Combinatorial targeting of angiogenesis with inducers of autophagy in the treatment of glioblastoma multiforme

Malignant gliomas represent 80% of tumors developing in the central nervous system, with 50% being glioblastomas (GBM), the most aggressive form of the disease. The benefit from current therapies is very limited, the overall 5-year survival rate of patients with GBM being less than 5%. Thus, there is an urgent need for better therapies. It has been reported that the use of tricyclic antidepressants (TCAs) is linked with reduced incidence of gliomas. In line with these observations, our lab previously demonstrated that combining imipramine (IM), a TCA that activates adenylate cyclase and elevates production of cAMP, with ticlopidine (TIC), an inhibitor of the purinergic receptor P2Y12 that otherwise suppresses cAMP, results in a concerted increase in the levels of intracellular cAMP, which consequently stimulates increased autophagy in glioma cells, leading to autophagy-associated cell death (AACD) in culture and during orthotopic tumor growth. In several mouse models of glioma, the combination improved survival and reduced malignant grade. Seeking to assess the therapeutic translational potential of these repurposed drugs, particularly IM, we performed pre-clinical trials combining IM+/-TIC with anti-VEGF therapy, which is commonly used in patients with GBM; such anti-angiogenic therapy is associated with a transitory improvement in progression-free survival and quality of life, but does not translate into an overall survival benefit. Using genetic mouse models with enabling bioluminescent monitoring of the cancer cells, we treated established GBMs with imipramine, ticlopidine, and B20S, an anti-mouse VEGF monoclonal antibody. We observed transient stabilization of tumor growth, and the overall survival was significantly extended in the regimens combining B20S with IM +/- TIC compared to control and single treatments. Similar to clinical observations in GBM patients treated with anti-angiogenic therapy, mice treated with B20S alone had no survival benefit, implicating a synergetic effect for the combination of anti-VEGF treatment with imipramine and ticlopidine. We found that the combinatorial therapy elicited further increases in autophagy and displayed some indication of blood vessel normalization, concomitant with an elevation of CD8+T lymphocyte infiltration into the orthotopic tumors. Notably, we could partially reverse the beneficial effect of the combinations using a CD8+T cell-depleting antibody, revealing the involvement of the recruited CD8+T cells in the efficacy of the combinatorial treatment, in addition to the cell-intrinsic autophagy-associated death that is demonstrable in culture and in tumors. Together, these results suggest a strategy to improve anti-angiogenic therapy in patients with glioblastoma, by using repurposed FDA-approved drugs that markedly elevate autophagy in concert with anti-VEGF therapy. The recruitment of CD8+ T cells into the treated tumors and their evident contribution to therapeutic efficacy motivates ongoing experiments aimed to heighten and sustain the induced anti-tumor immune response.

Anti-angiogenic immunotherapy for lung cancer

Ece Kadioglu

Lung cancer is the leading cause of cancer-associated deaths worldwide. Platinum-based chemotherapy is the most common therapeutic approach in advanced non-small cell lung cancer (NSCLC), particularly for tumors that carry non-druggable activating mutations. However, these tumors are generally not eradicated when diagnosed at an advanced stage and are frequently resistant to chemotherapy. Therefore, there is an urgent medical need for new treatments for this frequent cancer type. Immunotherapies are treatments based on increasing the strength of immune responses against cancer. In particular, monoclonal antibodies that block inhibitory "immune-checkpoints" expressed on T cells (e.g., programmed cell death protein 1, or PD1) show clinical efficacy in increasing tumor types. However, these therapies improve survival in only a minority of NSCLC patients, and ongoing efforts are being made for increasing their efficacy through combinatorial treatments. In this regard, growing evidence indicates that tumor angiogenesis is closely associated with immunosuppression. Pro-angiogenic factors, such as vascular endothelial growth factor A (VEGFA) and angiopoietin 2 (ANG2), cause abnormalities and dysfunctions in tumor blood vessels that impair T cell extravasation and anti-tumoral activity. Recent findings in preclinical cancer models show that reprogramming the features of tumor-associated blood vessels by anti-angiogenic drugs may help to increase intratumoral T cell abundance and sensitize refractory tumors to immunotherapy. Nevertheless, the potential of anti-angiogenics in the context of lung cancer immunotherapy is poorly understood. Therefore, the main objective of my project was to audit combinations of anti-angiogenic therapy and immune checkpoint blockade in a genetically engineered mouse model of NSCLC. I first studied the anti-tumoral activity of dual VEGFA and ANG2 inhibition using a bispecific antibody (A2V) in the KrasLSL-G12D/+;p53fl/fl (KP) NSCLC model. I found that A2V, but not single VEGFA or ANG2 blockade, had robust tumor-inhibitory activity in KP mice, which was at least comparable to standard-of-care chemotherapy. However, A2V only moderately increased cytotoxic T cells in the tumors. To potentially increase the immunogenicity of KP tumors, I also generated two KP mouse variants by either co-expressing a surrogate neoantigen or genetically deleting a DNA mismatch repair enzyme in the cancer cells. However, the therapeutic efficacy of A2V was not further improved in spite of a slightly more activated immune response. I next investigated combination therapies involving A2V and PD1 blockade. In contrast to previous findings in other cancer types, the addition of PD1 antibodies deteriorated the efficacy of A2V, likely through enhancing immunosuppressive mechanisms sustained, at least in part, by regulatory T cells and T cell-antagonizing cytokines. In summary, my work supports the potential clinical efficacy of combined VEGFA and ANG2 blockade for lung cancer therapy but provided little evidence for its synergy in association with anti-PD1 therapy.

