soluble factors from the collagen-fibrin gel cultures did not inhibit tumor cell killing by T cells. Moreover, intravital, two-photon microscopy showed that tumor-specific Pmel-1 effector T cells physically interacted with tumor-resident Tregs in mice. Tregs isolated from B16 tumors alone were sufficient to suppress CD8+ T cell–mediated killing, which depended on surface-bound TGF-β on the Tregs. Immunosuppression of CD8+ T cells correlated with a decrease in the abundance of the cytolytic protein granzyme B and an increase in the cell surface amount of the immune checkpoint receptor programmed cell death protein 1 (PD-1). These findings suggest that contact between Tregs and antitumor T cells in the tumor microenvironment inhibits antimelanoma immunity in a TGF-β–dependent manner and highlight potential ways to inhibit intratumoral Tregs therapeutically.

Clonal Abundance of Tumor-Specific CD4(+) T Cells Potentiates Efficacy and Alters Susceptibility to Exhaustion.

Current approaches to cancer immunotherapy aim to engage the natural T cell response against tumors. One limitation is the elimination of self-antigen-specific T cells from the immune repertoire. Using a system in which precursor frequency can be manipulated in a murine melanoma model, we demonstrated that the clonal abundance of CD4(+) T cells specific for self-tumor antigen positively correlated with antitumor efficacy. At elevated precursor frequencies, intraclonal competition impaired initial activation and overall expansion of the tumor-specific CD4(+) T cell population. However, through clonally derived help, this population acquired a polyfunctional effector phenotype and antitumor immunity was enhanced. Conversely, development of effector function was attenuated at low precursor frequencies due to irreversible T cell exhaustion. Our findings assert that the differential effects of T cell clonal abundance on phenotypic outcome should be considered during the design of adoptive T cell therapies, including use of engineered T cells.