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The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction–mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor–treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanoma. Implications: MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II–production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti–PD-1 immunotherapy for metastatic melanoma is planned.

The treatment of patients with metastatic melanoma with immune checkpoint inhibitors (ICI) leads to impressive response rates but primary and secondary resistance to ICI reduces progression-free survival. Novel strategies that interfere with resistance mechanisms are key to further improve patient outcome during ICI therapy. P53 is often inactivated by mouse-double-minute-2 (MDM2), which may decrease immunogenicity of melanoma cells. We analyzed primary patient-derived melanoma cell lines, performed bulk sequencing analysis of patient-derived melanoma samples, and used melanoma mouse models to investigate the role of MDM2-inhibition for enhanced ICI therapy. We found increased expression of IL15 and MHC-II in murine melanoma cells upon p53 induction by MDM2-inhibition. MDM2-inhibitor induced MHC-II and IL15-production, which was p53 dependent as Tp53 knockdown blocked the effect. Lack of IL15-receptor in hematopoietic cells or IL15 neutralization reduced the MDM2-inhibition/p53-induction–mediated antitumor immunity. P53 induction by MDM2-inhibition caused anti-melanoma immune memory as T cells isolated from MDM2-inhibitor–treated melanoma-bearing mice exhibited anti-melanoma activity in secondary melanoma-bearing mice. In patient-derived melanoma cells p53 induction by MDM2-inhibition increased IL15 and MHC-II. IL15 and CIITA expressions were associated with a more favorable prognosis in patients bearing WT but not TP53-mutated melanoma.Implications:MDM2-inhibition represents a novel strategy to enhance IL15 and MHC-II–production, which disrupts the immunosuppressive tumor microenvironment. On the basis of our findings, a clinical trial combining MDM2-inhibition with anti–PD-1 immunotherapy for metastatic melanoma is planned.

Supplementary Figures Legends and Methods

The bone marrow microenvironment (BMME) is key player in regulation and maintenance of hematopoiesis. Oncogenic RAS mutations, causing constitutive activation of multiple tumor-promoting pathways, are frequently found in human cancer. So far in hematologic malignancies, RAS mutations have only been reported to occur in hematopoietic cells. In this study, we investigated the effect of oncogenic Kras expression in the BMME in a chimeric mouse model. We observed that an activating mutation of Kras in the nonhematopoietic system leads to a phenotype resembling myelodysplastic syndrome (MDS) characterized by peripheral cytopenia, marked dysplasia within the myeloid lineage as well as impaired proliferation and differentiation capacity of hematopoietic stem and progenitor cells. The phenotypic changes could be reverted when the BM was re-isolated and transferred into healthy recipients, indicating that the KrasG12D-activation in the nonhematopoietic BMME was essential for the MDS phenotype. Gene expression analysis of sorted nonhematopoietic BM niche cells from KrasG12D mice revealed upregulation of multiple inflammation-related genes including IL1-superfamily members (Il1α, Il1β, Il1f9) and the NLPR3 inflammasome. Thus, pro-inflammatory IL1-signaling in the BMME may contribute to MDS development. Our findings show that a single genetic change in the nonhematopoietic BMME can cause an MDS phenotype. Oncogenic Kras activation leads to pro-inflammatory signaling in the BMME which impairs HSPCs function.Implications:These findings may help to identify new therapeutic targets for MDS.

S1. KrasG12D recipient mice develop a myelodysplastic syndrome within the transferred WT hematopoietic system. S2. Gating strategy used for the isolation of bone marrow (BM) niche cells. S3. The frequency of BM niche cells is not affected in KrasG12D recipient mice. S4. Differential pathway analysis in MSPCs and endothelial cells of KrasG12D versus WT mice. S5. Differential pathways analysis in CaR and osteoblast cells of KrasG12D versus WT recipient mice. S6. Expression of phosphorylated H2AX (γ-H2AX) in BM of WT or KrasG12D recipient mice. S7. Analysis of patient-derived BM for KRAS mutations by digital droplet polymerase chain reaction (ddPCR).

