Blockade of Discoidin Domain Receptor Signaling with Sitravatinib Reveals DDR2 as a Mediator of Neuroblastoma Pathogenesis and Metastasis.

Oncogene-driven expression and activation of receptor tyrosine kinases (RTK) promotes tumorigenesis and contributes to drug resistance. Increased expression of the kinases DDR2 (Discoid Domain Receptor 2), RET, PDGFRA, KIT, MET, and ALK (Anaplastic Lymphoma Kinase) independently correlate with decreased overall survival (OS) and event free survival (EFS) of pediatric neuroblastoma. The multikinase inhibitor sitravatinib targets DDR2, RET, PDGFRA, KIT and MET with low nanomolar activity and we therefore tested its efficacy against orthotopic and syngeneic tumor models. Sitravatinib markedly reduced cell proliferation and migration in vitro independently of MYCN (N-Myc proto-oncogene), ALK, or MYC (c-Myc proto-oncogene) status, and inhibited proliferation and metastasis of human orthotopic xenografts. Oral administration of sitravatinib to homozygous Th-MYCN transgenic mice (Th-MYCN+/+) after tumor initiation completely arrested further tumor development with no mice dying of disease while maintained on sitravatinib treatment (control cohort 57 days median time to sacrifice). Among these top kinases, DDR2 expression has the strongest correlation with poor survival and high stage at diagnosis, and the highest sensitivity to the drug. We confirmed on-target inhibition of collagen-mediated activation of DDR2. Genetic knockdown of DDR2 partially phenocopies Sitravatinib treatment, limiting tumor development and metastasis across tumor models. Analysis of single cell sequencing data demonstrated that DDR2 is restricted to mesenchymal-type tumor subpopulations and is enriched in Schwann Cell Precursor (SCP) subpopulations found in high-risk disease. These data define an unsuspected role for sitravatinib as a therapeutic agent in neuroblastoma and reveal a novel function for DDR2 as a driver of tumor growth and metastasis.

A Novel Druggable Dual-Specificity tYrosine-Regulated Kinase3/Calmodulin Kinase-like Vesicle-Associated Signaling Module with Therapeutic Implications in Neuroblastoma.

High-risk neuroblastoma is a very aggressive pediatric cancer, accounting for ~15% of childhood cancer mortality. Therefore, novel therapeutic strategies for the treatment of neuroblastoma are urgently sought. Here, we focused on the potential implications of the Dual-specificity tYrosine-Regulated Kinase (DYRK) family and downstream signaling pathways. We used bioinformatic analysis of public datasets from neuroblastoma cohorts and cell lines to search correlations between patient survival and expression of DYRK kinases. Additionally, we performed biochemical, molecular, and cellular approaches to validate and characterize our observations, as well as an in vivo orthotopic murine model of neuroblastoma. We identified the DYRK3 kinase as a critical mediator of neuroblastoma cell proliferation and in vivo tumor growth. DYRK3 has recently emerged as a key regulator of several biomolecular condensates and has been linked to the hypoxic response of neuroblastoma cells. Our data suggest a role for DYRK3 as a regulator of the neuroblastoma-specific protein CAMKV, which is also required for neuroblastoma cell proliferation. CAMKV is a very understudied member of the Ca2+/calmodulin-dependent protein kinase family, originally described as a pseudokinase. We show that CAMKV is phosphorylated by DYRK3, and that inhibition of DYRK3 kinase activity induces CAMKV aggregation, probably mediated by its highly disordered C-terminal half. Importantly, we provide evidence that the DYRK3/CAMKV signaling module could play an important role for the function of the mitotic spindle during cell division. Our data strongly support the idea that inhibition of DYRK3 and/or CAMKV in neuroblastoma cells could constitute an innovative and highly specific intervention to fight against this dreadful cancer.

PRMT5 activates AKT via methylation to promote tumor metastasis.

Protein arginine methyltransferase 5 (PRMT5) is the primary methyltransferase generating symmetric-dimethyl-arginine marks on histone and non-histone proteins. PRMT5 dysregulation is implicated in multiple oncogenic processes. Here, we report that PRMT5-mediated methylation of protein kinase B (AKT) is required for its subsequent phosphorylation at Thr308 and Ser473. Moreover, pharmacologic or genetic inhibition of PRMT5 abolishes AKT1 arginine 15 methylation, thereby preventing AKT1 translocation to the plasma membrane and subsequent recruitment of its upstream activating kinases PDK1 and mTOR2. We show that PRMT5/AKT signaling controls the expression of the epithelial-mesenchymal-transition transcription factors ZEB1, SNAIL, and TWIST1. PRMT5 inhibition significantly attenuates primary tumor growth and broadly blocks metastasis in multiple organs in xenograft tumor models of high-risk neuroblastoma. Collectively, our results suggest that PRMT5 inhibition augments anti-AKT or other downstream targeted therapeutics in high-risk metastatic cancers.

