Autologous stem-cell transplantation (ASCT) for multiple myeloma continues to confer benefit in the ‘novel agent’ era [1]. However, most patients relapse, risk stratification is inadequate and opti...
ABSTRACT High-dose alkylator-based conditioning followed by autologous stem-cell transplantation (ASCT) is a therapeutic mainstay for eligible patients with multiple myeloma. However, post-transplant relapses are common and prognostic biomarkers are scarce. Relapses are characterized by the influx of regulatory myeloid cells and dysfunctional T effectors. We have shown that myeloma-infiltrating myeloid cells produce versican (VCAN), a large matrix proteoglycan with tolerogenic activities. VCAN proteolysis by a-disintegrin-and-metalloproteinase-with-thrombospondin-motifs (ADAMTS) proteases generates versikine, a bioactive fragment (“matrikine”) that regulates Batf3-dendritic cells, known to control CD8+-attracting chemokine networks. Here we demonstrate that intense VCAN proteolysis predicts CD8+ infiltration post-transplant and paradoxically portends significantly inferior survival outcomes. Our data suggest that VCAN proteolysis promotes the influx of CD8+ effectors that are rendered overwhelmingly dysfunctional and/or frankly immunoregulatory (CD8+ Treg) at the tumor site. Thus, complex immunosuppressive circuits orchestrated through VCAN accumulation and turnover generate conditions favorable for myeloma tumor regrowth and point to a readily-assayed biomarker to identify the patients at risk for relapse and early death. The dismal outcomes associated with VCAN proteolysis may be rationally overcome through immunotherapies such as checkpoint inhibition (e.g., anti-TIGIT), tumor vaccines or anti-myeloid (e.g., anti-CSF-1R) approaches.
Lupus is experienced as an “aggressive and expansive” disease affecting patients’ activities of daily living, sense of self, and social functioning [1]. Currently there is no cure. Despite substantial efforts, the lupus drug development field has witnessed only one FDA-approved therapy in the last 50 years. There is an urgent need to better our understanding of the pathogenic mechanisms for lupus, and to develop novel therapeutic strategies for both cutaneous and systemic lupus erythematosus.
Abstract The impact of tumor matrix remodeling to the generation of an “inflamed” microenvironment that modulates responses to immunotherapy is unclear. Versican (VCAN) is a chondroitin sulphate matrix proteoglycan that promotes tolerogenic polarization of intratumoral DC through Toll-like receptor 2 (TLR2). Proteolytic processing of VCAN releases a bioactive N-terminal fragment (matrikine), versikine. In contrast to the tolerogenic actions of parental VCAN, versikine triggers IRF8-dependent transcription in myeloid cells and promotes Batf3-dendritic cell (DC) generation from FLT3L-mobilized bone marrow progenitors in vitro. Consistent with the Batf3-promoting effects of versikine, VCAN proteolysis correlates with T-cell infiltration across multiple cancers.The aims were to 1. define the impact of versikine on the intratumoral myeloid repertoire in vivo and 2. to define the efficacy of versikine as a vaccine adjuvant.4T1 breast carcinoma and Lewis Lung Carcinoma (LLC) empty vector (EV)- and versikine-expressing cells were implanted subcutaneously in syngeneic recipients. 1000mm3 tumors were harvested and intratumoral DC subsets were enumerated. Versikine-expressing tumors were characterized by significantly enhanced Batf3-DC (CD11chigh,MHC IIhigh Ly6C-, CD64-, CD24high,CD11blow) (p =0.0079 for 4T1 model and <0.0001 for LLC model), whereas cDC2 (CD11chigh,MHC IIhigh Ly6C-, CD64-, CD24low, CD11bhigh ) frequency was diminished (p= 0.0079 and <0.0001 respectively). Monocytic-derived DC (Mo-DC: CD11chigh, MHC IIhigh, Ly6C+, CD64+) remained unchanged. To determine the impact of versikine on responses to in situ vaccination using STING agonists, EV- and versikine-replete tumors (B16 and 4T1; 150 mm3), were injected intratumorally (IT) with a single