Acute graft-versus-host disease (aGVHD), which is driven by allogeneic T cells, has a high mortality rate and limited treatment options. Human β-defensin 2 (hBD-2) is an endogenous epithelial cell–derived host-defense peptide. In addition to its antimicrobial effects, hBD-2 has immunomodulatory functions thought to be mediated by CCR2 and CCR6 in myeloid cells. In this study, we analyzed the effect of recombinant hBD-2 on aGVHD development. We found that intestinal β-defensin expression was inadequately induced in response to inflammation in two independent cohorts of patients with aGVHD and in a murine aGVHD model. Treatment of mice with hBD-2 reduced GVHD severity and mortality and modulated the intestinal microbiota composition, resulting in reduced neutrophil infiltration in the ileum. Furthermore, hBD-2 treatment decreased proliferation and proinflammatory cytokine production by allogeneic T cells in vivo while preserving the beneficial graft-versus-leukemia effect. Using transcriptome and kinome profiling, we found that hBD-2 directly dampened primary murine and human allogeneic T cell proliferation, activation, and metabolism in a CCR2- and CCR6-independent manner by reducing proximal T cell receptor signaling. Furthermore, hBD-2 treatment diminished alloreactive T cell infiltration and the expression of genes involved in T cell receptor signaling in the ilea of mice with aGVHD. Together, we found that both human and murine aGVHD were characterized by a lack of intestinal β-defensin induction and that recombinant hBD-2 represents a potential therapeutic strategy to counterbalance endogenous hBD-2 deficiency.

Patients with acute myeloid leukemia (AML) often achieve remission after allogeneic hematopoietic cell transplantation (allo-HCT) but subsequently die of relapse driven by leukemia cells resistant to elimination by allogeneic T cells based on decreased major histocompatibility complex II (MHC-II) expression and apoptosis resistance. Here we demonstrate that mouse-double-minute-2 (MDM2) inhibition can counteract immune evasion of AML. MDM2 inhibition induced MHC class I and II expression in murine and human AML cells. Using xenografts of human AML and syngeneic mouse models of leukemia, we show that MDM2 inhibition enhanced cytotoxicity against leukemia cells and improved survival. MDM2 inhibition also led to increases in tumor necrosis factor-related apoptosis-inducing ligand receptor-1 and -2 (TRAIL-R1/2) on leukemia cells and higher frequencies of CD8+CD27lowPD-1lowTIM-3low T cells, with features of cytotoxicity (perforin+CD107a+TRAIL+) and longevity (bcl-2+IL-7R+). CD8+ T cells isolated from leukemia-bearing MDM2 inhibitor-treated allo-HCT recipients exhibited higher glycolytic activity and enrichment for nucleotides and their precursors compared with vehicle control subjects. T cells isolated from MDM2 inhibitor-treated AML-bearing mice eradicated leukemia in secondary AML-bearing recipients. Mechanistically, the MDM2 inhibitor-mediated effects were p53-dependent because p53 knockdown abolished TRAIL-R1/2 and MHC-II upregulation, whereas p53 binding to TRAILR1/2 promotors increased upon MDM2 inhibition. The observations in the mouse models were complemented by data from human individuals. Patient-derived AML cells exhibited increased TRAIL-R1/2 and MHC-II expression on MDM2 inhibition. In summary, we identified a targetable vulnerability of AML cells to allogeneic T-cell-mediated cytotoxicity through the restoration of p53-dependent TRAIL-R1/2 and MHC-II production via MDM2

This article has been retracted: please see Elsevier Policy on Article Withdrawal (https://www.elsevier.com/about/our-business/policies/article-withdrawal). This article has been retracted at the request of the Editor-in-Chief. Following the publication of the above article, the Editor was notified that an error occurred in which all images were published with incorrect versions. The Editor has taken the decision that the manuscript is no longer acceptable in its current form, nor with a corrigendum, as the extensive changes to the figures and publication would lead to ambiguity for our readers. We have therefore made the decision to retract this manuscript from Cancer Letters with the possibility of resubmission and republication of the manuscript in its corrected form after peer review.