AGI-134: a fully synthetic α-Gal glycolipid that converts tumors into in situ autologous vaccines, induces anti-tumor immunity and is synergistic with an anti-PD-1 antibody in mouse melanoma models.

BACKGROUND: Treatments that generate T cell-mediated immunity to a patient's unique neoantigens are the current holy grail of cancer immunotherapy. In particular, treatments that do not require cumbersome and individualized ex vivo processing or manufacturing processes are especially sought after. Here we report that AGI-134, a glycolipid-like small molecule, can be used for coating tumor cells with the xenoantigen Galα1-3Galß1-4GlcNAc (α-Gal) in situ leading to opsonization with pre-existing natural anti-α-Gal antibodies (in short anti-Gal), which triggers immune cascades resulting in T cell mediated anti-tumor immunity. METHODS: Various immunological effects of coating tumor cells with α-Gal via AGI-134 in vitro were measured by flow cytometry: (1) opsonization with anti-Gal and complement, (2) antibody-dependent cell-mediated cytotoxicity (ADCC) by NK cells, and (3) phagocytosis and antigen cross-presentation by antigen presenting cells (APCs). A viability kit was used to test AGI-134 mediated complement dependent cytotoxicity (CDC) in cancer cells. The anti-tumoral activity of AGI-134 alone or in combination with an anti-programmed death-1 (anti-PD-1) antibody was tested in melanoma models in anti-Gal expressing galactosyltransferase knockout (α1,3GT-/-) mice. CDC and phagocytosis data were analyzed by one-way ANOVA, ADCC results by paired t-test, distal tumor growth by Mantel-Cox test, C5a data by Mann-Whitney test, and single tumor regression by repeated measures analysis. RESULTS: In vitro, α-Gal labelling of tumor cells via AGI-134 incorporation into the cell membrane leads to anti-Gal binding and complement activation. Through the effects of complement and ADCC, tumor cells are lysed and tumor antigen uptake by APCs increased. Antigen associated with lysed cells is cross-presented by CD8α+ dendritic cells leading to activation of antigen-specific CD8+ T cells. In B16-F10 or JB/RH melanoma models in α1,3GT-/- mice, intratumoral AGI-134 administration leads to primary tumor regression and has a robust abscopal effect, i.e., it protects from the development of distal, uninjected lesions. Combinations of AGI-134 and anti-PD-1 antibody shows a synergistic benefit in protection from secondary tumor growth. CONCLUSIONS: We have identified AGI-134 as an immunotherapeutic drug candidate, which could be an excellent combination partner for anti-PD-1 therapy, by facilitating tumor antigen processing and increasing the repertoire of tumor-specific T cells prior to anti-PD-1 treatment.

Rapid recruitment and activation of macrophages by anti-Gal/α-Gal liposome interaction accelerates wound healing.

Macrophages are pivotal in promoting wound healing. We hypothesized that topical application of liposomes with glycolipids that carry Galα1-3Galß1-4GlcNAc-R epitopes (α-gal liposomes) on wounds may accelerate the healing process by rapid recruitment and activation of macrophages in wounds. Immune complexes of the natural anti-Gal Ab (constituting ∼1% of Ig in humans) bound to its ligand, the α-gal epitope on α-gal liposomes would induce local activation of complement and generation of complement chemotactic factors that rapidly recruit macrophages. Subsequent binding of the Fc portion of anti-Gal coating α-gal liposomes to FcγRs on recruited macrophages may activate macrophage genes encoding cytokines that mediate wound healing. We documented the efficacy of this treatment in α1,3galactosyltrasferase knockout mice. In contrast to wild-type mice, these knockout mice lack α-gal epitopes and can produce the anti-Gal Ab. The healing time of excisional skin wounds treated with α-gal liposomes in these mice is twice as fast as that of control wounds. Moreover, scar formation in α-gal liposome-treated wounds is much lower than in physiologic healing. Additional sonication of α-gal liposomes resulted in their conversion into submicroscopic α-gal nanoparticles. These α-gal nanoparticles diffused more efficiently in wounds and further increased the efficacy of the treatment, resulting in 95-100% regeneration of the epidermis in wounds within 6 d. The study suggests that α-gal liposome and α-gal nanoparticle treatment may enhance wound healing in the clinic because of the presence of high complement activity and high anti-Gal Ab titers in humans.

