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Immune escape represents a major driver of acute myeloid leukemia (AML) reemergence after allogeneic hematopoietic cell transplantation (allo-HCT), with up to 40% of relapses prompted by nongenomic loss of HLA class II expression in leukemia cells. By integrative analysis of gene expression, DNA methylation, and chromatin accessibility in paired diagnosis/relapse primary samples and in the respective patient-derived xenografts (PDX), we identify the polycomb repressive complex 2 (PRC2) as a key epigenetic driver of this immune escape modality. We report that loss of expression of HLA class II molecules is accompanied by a PRC2-dependent reduction in chromatin accessibility. Pharmacologic inhibition of PRC2 subunits rescues HLA class II expression in AML relapses in vitro and in vivo, with consequent recovery of leukemia recognition by CD4+ T cells. Our results uncover a novel link between epigenetics and leukemia immune escape, which may rapidly translate into innovative strategies to cure or prevent AML posttransplantation relapse. Significance: Loss of HLA class II expression represents a frequent mechanism of leukemia posttransplantation relapse. Here we identify PRC2 as the main epigenetic driver of this immune escape modality and show that its chemical inhibition can reinstate a proficient graft-versus-leukemia effect, providing an innovative rationale for personalized epigenetic immunotherapies. See related commentary by Köhler and Zeiser, p. 1410. This article is highlighted in the In This Issue feature, p. 1397

Increasing clinical use of immune checkpoint inhibitors testifies to the importance of modulating the immune TME to obtain meaningful anti-tumor immune responses. Acting only on T lymphocytes may, however, not be sufficient, e.g. in immunologically-cold tumors or due to de novo or acquired resistance. Moreover, immune-related AEs remain hurdles of T cell therapies. To overcome these limitations and to awaken the immune system in an agnostic way against the tumor, we have developed a genetically modified cell-based autologous hematopoietic stem cell platform (Temferon) delivering immunotherapeutic payloads into the TME through Tie-2 expressing monocytes (TEMs), a subset of tumor infiltrating macrophages. TEM-GBM is an ongoing open-label, Phase 1/2a dose-escalating study evaluating the safety & efficacy of Temferon in up to 21 newly diagnosed patients with glioblastoma & unmethylated MGMT promoter assigned to 7 different cohorts (3 pts each) differing by Temferon dose (0.5-4.0x106/kg) and conditioning regimen (BCNU+ or Busulfan+Thiotepa). By Oct 15th, 2021, 15 pts (cohort 1-5) had received escalating doses of Temferon with a median follow up of 267 days (range: 60-749). Rapid engraftment and hematological recovery from nonmyeloablative conditioning occurred in all pts. Temferon-derived differentiated cells, as determined by the presence of vector genomes in the DNA, were found at increasing proportions in PB and BM, reaching up to 30% at 1 month for the highest cohorts tested (2.0x106/kg) and persisting up to 18 months, albeit at lower levels. Despite the significant proportion of engineered cells, only very low median concentrations of IFNα were detected in the plasma (D+30, 5.9; D+90, 8.8pg/mL) and in the cerebrospinal fluid (D+30, 1.5; D+90, 2.4pg/mL), indicating tight regulation of vector expression. SAEs were mostly attributed to conditioning chemotherapy (e.g. infections) or disease progression (e.g. seizures). 1 SUSAR (persistent GGT elevation) has occurred. Median OS is 14 mth from surgery (11 mth post Temferon). Four pts from the low dose cohorts underwent 2nd surgery. These recurrent tumors contained gene-marked cells and expressed IFN-responsive genes, indicative of local IFNα release by TEMs. In 1 pt, a stable lesion (as defined by MRI) had a higher proportion of T cells & TEMs, an increased IFN-response signature and myeloid re-programming revealed by scRNAseq, as compared to a synchronous, progressing tumor. TCR sequencing of blood and tumor samples showed a post-treatment increase in the cumulative frequency of tumor-associated T cell clones identified in 1st and 2nd surgery specimens (up to 4 out of 9 subjects). These results provide initial evidence for on-target activity of Temferon in GBM, to be consolidated with longer follow up in the higher dose cohorts. Citation Format: Bernhard Gentner, Gaetano Finocchiaro, Francesca Farina, Marica Eoli, Alessia Capotondo, Elena Anghileri, Matteo Barcella, Maria Grazia Bruzzone, Matteo Giovanni Carrabba, Valeria Cuccarini, Giorgio D'Alessandris, Francesco Di Meco, Valeria Ferla, Paolo Ferroli, Filippo Gagliardi, Federico Legnani, Pietro Mortini, Matteo Maria Naldini, Alessandro Olivi, Roberto Pallini, Monica Patanè, Rosina Paterra, Bianca Pollo, Marco Saini, Silvia Snider, Valentina Brambilla, Stefania Mazzoleni, Andrew Zambanini, Carlo Russo, Luigi Naldini, Fabio Ciceri. Genetically modified Tie-2 expressing monocytes target IFN-α2 to the glioblastoma tumor microenvironment (TME): Preliminary data from the TEM-GBM Phase 1/2a study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5213.

Infections are a major cause of morbidity and mortality in hematological patients. We prospectively tested a new molecular assay (Verigene®) in 79 consecutive hematological patients, with sepsis by gram-negative bacteria. A total of 82 gram-negative microorganisms were isolated by blood cultures, of which 76 cases were mono-microbial. Considering the bacteria detectable by the system, the concordance with standard blood cultures was 100%. Resistance genes were detected in 20 of the isolates and 100% were concordant with the phenotypic antibiotic resistance. Overall, this new assay correctly identified 66/82 of all the gram-negative pathogens, yielding a general sensitivity of 80.5%, and providing information on genetic antibiotic resistance in a few hours. This new molecular assay could ameliorate patient management, resulting in a more rational use of antibiotics.