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Blood cell formation must be appropriately maintained throughout life to provide robust immune function, hemostasis, and oxygen delivery to tissues, and to prevent disorders that result from over- or underproduction of critical lineages. Persistent inflammation deregulates hematopoiesis by damaging hematopoietic stem and progenitor cells (HSPCs), leading to elevated myeloid cell output and eventual bone marrow failure. Nonetheless, antiinflammatory mechanisms that protect the hematopoietic system are understudied. The transcriptional regulator STAT3 has myriad roles in HSPC-derived populations and nonhematopoietic tissues, including a potent antiinflammatory function in differentiated myeloid cells. STAT3 antiinflammatory activity is facilitated by STAT3-mediated transcriptional repression of Ube2n, which encodes the E2 ubiquitin-conjugating enzyme Ubc13 involved in proinflammatory signaling. Here we demonstrate a crucial role for STAT3 antiinflammatory activity in preservation of HSPCs and lineage-balanced hematopoiesis. Conditional Stat3 removal from the hematopoietic system led to depletion of the bone marrow lineage− Sca-1+ c-Kit+ CD150+ CD48− HSPC subset (LSK CD150+ CD48− cells), myeloid-skewed hematopoiesis, and accrual of DNA damage in HSPCs. These responses were accompanied by intrinsic transcriptional alterations in HSPCs, including deregulation of inflammatory, survival and developmental pathways. Concomitant Ube2n/Ubc13 deletion from Stat3-deficient hematopoietic cells enabled lineage-balanced hematopoiesis, mitigated depletion of bone marrow LSK CD150+ CD48− cells, alleviated HSPC DNA damage, and corrected a majority of aberrant transcriptional responses. These results indicate an intrinsic protective role for STAT3 in the hematopoietic system, and suggest that this is mediated by STAT3-dependent restraint of excessive proinflammatory signaling via Ubc13 modulation.

Introduction: Cancer immunotherapy is a modern strategy aiming at restoring the capacity of the immune system to target tumors in cancer patients. Toll-like receptor (TLR) agonists may enhance vaccination or direct immune activation at the tumor microenvironment. This clinical trial evaluated the biologic effects of Resiquimod, a TLR agonist that can activate both myeloid (TLR 8) and plasmacytoid (TLR 7) dendritic cells, on advanced stage melanoma. Methods: Subjects with in-transit melanoma metastases or high risk for recurrence and appropriate HLA were treated with peptide vaccination (class 1 restricted peptide GP100(g209-2m) and, if HLA-DP4+, class 2 restricted peptide MAGE-3243-258). Half of the patients were randomized to receive Resiquimod as an adjuvant applied to the GP100 vaccination site. Subjects with in-transit disease were then treated with resiquimod topically on half of the target lesions. To evaluate the T cell function, fresh PBMC and single cell tumor suspension were analyzed by flow cytometry using gp100-specific dextramer staining. RNA from the vaccination site was also analyzed using real-time PCR. Results: All patients (n=47) underwent GP100(g209-2m) vaccination, a majority (39) also received the MAGE-3243-258 peptide. Type 1 interferon pathway protein profiles of vaccination sites showed activation of plasmacytoid dendritic cells in patients with Resiquimod, but not in its absence. Nineteen subjects had in-transit disease at entry into the trial. In response to peptide vaccination alone, tumor regression was more likely in patients who received Resiquimod (group A) compared to those who did not (group B). (4/9 vs 0/10). In group A, 5 patients continued treatment with Resiquimod topically on the tumors, and all had tumor response (4PR, 1CR). In group B, 5 continued to tumoral resiquimod and 3 had regression (3 PR). Type I interferon (as measured by MxA and IRF7) IFN-gamma and TNF-alpha increased at the vaccination site 24 hrs after vaccination only at the sites where Resiquimod was applied. In blood, Resiquimod increased gp100-specific CD8 T cells frequency at week 8 (p=0.03) only in patients who received Resiquimod at the vaccination site. Conclusions: Resiquimod activates plasmacytoid dendritic cells at a peptide vaccination site and augments peptide vaccination sufficiently to mediate regression of in-transit melanoma metastasis. Resiquimod on in-transit melanoma, in vaccinated hosts, drives regression of metastases, regardless of previous exposure at vaccination site. An increased amount of cytokines such type I interferon, IFN-gamma, TNF-alpha, and T specific cytotoxic frequency were increased at the vaccination site after patients received Resiquimod. Citation Format: Jorge A. Borin Scutti, Luis M. Vence, Richard E. Royal, Tara C. Wray, Janice N. Cormier, Jeffrey E. Lee, Anthony Lucci, Jeffrey E. Gershenwald, Merrick I. Ross, Jennifer Wargo, Karen A. Millerchip, Rodabe N. Amaria, Michael A. Davis, Adi Diab, Isabella C. Glitza, Wen Hwu, Sapna Patel, Scott E. Woodman, Willem W. Overwijk, Patrick Hwu. Resiquimod, a Toll-like receptor agonist promotes melanoma regression by enhancing plasmacytoid dendritic cells and T cytotoxic activity as a vaccination adjuvant and by direct tumor application [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 614.