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Cancer progression entails close crosstalk between tumor cells and immune system. Recent advances in understanding mutual tumor-immune interactions translated into the development of cancer immunotherapy, changing the cancer treatment paradigm. Nevertheless, despite achieving long-term effect even in advanced malignancies, only a minor subset of patients responds to immunotherapy. The activation of the immune system, as well as good response to the immunotherapy strongly depends on tumor immunogenicity and effective presentation of tumor-associated antigens by conventional dendritic cells type 1 (cDC1). However, downregulation of antigen-presentation machinery and cDC1 exclusion represent major mechanisms of cancer immune evasion, leading to immunotherapy failure. Previously, we identified a combination of 3 transcription factors, PU.1, IRF8, and BATF3 (PIB) that instructed direct reprogramming of fibroblasts into cDC1-like cells endowed with the ability to present and cross-present antigens. Here, we hypothesize that cDC1-direct reprogramming employing PIB transcription factors can impose antigen-presentation directly in human tumor cells, generating functional tumor-antigen presenting cells (tumor-APCs) able to prime antigen-specific T cell responses. Here, we used a large panel of human cell lines (>30) derived from leukemia and multiple solid tumors including glioblastoma, melanoma, breast, pancreatic, ovarian, prostate, head and neck, and lung adenocarcinoma and evaluated dendritic cell reprogramming efficiency. Enforced expression of PIB induced a cDC1-phenotype in tumor cells, as demonstrated by an emerging CD45+HLA-DR+ population and the activation of the cDC1-markers CD11c, CD141, and CLEC9A. Induced cells showed global transcriptional reprogramming towards cDC1 fate imposing a tumor-APC signature in cancer cells at varying efficiencies ranging between 0.2±0.1% to 94.5±7.6% according to the tumor cell of origin. PIB induces rapid transcriptomic and epigenetic rewiring of cancer cells towards cDC1-fate, activating antigen presentation and processing machinery. This reprogramming process is asynchronous and leads to the generation of intermediate populations with active tumor-APC gene expression signatures. Furthermore, tumor-APCs upregulated expression of HLA-ABC and the co-stimulatory molecules CD40, CD80, and CD86, suggesting the acquired ability to prime immune-effectors. Functionally, tumor-APCs secrete pro-inflammatory cytokines including IL12p70, TNFα, CXCL10, and IL-29 upon TLR3/4 stimulation with Poly:IC and LPS, are competent in phagocytosis of dead cells and proteins, and can activate antigen-specific CD8+ T-cells. Importantly, cDC1-cell fate adoption was accompanied by downregulation of proliferation and diminished tumorigenicity, as evidenced by the loss of anchorage-independent growth. Importantly, cDC1-reprogramming was efficient in primary tissues obtained from head and neck, breast, urothelial and pancreatic carcinoma, as well as melanoma patients. Notably, PIB-mediated cDC1 reprogramming of tumor cells is substantially augmented when combined with a histone deacetylase inhibitor, valproic acid. In summary, PIB expression imposes cDC1 fate and function in a broad range of human cancer cells suggesting a capacity to elicit anti-tumor responses by orchestrating innate and adaptive immunity. Our strategy combines cDC1 antigen-processing and presenting abilities with the endogenous generation of tumor-antigens. This approach merges cell fate reprogramming with cancer immunotherapy, paving the way for the development of novel gene therapy for cancer. Disclosures No relevant conflicts of interest to declare.

An isochromosome of the long arm of chromosome 17, i(17q), is the most frequent genetic abnormality observed during the disease progression of Philadelphia chromosome–positive chronic myeloid leukemia (CML), and has been described as the sole anomaly in various other hematologic malignancies. The i(17q) hence plays a presumably important pathogenetic role both in leukemia development and progression. This notwithstanding, the molecular consequences of this abnormality have not been investigated in detail. We have analyzed 21 hematologic malignancies (8 CML in blast crisis, 8 myelodysplastic syndromes [MDS], 2 acute myeloid leukemias, 2 chronic lymphocytic leukemias, and 1 acute lymphoblastic leukemia) with i(17q) by fluorescence in situ hybridization (FISH). Using a yeast artificial chromosome (YAC) contig, derived from the short arm of chromosome 17, all cases were shown to have a breakpoint in 17p. In 12 cases, the breaks occurred within the Smith-Magenis Syndrome (SMS) common deletion region in 17p11, a gene-rich region which is genetically unstable. In 10 of these 12 cases, we were able to further map the breakpoints to specific markers localized within a single YAC clone. Six other cases showed breakpoints located proximally to the SMS common deletion region, but still within 17p11, and yet another case had a breakpoint distal to this region. Furthermore, using chromosome 17 centromere-specific probes, it could be shown that the majority of the i(17q) chromosomes (11 of 15 investigated cases) were dicentric, ie, they contained two centromeres, strongly suggesting that i(17q) is formed through an intrachromosomal recombination event, and also implicating that the i(17q), in a formal sense, should be designated idic(17)(p11). Because i(17q) formation results in loss of 17p material, potentially uncovering the effect of a tumor suppressor on the remaining 17p, the occurrence of TP53 mutations was studied in 17 cases by sequencing the entire coding region. In 16 cases, noTP53 mutations were found, whereas one MDS displayed a homozygous deletion of TP53. Thus, our data suggest that there is no association between i(17q) and coding TP53 mutations, and that another tumor suppressor gene(s), located in proximity of the SMS common deletion region, or in a more distal location, is of pathogenetic importance in i(17q)-associated leukemia.

Hematologic malignancies are characterized by fusion genes of biological/clinical importance. Immortalized cell lines with such aberrations are today widely used to model different aspects of leukemogenesis. Using cDNA microarrays, we determined the gene expression profiles of 40 cell lines as well as of primary leukemias harboring 11q23/MLL-rearrangements, t(1;19)[TCF3/PBX1], t(12;21)[ETV6/RUNX1], t(8;21)[RUNX1/CBFA2T1], t(8;14)[IGH@/MYC], t(8;14)[TRA@/MYC], t(9;22)[BCR/ABL1], t(10;11)[PICALM/MLLT10], t(15;17)[PML/RARA], or inv(16)[CBFB/MYH11]. Unsupervised classification revealed that hematopoietic cell lines of diverse origin, but with the same primary genetic changes, segregated together, suggesting that pathogenetically important regulatory networks remain conserved despite numerous passages. Moreover, primary leukemias co-segregated with cell lines carrying identical genetic rearrangements, further supporting that critical regulatory pathways remain intact in hematopoietic cell lines. Transcriptional signatures correlating with clinical subtypes/primary genetic changes were identified and annotated based on their biological/molecular properties and chromosomal localization. Furthermore, the expression profile of tyrosine kinase-encoding genes was investigated, identifying several differentially expressed members, segregating with primary genetic changes, which may be targeted with tyrosine kinase inhibitors. The identified conserved signatures are likely to reflect regulatory networks of importance for the transforming abilities of the primary genetic changes and offer important pathogenetic insights as well as a number of targets for future rational drug design.