Abstract
Balancing safety and efficacy is a major consideration for cancer treatments, especially when combining cancer immunotherapy with other treatment modalities such as chemotherapy. Approaches ...that induce immunogenic cell death (ICD) are expected to eliminate cancer cells by direct cell killing as well as activation of an antitumor immune response. We have developed a gene therapy approach based on p19Arf and interferon-β gene transfer that, similar to conventional inducers of ICD, results in the release of DAMPS and immune activation. Here, aiming to potentiate this response, we explore whether association between our approach and treatment with doxorubicin (Dox), a known inducer of ICD, could further potentiate treatment efficacy without inducing cardiotoxicity, a critical side effect of Dox. Using central composite rotational design analysis, we show that cooperation between gene transfer and chemotherapy killed MCA205 and B16F10 cells and permitted the application of reduced viral and drug doses. The treatments also cooperated to induce elevated levels of ICD markers in MCA205, which correlated with improved efficacy of immunotherapy in vivo. Treatment of subcutaneous MCA205 tumors associating gene transfer and low dose (10 mg/kg) chemotherapy resulted in inhibition of tumor progression. Moreover, the reduced dose did not cause cardiotoxicity as compared to the therapeutic dose of Dox (20 mg/kg). The association of p19Arf/interferon-β gene transfer and Dox chemotherapy potentiated antitumor response and minimized cardiotoxicity.
Immunogenic cell death induced by anticancer chemotherapy is characterized by a series of molecular hallmarks that include the exodus of high-mobility group box 1 protein (HMGB1) from dying cells. ...HMGB1 is a nuclear nonhistone chromatin-binding protein. It is secreted at the late stages of cellular demise and engages Toll-like receptor4 (TLR4) on dendritic cells (DCs) to accelerate the processing of phagocytic cargo in the DC and to facilitate antigen presentation by DC to T cells. The absence of HMGB1 expression by dying tumor cells exposed to anthracyclines or oxaliplatin compromises DC-dependent T-cell priming by tumor-associated antigens. Here, we show that transplantable tumors exhibiting weak expression of nuclear HMGB1 respond to chemotherapy more effectively if the treatment is combined with the local or systemic administration of a highly purified and physiochemically defined and standardized lipopolysaccharide solution, which acts as a high-potency and exclusive TLR4 agonist, called Dendrophilin (DEN). The synergistic antitumor effects mediated by the combination of chemotherapy and immunotherapy relied upon the presence of the MyD88 (myeloid differentiation primary response gene) adapter of TLR4 (but not that of the TIR-domain-containing adapter-inducing interferon-β adapter), in line with the well-characterized action of DEN on the MyD88 signaling pathway. DEN and anthracyclines synergized to induce intratumoral accumulation of interferon-γ-producing CD4(+) and CD8(+) T lymphocytes. Moreover, DEN could restore the immunogenicity of dying tumor cells from which HMGB1 had been depleted by RNA interference. These findings underscore the potential clinical utility of combination regimens involving immunogenic chemotherapy and certain TLR4 agonists in advanced HMGB1-deficient cancers.
Simultaneous reestablishment of p53/p19
Arf
and interferon-β pathways in melanoma cells culminates in a cell death process that displays features of necroptosis along with the release of immunogenic ...cell death molecules and unleashes an antitumor immune response mediated by natural killer cells, neutrophils as well as CD4
+
and CD8
+
T lymphocytes.
The success of immunotherapies brings hope for the future of cancer treatment. Even so, we are faced with a new challenge, that of understanding which patients will respond initially and, possibly, ...develop resistance. The examination of the immune profile, especially approaches related to the immunoscore, may foretell which tumors will have a positive initial response. Ideally, the mutation load would also be analyzed, helping to reveal tumor associated antigens that are predictive of an effective cytolytic attack. However, the response may be hindered by changes induced in the tumor and its microenvironment during treatment, perhaps stemming from the therapy itself. To monitor such alterations, we suggest that minimally invasive approaches should be explored, such as the analysis of circulating tumor DNA. When testing new drugs, the data collected from each patient would initially represent an N of 1 clinical trial that could then be deposited in large databases and mined retrospectively for trends and correlations between genetic alterations and response to therapy. We expect that the investment in personalized approaches that couple molecular analysis during clinical trials will yield critical data that, in the future, may be used to predict the outcome of novel immunotherapies.