In Situ Conversion of Melanoma Lesions into Autologous Vaccine by Intratumoral Injections of α-gal Glycolipids.

Autologous melanoma associated antigens (MAA) on murine melanoma cells can elicit a protective anti-tumor immune response following a variety of vaccine strategies. Most require effective uptake by antigen presenting cells (APC). APC transport and process internalized MAA for activation of anti-tumor T cells. One potential problem with clinical melanoma vaccines against autologous tumors may be that often tumor cells do not express surface markers that label them for uptake by APC. Effective uptake of melanoma cells by APC might be achieved by exploiting the natural anti-Gal antibody which constitutes ~1% of immunoglobulins in humans. This approach has been developed in a syngeneic mouse model using mice capable of producing anti-Gal. Anti-Gal binds specifically to α-gal epitopes (Galα1-3Galß1-4GlcNAc-R). Injection of glycolipids carrying α-gal epitopes (α-gal glycolipids) into melanoma lesions results in glycolipid insertion into melanoma cell membranes, expression of α-gal epitopes on the tumor cells and binding of anti-Gal to these epitopes. Interaction between the Fc portions of bound anti-Gal and Fcγ receptors on APC induces effective uptake of tumor cells by APC. The resulting anti-MAA immune response can be potent enough to destroy distant micrometastases. A clinical trial is now open testing effects of intratumoral α-gal glycolipid injections in melanoma patients.

Increased immunogenicity of HIV-1 p24 and gp120 following immunization with gp120/p24 fusion protein vaccine expressing alpha-gal epitopes.

Developing an effective HIV-1 vaccine will require strategies to enhance antigen presentation to the immune system. In a previous study we demonstrated a marked increase in immunogenicity of the highly glycosylated HIV-1 gp120 protein following enzymatic addition of alpha-gal epitopes to the carbohydrate chains. In the present study we determined whether gp120(alphagal) can also serve as an effective platform for targeting other HIV-1 proteins to APC and thus increase immunogenicity of both proteins. For this purpose we produced a recombinant fusion protein between gp120 and the HIV-1 matrix p24 protein (gp120/p24). Multiple alpha-gal epitopes were synthesized enzymatically on the gp120 portion of the fusion protein to generate a gp120(alphagal)/p24 vaccine. Immune responses to gp120(alphagal)/p24 compared to gp120/p24 vaccine lacking alpha-gal epitopes were evaluated in alpha1,3galactosyltransferase knockout (KO) mice. These mice lack alpha-gal epitopes and, therefore, are capable of producing the anti-Gal antibody. T cell responses to p24, as assessed by ELISPOT and by CD8+ T cells intracellular staining assays for IFNgamma, was on average 12- and 10-fold higher, respectively, in gp120(alphagal)/p24 immunized mice than in mice immunized with gp120/p24. In addition, cellular and humoral immune responses against gp120 were higher by 10-30-fold in mice immunized with gp120(alphagal)/p24 than in gp120/p24 immunized mice. Our data suggest that the alpha-gal epitopes on the gp120 portion of the fusion protein can significantly augment the immunogenicity of gp120, as well as that of the fused viral protein which lacks alpha-gal epitopes. This strategy of anti-Gal mediated targeting to APC may be used for production of effective HIV-1 vaccines comprised of various viral proteins fused to gp120.

Accelerated healing of skin burns by anti-Gal/alpha-gal liposomes interaction.

Topical application of alpha-gal liposomes on burns results in rapid local recruitment of neutrophils and macrophages. Recruited macrophages are pivotal for healing of burns because they secrete cytokines/growth factors that induce epidermis regeneration and tissue repair. alpha-Gal liposomes have glycolipids with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R) which bind anti-Gal, the most abundant natural antibody in humans constituting approximately 1% of immunoglobulins. Interaction of alpha-gal liposomes with anti-Gal within the fluid film formed on burns, activates complement and generates chemotactic complement cleavage peptides which effectively recruit neutrophils and macrophages. Anti-Gal IgG coating alpha-gal liposomes further binds to Fcgamma receptors on macrophages and activates them to secrete cytokines/growth factors. Efficacy of alpha-gal liposomes treatment in accelerating burn healing is demonstrated in the experimental model of alpha1,3galactosyltransferase knockout mice. These mice are the only available nonprimate mammals that can produce anti-Gal in titers similar to those in humans. Pairs of burns in mice were covered either with a spot bandage coated with 10mg alpha-gal liposomes, or with a control spot bandage coated with saline. On Day 3 post-treatment, the alpha-gal liposomes treated burns contained approximately 5-fold as many neutrophils as control burns, whereas macrophages were found only in alpha-gal liposomes treated burns. On Day 6, 50-100% of the surface area of alpha-gal liposomes treated burns were covered with regenerating epidermis (re-epithelialization), whereas almost no epidermis was found in control burns. The extensive recruitment of macrophages by anti-Gal/alpha-gal liposomes interaction was further demonstrated in vivo with polyvinyl alcohol (PVA) sponge discs containing alpha-gal liposomes, implanted subcutaneously. Since anti-Gal is abundant in all humans, it is suggested that treatment with alpha-gal liposomes will be effective also in patients with burns and other skin wounds.