subtherapeutic dose (200 μg) of DMXAA (murine STING agonist) or vehicle. EV-tumors did not significantly respond to 200μg DMXAA, whereas many B16-versikine and 4T1-versikine tumors regressed or growth was inhibited (p<0.001 and p=0.014 respectively). Necrosis was frequently observed in 4T1 versikine-secreting tumors (6/9 mice) within 24 hours after treatment. Versikine extended survival after subtherapeutic DMXAA treatment in 4T1; log rank=p=0.01. Versikine's effects were abrogated in Batf3-null mice. To quantitate antigen-specific responses in the presence or absence of versikine, EV- and versikine-replete LLC tumors were injected IT with 500 μg DMXAA (therapeutic dose) or vehicle. We observed a significant increase in the frequency of CD8+ MHCI:SIINFEKL tetramer+ splenocytes in LLC-versikine-bearing animals as well as a marked increase in central memory T splenocytes (TCM) (CD62LhighCD44high). VCAN matrikines may generate effective adjuvants for in situ vaccination strategies across diverse solid and hematopoietic tumor types. Citation Format: Athanasios Papadas, Evan Flietner, Zachary Morrow, Joshua Wiesner, Alexander Cicala, Adam Pagenkopf, Chelsea Hope, Philip Emmerich, Dustin Deming, Jing Zhang, Peiman Hematti, Natalie Callander, Alexander Rakhmilevich, Mario Otto, Christian Capitini, Fotis Asimakopoulos. Versican proteolytic fragments (matrikines) synergize with STING agonists to elicit robust anti-tumor CD8+ T cell responses [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5733.
Abstract Colorectal cancer (CRC) originates within immunologically complex microenvironments. To date the benefits of immunotherapy have been modest except in neoantigen-laden mismatch repair (MMR)-deficient tumors. Approaches to enhance tumor-infiltrating lymphocytes in the tumor bed may substantially augment clinical immunotherapy responses. We recently reported that proteolysis of the tolerogenic matrix proteoglycan versican (VCAN), in myeloma tumors, generates a bioactive fragment, versikine, with putative immunostimulatory activities. We show that VCAN proteolysis strongly correlated with CD8+ T-cell infiltration in CRC. Tumors displaying active VCAN proteolysis and low total VCAN were associated with robust (10-fold) CD8+ T-cell infiltration. The correlation between VCAN proteolysis and CD8+ T-cell infiltration was maintained in MMR-proficient and -deficient CRCs. Tumor-intrinsic WNT pathway activation was associated with CD8+ T-cell exclusion and correlated with VCAN accumulation. Our findings indicate VCAN proteolysis may shape CRC immune contexture and provide a rationale for testing VCAN proteolysis as a predictive and/or prognostic immune biomarker. Therapeutic manipulation of the VCAN-versikine axis may augment immunotherapy efficacy against CRC.
Stimulatory type 1 conventional dendritic cells (cDC1s) engage in productive interactions with CD8+ effectors along tumor-stroma boundaries. The paradoxical accumulation of "poised" cDC1s within stromal sheets is unlikely to simply reflect passive exclusion from tumor cores. Drawing parallels with embryonic morphogenesis, we hypothesized that invasive margin stromal remodeling generates developmentally conserved cell fate cues that regulate cDC1 behavior. We find that, in human T cell-inflamed tumors, CD8+ T cells penetrate tumor nests, whereas cDC1s are confined within adjacent stroma that recurrently displays site-specific proteolysis of the matrix proteoglycan versican (VCAN), an essential organ-sculpting modification in development. VCAN is necessary, and its proteolytic fragment (matrikine) versikine is sufficient for cDC1 accumulation. Versikine does not influence tumor-seeding pre-DC differentiation; rather, it orchestrates a distinctive cDC1 activation program conferring exquisite sensitivity to DNA sensing, supported by atypical innate lymphoid cells. Thus, peritumoral stroma mimicking embryonic provisional matrix remodeling regulates cDC1 abundance and activity to elicit T cell-inflamed tumor microenvironments.