EPFL2019

Targeting and Modulating Tumor-Associated Sites with Novel Nanoparticle-Based Immunotherapies

Laura Jeanbart

Cancer is a global disease and a leading cause of death worldwide. While surgery, radiotherapy and chemotherapy comprise the classical tools to eradicate tumors, they do not cure cancer, cannot prevent metastases, lead to side effects, and most importantly they do not instruct the patientʼs immune system to recognize and fight tumor cells. Since the discovery of the immune systemʼs implication in cancer, immunotherapies, which aim at using and educating the immune system to fight and reject cancer, have come to complement classical tumor-debulking methods. A major goal of immunotherapy is to induce or activate effector cytotoxic CD8+ T lymphocytes that can infiltrate and kill the tumor while overcoming immune suppressive mechanisms, which impede their efficacy. Moreover, cancer immunotherapies aim at inducing memory to tumor antigens to prevent relapse or metastases, which traditional therapies cannot do. Dendritic cell (DC) targeting and activation are key for inducing adaptive immunity. Our laboratory has developed synthetic nanoparticles (NPs) capable of draining through lymphatics to target skin-draining lymph nodes (LNs) and being phagocytosed by resident antigen-presenting cells (APCs) upon intradermal injection. We developed a reproducible method to conjugate tumor antigens to NPs and co-delivered them with CpG-conjugated NPs. We found that a NP vaccine composed of a single melanoma-derived peptide (TRP- 2180-188) led to dramatic tumor growth delay in melanoma and was more efficient than a NP vaccine containing several tumor antigens from whole tumor cell lysate (TL). These findings contradicted our hypothesis that immunization with TL might lead to a broader T cell response and hence improved therapeutic outcomes. Additionally, we found that the dose of CpG could modulate the magnitude of the therapeutic outcomes in both single epitope and multiple antigen based NP-vaccines. Considering the efficacy of the developed NP vaccines, we used them as a tool to ask a fundamental and extremely relevant question regarding the influence of vaccine administration site on clinical efficacy: is it therapeutically more beneficial to target a tumor- draining lymph node (tdLN), which is immune suppressed but tumor antigen-primed, or a non-tdLN, which is neither suppressed nor primed by the tumor? We found that targeting the tdLN with NP vaccines led to significantly enhanced therapeutic outcomes in two different tumor models over targeting a non-tdLN, and that efficacy relied on NP conjugation of antigen and adjuvant. This suggested that antigen drainage from the tumor might be available to APCs in the tdLN and we further hypothesized that therapies that lead to immunogenic tumor cell death might synergize with a tdLN-targeted adjuvant approach. While cytotoxic modalities, such as chemotherapy and radiotherapy, have traditionally been used for their ability to kill tumor cells, tumor cells shed antigens and debris upon cell death that drain to the tdLN. Consistent with our hypothesis, targeting an adjuvant to the tdLN enhanced therapeutic benefits of chemo- and radio- therapy, while targeting a non-tdLN did not. We hypothesized that targeting the tdLN with NP vaccines might switch the tdLN microenvironment from a suppressive to an immunogenic one. This suggested that direct targeting of active suppressive mechanisms in the tumor might further improve immunotherapy. We thus engineered a nano-sized micellar carrier loaded with 6-thioguanine (MC-6TG), a cytotoxic drug used in leukemia, and explored its use in targeting T cell- impairing myeloid-derived suppressor cells (MDSCs) in order to enhance the efficacy of therapeutic vaccines. MC-6TG entirely eradicated both Ly6c+ monocytic and Ly6g+ granulocytic MDSCs for up to 7 d in the blood, tumor and tdLN of tumor-bearing mice. Since MC-6TG also targeted certain subsets of macrophages and DCs, depleting MDSCs did not have an impact on the efficacy of a NP vaccine, which relies on APCs for efficacy. However, MC-6TG synergized with adoptively transferred CD8+ T cells, significantly delaying tumor growth and enhancing survival in a murine model of implantable melanoma. Overall, this thesis describes the design and implementation of a novel approach to immunotherapy: we engineered NP-based therapeutic vaccines, optimized their delivery route to enhance efficacy, and simultaneously tackled immune suppressive cells in the tumor microenvironment to allow optimal therapeutic outcomes. The technologies and methods developed in this work are particularly relevant to clinical oncology and have the potential to benefit cancer patients by improving cancer therapy efficacy.

EPFL2013

Dual Targeting of the Autophagic Regulatory Circuitry in Gliomas with Repurposed Drugs Elicits Cell-Lethal Autophagy and Therapeutic Benefit

Douglas Hanahan, Aristea Massaras, Ksenya Shchors

The associations of tricyclic antidepressants (TCAs) with reduced incidence of gliomas and elevated autophagy in glioma cells motivated investigation in mouse models of gliomagenesis. First, we established that imipramine, a TCA, increased autophagy and conveyed modest therapeutic benefit in tumor-bearing animals. Then we screened clinically approved agents suggested to affect autophagy for their ability to enhance imipramine-induced autophagy-associated cell death. The anticoagulant ticlopidine, which inhibits the purinergic receptor P2Y(12), potentiated imipramine, elevating cAMP, a modulator of autophagy, reducing cell viability in culture, and increasing survival in glioma-bearing mice. Efficacy of the combination was obviated by knockdown of the autophagic regulatory gene ATG7, implicating cell-lethal autophagy. This seemingly innocuous combination of TCAs and P2Y(12) inhibitors may have applicability for treating glioma.