Abstract
Association of immunotherapies with chemotherapy can significantly improve cancer treatment, either by enhancing cell death or by superior immune stimulation. However, as reported in some ...studies, combinatorial strategies are hampered by a strong increase of severe treatment-related toxicity. Previously, we have developed specialized adenoviral vectors for the delivery of p19Arf (tumor suppressor protein) and Interferon-beta (IFN-beta immunomodulatory cytokine) to p53wt tumor cells, a unique combination capable of promoting immunogenic cell death and immune activation. Here, we explore potential benefits of the association of p19/IFN-beta immunotherapy with doxorubicin chemotherapy, also an inducer of immunogenic cell death. First, we observed that single-agent treatment of s.c. MCA205 sarcomas was successful only in immunocompetent, but not immunodeficient, mice, indicating that each treatment depends, at least in some part, on the immune system. Next, using factorial experimental design and propidium iodide staining, we revealed that the combined therapies augment cell death at lower doses of virus and doxorubicin than those needed for the individual treatments. This observation was confirmed by MTT assay, caspase 3 activity, calreticulin exposure, and also in the B16F10 cell line. In order to investigate the in vivo effects of p19Arf/IFN-beta and doxorubicin association, tumor-bearing mice were treated with the single or combined agents. Molecular imaging indicated a prolonged caspase 3 activation in situ only in the p19Arf/IFN-beta+dox group, even though doxorubicin alone (20 mg/kg) achieved the greatest extension of survival, with no evident benefit from its association with p19/IFN-beta immunotherapy. Furthermore, we asked if by using the combined treatment we could reduce the dose of doxorubicin and still maintain tumor control, an application that would be truly interesting since doxorubicin has been shown to severely affect heart function. Indeed, the subtherapeutic dose of doxorubicin (10 mg/kg) extended survival only in association with p19/IFN-beta and, most importantly, echocardiogram analysis revealed the elimination of the cardiotoxicity seen with the 20-mg/kg dose. The results obtained so far indicate that pretreatment with p19Arf and IFN-beta immunotherapy restores therapeutic efficacy of subtherapeutic dose of doxorubicin, preserving cardiac function. More studies are on the way to reveal the immunologic impact of this association.
Citation Format: Ruan F. V. Medrano, Sr., Samir A. Mendonca, Sr., Aline H. Ribeiro, Sr., João PP Catani, Sr., Valker A. Feitosa, Sr., Elaine G. Rodrigues, Sr., Bryan E. Strauss, Sr.. Potentiation of doxorubicin low-dose efficacy through its association with p19Arf/Interferon-beta immunotherapy: Combining two immunogenic cell death inducers for the treatment of cancer abstract. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; São Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr B39.
The small heat shock proteins (smHSPs) belong to a family of proteins that function as molecular chaperones by preventing protein aggregation and are also known to contain a conserved region termed ...α-crystallin domain. Here, we report the expression, purification, and partial characterization of a novel smHSP (HSP17.9) from the phytopathogen
Xylella fastidiosa, causal agent of the citrus variegated chlorosis (CVC). The gene was cloned into a pET32-Xa/LIC vector to over-express the protein coupled with fusion tags in
Escherichia coli BL21(DE3). The expressed HSP17.9 was purified by immobilized metal affinity chromatography (IMAC) and had its identity determined by mass spectrometry (MALDI-TOF). The correct folding of the purified recombinant protein was verified by circular dichroism spectroscopy. Finally, the HSP17.9 protein also proved to efficiently prevent induced aggregation of insulin, strongly indicating a chaperone-like activity.
Introdução: As síndromes hemorrágicas no intra e pós-operatório de operações com circulação extracorpórea (CEC) constituem motivo de preocupação e, parte delas, pode ser atribuída à heparina não ...fracionada (HNF), droga indispensável e, até hoje, insubstituível nesse tipo de procedimento. Alguns autores consideram a ação anticoagulante da HNF como o principal responsável pelo sangramento e investem em drogas antifibrinolíticas ou que alteram a atividade plaquetária para tentar substituí-la. Toda HNF contém frações de heparina de baixo peso molecular (HBPM), não neutralizáveis pela protamina, que, em doses elevadas, e/ou em pacientes heparino-sensíveis, podem causar vasoplegia e aumento no sangramento pós-operatório em operações com CEC. Material e Métodos: Isolamos uma heparina de alto peso molecular (HAPM - peso modal de 25.000 Daltons), com 11% de frações de HBPM (< 7.000 Daltons), para experiências "in vitro" e "in vivo", e comparamos com HNF (peso modal de 15.000 Daltons), com 21% de frações de HBPM. Resultados: A atividade específica anticoagulante, por massa, foi superior quando comparada com a HNF tanto "in vitro", 273 ui/mg contra 181 ui/mg e TTPA mais elevado nas várias diluições, como "in vivo", em cães, durante CEC, comprovado pelo TCA, TTPA e heparinemia. A vida média da HNF foi de 60 minutos e acima de 90 minutos para a HAPM, na situação de experimentação. Conclusão: Acreditamos que esta experiência, inédita na literatura indexada, nos habilite ao uso da HAPM, em seres humanos, para averiguação da sua melhor neutralização pela protamina e menor incidência de hemorragia.
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