Metastatic small cell lung cancer (SCLC) is not curable. While SCLC is initially sensitive to chemotherapy, remissions are short-lived. The relapse is induced by chemotherapy-selected tumor stem cells, which express the AC133 epitope of the CD133 stem cell marker. We studied the effectiveness of AC133-specific CAR T cells post-chemotherapy using human primary SCLC and an orthotopic xenograft mouse model. AC133-specific CAR T cells migrated to SCLC tumor lesions, reduced the tumor burden, and prolonged survival in a humanized orthotopic SCLC model, but were not able to entirely eliminate tumors. We identified CD73 and PD-L1 as immune-escape mechanisms and combined PD-1-inhibition and CD73-inhibition with CAR T cell treatment. This triple-immunotherapy induced cures in 25% of the mice, without signs of graft-versus-host disease or bone marrow failure. AC133

The bone marrow microenvironment (BMME) is key player in regulation and maintenance of hematopoiesis. Oncogenic

Cancers co-opt indoleamine 2,3-dioxygenase 1 (IDO1) as a mechanism for evading the immune system, and IDO1 inhibitors have emerged as a promising new approach for the treatment cancer through their potential to restore antitumor immunity and to synergise with existing therapies. As part of our investigations into novel small molecule inhibitors of IDO1, we screened a number of human and murine tumor lines for expression of tryptophan dioxygenases. All the human and murine tumor lines that we tested were found not to express tryptophan dioxygenases unless induced with IFN-gamma. Our screen included testing 50 primary human melanoma lines developed from tumor samples obtained New Zealand Melanoma patients (NZMel lines) for expression of IDO1 and IDO2. After co-culture with IFN-gamma, 84% of the NZMel lines were IDO1+ and IDO2+; 4% were IDO1+ only; 2 % were IDO2+ only; whilst 10% did not express either IDO1 or IDO2. Tryprophan dioxygenase (TDO) was not detected in any of the NZMel lines before or after IFN-gamma exposure. To overcome the need for IFN-gamma induction for our cell based assays of IDO1 inhibitory activity of our novel compounds, we transfected the wild-type murine Lewis Lung carcinoma line (LLTC) to constitutively express murine IDO1 (LLTC-mIDO1) or human IDO1 (LLTC-hIDO1), and used these engineered lines to assay for potential species selectivity of our inhibitors. IDO1 expression in the lines was consistent and stable, but when LLTC-hIDO1 cells were implanted subcutaneously into syngeneic mice for in vivo testing of inhibitors, we found considerable heterogeneity in IDO1 expression between tumors. Approximately half of the tumors from the same batch of implants were completely negative for IDO1 expression by Western blot analysis. More intriguingly, when fragments of an IDO1+ or an IDO1- donor tumor were implanted into new recipients, the same degree of intertumoral heterogeneity in IDO1 expression was seen in the subsequent generation of tumors. In contrast, subcutaneous tumors developing from murine GL261 glioma cells similarly engineered to over-express hIDO1 (GL261-hIDO1) were homogeneous in their expression of hIDO1, and were used for subsequent in vivo studies of intratumoral IDO1expression on its effects on immune cell infiltrates and tumor growth. GL261-hIDO1 tumors had a significantly faster growth rate than the wild-type tumors, and 24 days after implantation, had reached a mean tumor volume of 3600 mm3 compared to a mean tumor volume of 500 mm3 of wild-type tumors. Analysis of the immune cell infiltrates, showed that 14-day GL261 wild-type tumors had a 3-fold higher percentage of CD3+ T cells than that in GL261-hIDO1 tumors. CD8+ cells made up 55% and 25% of the CD3+ cells in wild-type and GL261-hIDO1 tumors, respectively. Percentage of Foxp3+ (Treg) cells was higher in GL261-hIDO1 tumors compared to that in wild-type tumors. Citation Format: Lai-Ming Ching, Petr Tomek, Brian D. Palmer, Sofian Tijono, Kimiora Henare, Lukas M. Braun. In vitro and in vivo models for evaluation of inhibitors of tryptophan dioxygenases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4138. doi:10.1158/1538-7445.AM2017-4138