MiRNAs as Anti-Angiogenic Adjuvant Therapy in Cancer: Synopsis and Potential

Angiogenesis is a key mechanism for tumor growth and metastasis and has been a therapeutic target for anti-cancer treatments. Intensive vascular growth is concomitant with the rapidly proliferating tumor cell population and tumor outgrowth. Current angiogenesis inhibitors targeting either one or a few pro-angiogenic factors or a range of downstream signaling molecules provide clinical benefit, but not without significant side effects. miRNAs are important post-transcriptional regulators of gene expression, and their dysregulation has been associated with tumor progression, metastasis, resistance, and the promotion of tumor-induced angiogenesis. In this mini-review, we provide a brief overview of the current anti-angiogenic approaches, their molecular targets, and side effects, as well as discuss existing literature on the role of miRNAs in angiogenesis. As we highlight specific miRNAs, based on their activity on endothelial or cancer cells, we discuss their potential for anti-angiogenic targeting in cancer as adjuvant therapy and the importance of angiogenesis being evaluated in such combinatorial approaches.

Signaling of MK2 sustains robust AP1 activity for triple negative breast cancer tumorigenesis through direct phosphorylation of JAB1

Triple negative breast cancer (TNBC) cells are generally more invasive than estrogen receptor-positive (ER + ) breast cancer cells. Consistent with the importance of activator protein 1 (AP1) transcription factors in invasion, AP1 activity is much higher in TNBC lines than ER + lines. In TNBC cells, robust AP1 activity is facilitated by both ERK and p38MAPK signaling pathways. While ERK signaling pathway regulates AP1 activity by controlling the abundance of AP1 transcription factors, p38MAPK signaling pathway does it by enhancing AP1 binding to AP1 sites without altering their abundance. Here, we show that p38MAPK regulation of AP1 activity involves both MAPKAPK2 (MK2) and JAB1, a known JUN-binding protein. MK2 not only interacts with JAB1 but also directly phosphorylates JAB1 at Ser177 in TNBC cells. Interestingly, Ser177 phosphorylation does not affect JAB1 and JUN interaction. Instead, interfering with p38MAPK signaling pathway or introducing an S to A point mutation at Ser177 of JAB1 reduces JUN recruitment to the AP1 sites in cyclin D1, urokinase plasminogen activator (uPA) and uPA receptor promoters. Moreover, knockdown of JAB1 diminishes >60% of AP1 transcriptional activity in TNBC cells. Taken together, these results indicate that MK2-mediated phosphorylation of JAB1 facilitates JUN recruitment to AP1 sites, thus augmenting AP1 activity. In line with the role of JAB1 in AP1 activity, silencing JAB1 leads to dramatic reduction in TNBC cell growth, in vitro invasion and in vivo tumor outgrowth. This study suggests that the p38MAPK-MK2 signaling pathway promotes TNBC tumorigenesis by sustaining robust AP1 activity.

Afucosylated IgG Targets FcγRIV for Enhanced Tumor Therapy in Mice

Promising strategies for maximizing IgG effector functions rely on the introduction of natural and non-immunogenic modifications. The Fc domain of IgG antibodies contains an N-linked oligosaccharide at position 297. Human IgG antibodies lacking the core fucose in this glycan have enhanced binding to human (FcγR) IIIa/b, resulting in enhanced antibody dependent cell cytotoxicity and phagocytosis through these receptors. However, it is not yet clear if glycan-enhancing modifications of human IgG translate into more effective treatment in mouse models. We generated humanized hIgG1-TA99 antibodies with and without core-fucose. C57Bl/6 mice that were injected intraperitoneally with B16F10-gp75 mouse melanoma developed significantly less metastasis outgrowth after treatment with afucosylated hIgG1-TA99 compared to mice treated with wildtype hhIgG1-TA99. Afucosylated human IgG1 showed stronger interaction with the murine FcγRIV, the mouse orthologue of human FcγRIIIa, indicating that this glycan change is functionally conserved between the species. In agreement with this, no significant differences were observed in tumor outgrowth in FcγRIV-/- mice treated with human hIgG1-TA99 with or without the core fucose. These results confirm the potential of using afucosylated therapeutic IgG to increase their efficacy. Moreover, we show that afucosylated human IgG1 antibodies act across species, supporting that mouse models can be suitable to test afucosylated antibodies.

Combinatorial immunotherapies overcome MYC-driven immune evasion

For many human cancers, including triple negative breast cancer (TNBC), a modest number of patients benefit from immune checkpoint inhibitors, and few experience cancer remission. Expression of programed death-ligand 1 (PD-L1), tumor immune infiltration, or tumor mutation burden have been widely investigated for predicting cancer immunotherapy response. Whether specific oncogenes diminish response to immunotherapy and whether these effects are reversible remains poorly understood. We predicted that MYC, an oncogene that is frequently overexpressed and is associated with worse prognosis, may predict immunotherapy response in patients with TNBC. Here, we report that MYC-elevated TNBCs are resistant to immune checkpoint inhibitors. Using mouse models of TNBC and patient data we report that MYC signaling is associated with low tumor cell PD-L1, low overall immune cell infiltration, and low tumor cell MHC-I expression. Restoring interferon signaling in the tumor reduces MYC expression and increases MHC-I expression. By combining a TLR9 agonist and an agonistic antibody against OX40 with anti-PD-L1, most mice experience complete tumor regression and are protected from new TNBC tumor outgrowth. Our findings demonstrate that MYC-dependent immune evasion is reversible and druggable, and if strategically targeted, may improve outcomes for patients treated with immune checkpoint inhibitors.