Mechanism for increased immunogenicity of vaccines that form in vivo immune complexes with the natural anti-Gal antibody.

Anti-Gal constitutes approximately 1% of circulating IgG in humans and interacts specifically with alpha-gal epitopes. We reported previously that expression of alpha-gal epitopes on HIV gp120 and influenza virus vaccines increases immunogenicity by approximately 100-fold. We hypothesize that immunogenicity of any microbial vaccine can be markedly increased by linked alpha-gal epitopes due to in vivo formation of immune complexes with anti-Gal and the effective internalization of such immune complexes by APC, via Fc/FcgammaR interaction. The increased transport to lymph nodes and processing of anti-Gal complexed vaccines internalized by APC, results in effective activation of vaccine specific CD4(+) and CD8(+) T cells, and high cellular and humoral immune response. This universal mechanism for anti-Gal mediated increased immunogenicity is demonstrated in alpha1,3galactosyltransferase knockout mice with ovalbumin as a model vaccine.

Intratumoral injection of alpha-gal glycolipids induces a protective anti-tumor T cell response which overcomes Treg activity.

alpha-Gal glycolipids capable of converting tumors into endogenous vaccines, have alpha-gal epitopes (Gal alpha 1-3 Gal beta 1-4GlcNAc-R) and are extracted from rabbit RBC membranes. alpha-Gal epitopes bind anti-Gal, the most abundant natural antibody in humans constituting 1% of immunoglobulins. alpha-Gal glycolipids insert into tumor cell membranes, bind anti-Gal and activate complement. The complement cleavage peptides C5a and C3a recruit inflammatory cells and APC into the treated lesion. Anti-Gal further opsonizes the tumor cells and targets them for effective uptake by recruited APC, via Fc gamma receptors. These APC transport internalized tumor cells to draining lymph nodes, and present immunogenic tumor antigen peptides for activation of tumor specific T cells. The present study demonstrates the ability of alpha-gal glycolipids treatment to prevent development of metastases at distant sites and to protect against tumor challenge in the treated mice. Adoptive transfer studies indicate that this protective immune response is mediated by CD8+ T cells, activated by tumor lesions turned vaccine. This T cell activation is potent enough to overcome the suppressive activity of Treg cells present in tumor bearing mice, however it does not elicit an autoimmune response against antigens on normal cells. Insertion of alpha-gal glycolipids and subsequent binding of anti-Gal are further demonstrated with human melanoma cells, suggesting that intratumoral injection of alpha-gal glycolipids is likely to elicit a protective immune response against micrometastases also in cancer patients.

Immunogenicity of influenza virus vaccine is increased by anti-gal-mediated targeting to antigen-presenting cells.

This study describes a method for increasing the immunogenicity of influenza virus vaccines by exploiting the natural anti-Gal antibody to effectively target vaccines to antigen-presenting cells (APC). This method is based on enzymatic engineering of carbohydrate chains on virus envelope hemagglutinin to carry the alpha-Gal epitope (Gal alpha 1-3Gal beta 1-4GlcNAc-R). This epitope interacts with anti-Gal, the most abundant antibody in humans (1% of immunoglobulins). Influenza virus vaccine expressing alpha-Gal epitopes is opsonized in situ by anti-Gal immunoglobulin G. The Fc portion of opsonizing anti-Gal interacts with Fc gamma receptors on APC and induces effective uptake of the vaccine virus by APC. APC internalizes the opsonized virus to transport it to draining lymph nodes for stimulation of influenza virus-specific T cells, thereby eliciting a protective immune response. The efficacy of such an influenza vaccine was demonstrated in alpha 1,3galactosyltransferase (alpha 1,3GT) knockout mice, which produce anti-Gal, using the influenza virus strain A/Puerto Rico/8/34-H1N1 (PR8). Synthesis of alpha-Gal epitopes on carbohydrate chains of PR8 virus (PR8(alpha gal)) was catalyzed by recombinant alpha1,3GT, the glycosylation enzyme that synthesizes alpha-Gal epitopes in cells of nonprimate mammals. Mice immunized with PR8(alpha gal) displayed much higher numbers of PR8-specific CD8(+) and CD4(+) T cells (determined by intracellular cytokine staining and enzyme-linked immunospot assay) and produced anti-PR8 antibodies with much higher titers than mice immunized with PR8 lacking alpha-Gal epitopes. Mice immunized with PR8(alpha gal) also displayed a much higher level of protection than PR8 immunized mice after being challenged with lethal doses of live PR8 virus. We suggest that a similar method for increasing immunogenicity may be applicable to avian influenza vaccines.