Sexual dimorphism is exhibited remarkably in the female predominance of autoimmune diseases (e.g. systemic lupus erythematosus, female‐to‐male ratio 9 : 1). To understand the female bias in autoimmunity, we focused on vestigial‐like family member 3 (VGLL3), a molecule with increased expression in females and known to promote autoimmunity. We report that VGLL3 mediates the cellular stress response by upregulating p53 and IL‐17C. Energy stress allows VGLL3 to be induced by IFNα, which ultimately leads to p53‐dependent, lupus‐associated, inflammatory cell death. Our results suggest that female‐biased expression of VGLL3 helps cells adapt to metabolic stress, which, intriguingly, is known as a significant challenge during the evolution of placental mammals due to the need to feed a developing embryo. The findings also uncover the importance of maintaining metabolic homeostasis in the prevention of autoimmunity.
Abstract Background: Infiltration of CD8+ T cells has shown important prognostic implications in colorectal cancers (CRC). Our group has recently correlated proteolysis of an extracellular matrix proteoglycan, versican (VCAN), with infiltration of CRCs by CD8+ T cells. Cleavage by ADAMTS proteases generates a bioactive fragment of versican, versikine (VKINE). VKINE stimulates Batf3-driven dendritic cell activation and maturation, which are critical for immunotherapy efficacy. Methods: CT26 murine CRC cells were transfected to constitutively express VKINE. BALB/c mice between 50 and 70 days of age were orthotopically injected with either 105 empty vector (CT26-EV) or VKINE-expressing (CT26-VKINE) cells. Tumor growth was tracked weekly with colonoscopy and growth rate assessed using percent lumen occlusion. Mice were euthanized and dissected once moribund. Tissues were formalin-fixed and paraffin-embedded (FFPE). Immunohistochemical (IHC) staining was performed using standard procedures. Additionally, FFPE samples from human CRC liver metastases (n=9) were obtained under the University of Wisconsin Translational Science Biocore Institutional Review Board-approved protocol and IHC performed for VCAN, VKINE and CD8. The number of CD8+ tumor-infiltrating lymphocytes (TILs) per high-powered field (HPF) within the malignant epithelium was calculated using four separate 400x magnification fields of view. Results: CT26-EV and CT26-VKINE injected mice both had a ~70% tumor initiation rate and these tumors averaged ~50% lumen occlusion by 14 days post injection. Survival data for each group were compared using a log-rank test. A trend towards improved survival was observed in those mice injected with CT26-VKINE (average survival of 27 days for CT26-VKINE compared to 22 days for CT26-EV, p=0.41, n=13). The average number of CD8+ TILs for each tumor were compared. For mice with CT26-VKINE, the average number of CD8+ TILs per HPF was 7.6, compared to 4.7 for mice with CT26-EV (p=0.39). Human CRC liver metastases were analyzed for VCAN, VKINE, and CD8+ T cells. Several lesions had little to no VCAN staining, whereas others had intense pockets or peripheral staining. VKINE staining was more abundant in metastatic CRC lesions than in primary lesions. CD8+ T cells were excluded in the setting of increased VCAN staining and infiltration was noted specifically in areas of VCAN proteolysis (low VCAN and high VKINE staining). These areas of proteolysis were more often present at the tumor margin and correlated with ADAMTS1 expression. Conclusions: Immunotherapies offer a unique treatment option for cancer patients, but their use is limited in CRC. VCAN proteolysis and the generation of bioactive VKINE has potential to influence the ability of CD8+ T cells to infiltrate the tumor in both primary and metastatic CRC, indicating the potential to utilize VCAN and VKINE as immune biomarkers or therapeutic targets. Citation Format: Philip B. Emmerich, Susan N. Payne, Connor J. Maloney, Rosabella T. Pitera, Hanna Rainiero, Gioia Sha, Athanasios T. Papadas, Adam C. Pagenkopf, Michael F. Bassetti, Kristina A. Matkowskyj, Fotis Asimakopoulos, Dustin A. Deming. Versican proteolysis: A novel immunostimulatory component of CD8+ T cell responses to colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3146.