Local IFNα enhances the anti-tumoral efficacy of systemic anti-PD1 to prevent tumor relapse

BackgroundTumor relapse constitutes a major challenge for anti-tumoral treatments, including immunotherapies. Indeed, most cancer-related deaths occur during the tumor relapse phase.MethodsWe designed a mouse model of tumor relapse in which mice transplanted with E7+ TC1 tumor cells received a single therapeutic vaccination of STxB-E7+IFNα. Unlike the complete regression observed after two vaccinations, such a treatment induced a transient shrinkage of the tumor mass, followed by a rapid tumor outgrowth. To prevent this relapse, we tested the efficacy of a local administration of IFNα together with a systemic therapy with anti-PD1 Ab. The immune response was analyzed during both the tumor regression and relapse phases.ResultsWe show that, during the regression phase, tumors of mice treated with a single vaccination of STxB-E7 + IFNα harbor fewer activated CD8 T cells and monocytes than tumors doomed to fully regress after two vaccinations. In contrast, the systemic injection of an anti-PD1 Ab combined with the peri-tumoral injection of IFNα in this time frame promotes infiltration of activated CD8 T cells and myeloid cells, which, together, exert a high cytotoxicity in vitro against TC1 cells. Moreover, the IFNα and anti-PD1 Ab combination was found to be more efficient than IFNα or anti-PD1 used alone in preventing tumor relapse and was better able to prolong mice survival.ConclusionsTogether, these results indicate that the local increase of IFNα in combination with an anti-PD1 therapy is an effective way to promote efficient and durable innate and adaptive immune responses preventing tumor relapse.

IMMU-32. NON-METABOLIC IDO ACTIVITY INCREASES COMPLEMENT FACTOR H LEVELS WHICH ENHANCES IMMUNOSUPPRESSION IN HUMAN GLIOBLASTOMA

Indoleamine 2,3 dioxygenase 1 (IDO) is an immunosuppressive factor expressed in ≥90% of patient resected glioblastoma (GBM). Canonically, IDO suppresses the immune response by metabolizing the essential amino acid, tryptophan, into the downstream metabolite, kynurenine. Based on the in vivo finding that the genetic knockout of tumor cell IDO does not change brain tumor tryptophan and kynurenine levels in syngeneic mice, we recently questioned the mechanistic role of IDO in GBM. To determine whether tumor cell tryptophan metabolism is responsible for the pathogenic effects of IDO, we created IDO-deficient murine GBM cells that were reconstituted with either (i) empty expression vector, (ii) a vector expressing wild-type IDO, (iii) or a vector expressing enzyme-inactive IDO. GBM cell IDO expression increased Tregs and decreased survival, but did so independent of tryptophan metabolism. To understand how IDO causes these effects, we performed Clariom D microarray analysis of human GBM cells and discovered that complement factor H (CFH) is significantly increased in GBM cells expressing IDO but decreased by treatment with IDO siRNA. Notably, CFH siRNA decreased CFH- but not IDO-expression in GBM cells. We next found that GBM cell IDO regulates CFH levels independent of tryptophan metabolism. To determine the effects of CFH on tumor outgrowth, we intracranially-engrafted syngeneic mice with IDO-deficient murine GBM cells reconstituted with either empty expression vector or a vector expressing CFH splice variant 2. Tumor CFH significantly decreased survival from GBM bearing mice similar to that of patient-resected GBM with high CFH levels whereby patients present with a faster rate to tumor recurrence. In summary, our study is the first to report an IDO-dependent non-metabolic regulation of CFH in tumor cells and highlights a new immunotherapeutic target in patients with incurable GBM.

Combination of thermally ablative focused ultrasound with gemcitabine controls breast cancer via adaptive immunity