Intratumoral injection of alpha-gal glycolipids induces xenograft-like destruction and conversion of lesions into endogenous vaccines.

This study describes a novel cancer immunotherapy treatment that exploits the natural anti-Gal Ab to destroy tumor lesions and convert them into an endogenous vaccine targeted to APC via FcgammaR. Anti-Gal constitutes 1% of immunoglobulins in humans and interacts specifically with alpha-gal epitopes (Galalpha1-3Galbeta1-4GlcNAc-R). The binding of anti-Gal to alpha-gal epitopes on pig cells mediates xenograft rejection. The proposed method uses glycolipid micelles with multiple alpha-gal epitopes (alpha-gal glycolipids). These glycolipids are extracted from rabbit red cell membranes and are comprised of ceramides with carbohydrate chains containing 5-25 carbohydrates, all capped with alpha-gal epitopes. Efficacy of this treatment was demonstrated in alpha1,3-galactosyltransferase knockout mice producing anti-Gal and bearing B16 melanoma or B16/OVA producing OVA as a surrogate tumor Ag. These mice are unique among nonprimate mammals in that, similar to humans, they lack alpha-gal epitopes and can produce the anti-Gal Ab. Intratumoral injection of alpha-gal glycolipids results in local inflammation mediated by anti-Gal binding to the multiple alpha-gal epitopes and activation of complement. These glycolipids spontaneously insert into tumor cell membranes. The binding of anti-Gal to alpha-gal expressing tumor cells induces the destruction of treated lesions as in anti-Gal-mediated xenograft rejection. Anti-Gal further opsonizes tumor cells within the lesion and, thus, targets them for effective uptake by APC that transport the tumor Ags to draining lymph nodes. APC further cross-present immunogenic tumor Ag peptides and elicit a systemic anti-tumor immune response. Similar intratumoral injection of alpha-gal glycolipids in humans is likely to induce the destruction of treated lesions and elicit a protective immune response against micrometastases.

Increased immunogenicity of human immunodeficiency virus gp120 engineered to express Galalpha1-3Galbeta1-4GlcNAc-R epitopes.

The glycan shield comprised of multiple carbohydrate chains on the human immunodeficiency virus (HIV) envelope glycoprotein gp120 helps the virus to evade neutralizing antibodies. The present study describes a novel method for increasing immunogenicity of gp120 vaccine by enzymatic replacement of sialic acid on these carbohydrate chains with Galalpha1-3Galbeta1-4GlcNAc-R (alpha-gal) epitopes. These epitopes are ligands for the natural anti-Gal antibody constituting approximately 1% of immunoglobulin G in humans. We hypothesize that vaccination with gp120 expressing alpha-gal epitopes (gp120(alphagal)) results in in vivo formation of immune complexes with anti-Gal, which targets vaccines for effective uptake by antigen-presenting cells (APC), due to interaction between the Fc portion of the antibody and Fcgamma receptors on APC. This in turn results in effective transport of the vaccine to lymph nodes and effective processing and presentation of gp120 immunogenic peptides by APC for eliciting a strong anti-gp120 immune response. This hypothesis was tested in alpha-1,3-galactosyltransferase knockout mice, which produce anti-Gal. Mice immunized with gp120(alphagal) produced anti-gp120 antibodies in titers that were >100-fold higher than those measured in mice immunized with comparable amounts of gp120 and effectively neutralized HIV. T-cell response, measured by ELISPOT, was much higher in mice immunized with gp120(alphagal) than in mice immunized with gp120. It is suggested that gp120(alphagal) can serve as a platform for anti-Gal-mediated targeting of additional vaccinating HIV proteins fused to gp120(alphagal), thereby creating effective prophylactic vaccines.