The tumor microenvironment counters antitumor T-cell responses, in part, by blunting their activation and infiltration. Ligands that engage Toll-like receptors (TLR) on T cells and antigen-presenting cells can act as potent immune adjuvants. In this study, we show how tumor-reactive T cells engineered to secrete bacterial flagellin, a TLR5 ligand (TLR5L), can engender a costimulatory signal that augments antitumor activity. Human T cells engineered to express TLR5L along with DMF5, a T-cell receptor that recognizes the melanoma antigen MART-127–35 (DMF5TLR5L T cells), displayed increased proliferation, cytokine production, and cytolytic activity against melanoma cells. In a xenogenetic model, adoptive transfer of DMF5TLR5L T cells reduced tumor growth kinetics and prolonged mouse survival. In a syngeneic model, similarly engineered melanoma-reactive T cells (pmelTLR5L) displayed a relative increase in antitumor activity against established tumors, compared with unmodified T cells. In this model, we documented increased T-cell infiltration associated with increased levels of CCR1 and CXCR3 levels on T cells, a reduction in PD-1+Lag3+ T cells and CD11+Gr1+ myeloid-derived suppressor cells, and changes in the chemokine/cytokine profile of tumors. Our findings show how T cell–mediated delivery of a TLR agonist to the tumor site can contribute to antitumor efficacy, in the context of adoptive T-cell immunotherapy. Cancer Res; 75(10); 1–13. ©2015 AACR.

The tumor microenvironment counters antitumor T cell responses in part by blunting their activation and infiltration. Ligands that engage Toll-like receptors (TLR) on T cells and antigen-presenting cells can act as potent immune adjuvants. In this study, we show how tumor-reactive T cells engineered to secrete bacterial flagellin, a TLR5 ligand (TLR5L), can engender a co-stimulatory signal that augments antitumor activity. Human T cells engineered to express TLR5L along with DMF5, a T cell receptor that recognizes the melanoma antigen MART-127-35 (DMF5TLR5L T cells), displayed increased proliferation, cytokine production and cytolytic activity against melanoma cells. In a xenogenetic model, adoptive transfer of DMF5TLR5L T cells reduced tumor growth kinetics and prolonged mouse survival. In a syngeneic model, similarly engineered melanoma-reactive T cells (pmelTLR5L) displayed a relative increase in antitumor activity against established tumors, compared to unmodified T cells. In this model, we documented increased T cell infiltration associated with increased levels of CCR1 and CXCR3 levels on T cells, a reduction in PD1+Lag3+ T cells and CD11+Gr1+ myeloid-derived suppressor cells, and changes in the chemokine/cytokine profile of tumors. Our findings show how T cell-mediated delivery of a TLR agonist to the tumor site can contribute to antitumor efficacy, in the context of adoptive T cell immunotherapy.

Acute myelogenous leukemia (AML) is a heterogeneous disorder characterized by clonal proliferation of stem cell-like blasts in bone marrow (BM); however, their unique cellular interaction within the BM microenvironment and its functional significance remain unclear. Here, we assessed the BM microenvironment of AML patients and demonstrate that the leukemia stem cells induce a change in the transcriptional programming of the normal mesenchymal stromal cells (MSC). The modified leukemic niche alters the expressions of cross-talk molecules (i.e., CXCL12 and JAG1) in MSCs to provide a distinct cross-talk between normal and leukemia cells, selectively suppressing normal primitive hematopoietic cells while supporting leukemogenesis and chemoresistance. Of note, AML patients exhibited distinct heterogeneity in the alteration of mesenchymal stroma in BM. The distinct pattern of stromal changes in leukemic BM at initial diagnosis was associated with a heterogeneous posttreatment clinical course with respect to the maintenance of complete remission for 5 to 8 years and early or late relapse. Thus, remodeling of mesenchymal niche by leukemia cells is an intrinsic self-reinforcing process of leukemogenesis that can be a parameter for the heterogeneity in the clinical course of leukemia and hence serve as a potential prognostic factor. Cancer Res; 75(11); 1–10. ©2015 AACR.

Acute myeloid leukemia (AML) is a heterogeneous disorder characterized by clonal proliferation of stem cell-like blasts in bone marrow; however, their unique cellular interaction within the bone marrow microenvironment and its functional significance remain unclear. Here, we assessed the bone marrow microenvironment of AML patients and demonstrate that the leukemia stem cells induce a change in the transcriptional programming of the normal mesenchymal stromal cells. The modified leukemic niche alters the expressions of cross-talk molecules (i.e., CXCL-12 and Jagged-1) in mesenchymal stromal cells to provide a distinct cross-talk between normal and leukemia cells, selectively suppressing normal primitive hematopoietic cells while supporting leukemogenesis and chemoresistance. Of note, AML patients exhibited distinct heterogeneity in the alteration of mesenchymal stroma in bone marrow. The distinct pattern of stromal changes in leukemic bone marrow at initial diagnosis was associated with a heterogeneous post-treatment clinical course with respect to the maintenance of complete remission for 5-8 years and early or late relapse. Thus, remodeling of mesenchymal niche by leukemia cells is an intrinsic self-reinforcing process of leukemogenesis that can be a parameter for the heterogeneity in the clinical course of leukemia and hence serve as a potential prognostic factor.

Assessing the functional significance of novel putative oncogenes remains a significant challenge given the limitations of current loss-of-function tools. Here, we describe a method that employs TALEN or CRISPR/Cas9-mediated knock-in of inducible degron tags (Degron-KI) that provides a versatile approach for the functional characterization of novel cancer genes and addresses many of the shortcomings of current tools. The Degron-KI system allows for highly specific, inducible, and allele-targeted inhibition of endogenous protein function, and the ability to titrate protein depletion with this system is able to better mimic pharmacologic inhibition compared with RNAi or genetic knockout approaches. The Degron-KI system was able to faithfully recapitulate the effects of pharmacologic EZH2 and PI3Kα inhibitors in cancer cell lines. The application of this system to the study of a poorly understood putative oncogene, SF3B1, provided the first causal link between SF3B1 hotspot mutations and splicing alterations. Surprisingly, we found that SF3B1-mutant cells are not dependent upon the mutated allele for in vitro growth, but instead depend upon the function of the remaining wild-type alleles. Collectively, these results demonstrate the broad utility of the Degron-KI system for the functional characterization of cancer genes. Cancer Res; 75(10); 1–10. ©2015 AACR.