BackgroundTriple-negative breast cancer (TNBC) remains recalcitrant to most targeted therapy approaches. However, recent clinical studies suggest that inducing tumor damage can render TNBC responsive to immunotherapy. We therefore tested a strategy for immune sensitization of murine TNBC (4T1 tumors) through combination of focused ultrasound (FUS) thermal ablation and a chemotherapy, gemcitabine (GEM), known to attenuate myeloid-derived suppressor cells (MDSCs).MethodsWe applied a sparse-scan thermally ablative FUS regimen at the tumor site in combination with systemically administered GEM. We used flow cytometry analysis to investigate the roles of monotherapy and combinatorial therapy in mediating local and systemic immunity. We also tested this combination in Rag1−/− mice or T cell-depleted wild-type mice to determine the essentiality of adaptive immunity. Further, we layered Programmed cell death protein 1 (PD-1) blockade onto this combination to evaluate its impact on tumor outgrowth and survival.ResultsThe immune-modulatory effect of FUS monotherapy was insufficient to promote a robust T cell response against 4T1, consistent with the dominant MDSC-driven immunosuppression evident in this model. The combination of FUS+GEM significantly constrained primary TNBC tumor outgrowth and extended overall survival of mice. Tumor control correlated with increased circulating antigen-experienced T cells and was entirely dependent on T cell-mediated immunity. The ability of FUS+GEM to control primary tumor outgrowth was moderately enhanced by either neoadjuvant or adjuvant treatment with anti-PD-1.ConclusionThermally ablative FUS in combination with GEM restricts primary tumor outgrowth, improves survival and enhances immunogenicity in a murine metastatic TNBC model. This treatment strategy promises a novel option for potentiating the role of FUS in immunotherapy of metastatic TNBC and is worthy of future clinical evaluation.Trial registration numbersNCT03237572 and NCT04116320.

ImmunoPET-informed sequence for focused ultrasound-targeted mCD47 blockade controls glioma

Phagocytic immunotherapies such as CD47 blockade have emerged as promising strategies for glioblastoma (GB) therapy, but the blood brain/tumor barriers (BBB/BTB) pose a persistent challenge for mCD47 delivery that can be overcome by focused ultrasound (FUS)-mediated BBB/BTB disruption. We here leverage immuno-PET imaging to determine how timing of [89Zr]-mCD47 injection relative to FUS impacts antibody penetrance into orthotopic murine gliomas. We then design and implement a rational paradigm for combining FUS and mCD47 for glioma therapy. We demonstrate that timing of antibody injection relative to FUS BBB/BTB disruption is a critical determinant of mCD47 access, with post-FUS injection conferring superlative antibody delivery to gliomas. We also show that mCD47 delivery across the BBB/BTB with repeat sessions of FUS can significantly constrain tumor outgrowth and extend survival in glioma-bearing mice. This study generates provocative insights for ongoing pre-clinical and clinical evaluations of FUS-mediated antibody delivery to brain tumors. Moreover, our results confirm that mCD47 delivery with FUS is a promising therapeutic strategy for GB therapy.

Type 1 conventional dendritic cells are systemically dysregulated early in pancreatic carcinogenesis

Type 1 conventional dendritic cells (cDC1s) are typically thought to be dysregulated secondarily to invasive cancer. Here, we report that cDC1 dysfunction instead develops in the earliest stages of preinvasive pancreatic intraepithelial neoplasia (PanIN) in the KrasLSL-G12D/+ Trp53LSL-R172H/+ Pdx1-Cre–driven (KPC) mouse model of pancreatic cancer. cDC1 dysfunction is systemic and progressive, driven by increased apoptosis, and results in suboptimal up-regulation of T cell–polarizing cytokines during cDC1 maturation. The underlying mechanism is linked to elevated IL-6 concomitant with neoplasia. Neutralization of IL-6 in vivo ameliorates cDC1 apoptosis, rescuing cDC1 abundance in tumor-bearing mice. CD8+ T cell response to vaccination is impaired as a result of cDC1 dysregulation. Yet, combination therapy with CD40 agonist and Flt3 ligand restores cDC1 abundance to normal levels, decreases cDC1 apoptosis, and repairs cDC1 maturation to drive superior control of tumor outgrowth. Our study therefore reveals the unexpectedly early and systemic onset of cDC1 dysregulation during pancreatic carcinogenesis and suggests therapeutically tractable strategies toward cDC1 repair.

Development of Tumor Cell-Based Vaccine with IL-12 Gene Electrotransfer as Adjuvant

Tumor cell-based vaccines use tumor cells as a source of tumor-associated antigens. In our study, we aimed to develop and test a tumor vaccine composed of tumor cells killed by irradiation combined with in vivo interleukin-12 gene electrotransfer as an adjuvant. Vaccination was performed in the skin of B16-F10 malignant melanoma or CT26 colorectal carcinoma tumor-bearing mice, distant from the tumor site and combined with concurrent tumor irradiation. Vaccination was also performed before tumor inoculation in both tumor models and tumor outgrowth was followed. The antitumor efficacy of vaccination in combination with tumor irradiation or preventative vaccination varied between the tumor models. A synergistic effect between vaccination and irradiation was observed in the B16-F10, but not in the CT26 tumor model. In contrast, up to 56% of mice were protected from tumor outgrowth in the CT26 tumor model and none were protected in the B16-F10 tumor model. The results suggest a greater contribution of the therapeutic vaccination to tumor irradiation in a less immunogenic B16-F10 tumor model, in contrast to preventative vaccination, which has shown greater efficacy in a more immunogenic CT26 tumor model. Upon further optimization of the vaccination and irradiation regimen, our vaccine could present an alternative tumor cell-based vaccine.