Study On The Mechanism Of Interferon-inducible Gene BACH1 Deletion Promoting The Antitumor Immune Response Of Oncolytic Virus HSV-1

Cancer is a major public health problem that threatens the health of the population.In recent years,the incidence rate and mortality rate of cancer have increased year by year.Cancer has become the leading cause of death from the disease.With the development of cancer immunotherapy,the survival period of patients has been prolonged and the quality of life after the treatment has also been significantly improved.However,cancer immunotherapy has no significant effect on patients with low lymphocyte infiltration in the tumor microenvironment.Due to the unique anti-tumor properties,oncolytic viruses change the tumor microenvironment and promote lymphocyte infiltration into the tumor microenvironment.In 2015,FDA approved the first oncolytic virus talimogene laherparepvec(T-VEC)for the treatment of melanoma.However,its durable response rate was 16%,which needed to be improved.T-VEC was modified based on HSV-1 which was sensitive to interferon.In order to further improve the therapeutic effect of T-VEC,we screened the interferon-stimulated genes(ISGs)against HSV-1 and clarified its mechanism.At the same time,we further studied the effect of the ISG on the anti-tumor immune response induced by HSV-1.Firstly,we overexpressed 288 ISGs in HEK293T cells,and then infected with HSV-1-Luc.By detecting the expression of luciferase,we found 8 ISGs that significantly inhibited the replication of HSV-1.And among them,we recognized BACH1 for the first time as a candidate for antiviral function.In clinical trials,the drug Panhemin has been proved to reduce the expression of BACH1;therefore we selected BACH1 for further research.Next,we decreased the expression level of BACH1 in HEK293T,MEF,MCA205 and B16 cells by shRNA or CRISPER-Cas9,and then infected HSV-1-luc.By detecting the genome copy number of the HSV-1,we found that HSV-1 replication increased.In addition,it was reported that Hemin promotes the ubiquitination and proteasome degradation of BACH1,thus reducing the protein level of BACH1 in cells.When we treated HEK293T cells with Hemin and found that the replication of HSV-1 increased.As a transcriptional regulator,BACHI inhibited the transcription of ICP4、ICP27 and UL39 by binding to their promoters which was independent on MAFK.In the mouse xenograft model,we found that reducing the expression of BACH1 increased the replication of HSV-1.Moreover,intratumoral injection of HSV-1 in BACH1 knockout tumors inhibited tumor growth,and significantly increased the infiltration of CD45+CD8+T lymphocytes into tumor microenvironment.Loss function of BACH1 promoted the anti-tumor immune response of HSV-1 due to the deficiency of BACH1 promoted HSV-1 tumor expansion and increased the turnor immunogenicity.When we infected HSV-1 in BACH1 knocked out cells,we found that deficiency of BACH1 promoted HSV-1 induced cell apoptosis,HMGB1 secretion and calreticulin exposure.We further treated mice with Hemin in the mouse tumor model,and found that Hemin treatment increased the replication of HSV-1.Then we combined Hemin with HSV-1 on tumor treatment and found that tumor growth was significantly slowed down,and the infiltration of CD45+CD8+T lymphocytes was also significantly increased.In summary,our research systematically screened ISGs against HSV-1 and clarified the mechanism of BACH1 against HSV-1.Knocking out BACH1 increased HSV-1 induced tumor immunogenicity and anti-tumor immune response.More importantly,the combined use of Hemin and HSV-1 on tumor treatment promoted T lymphocyte infiltration and inhibited tumor growth.This study demonstrates that Hemin improved the treatment effect of HSV-1 by down-regulating BACH1 and therefore provides a new strategy for improving the clinical efficiency of oncolytic virus T-VEC.