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We have shown that KRAS–TP53 genomic coalteration is associated with immune-excluded microenvironments, chemoresistance, and poor survival in pancreatic ductal adenocarcinoma (PDAC) patients. By treating KRAS–TP53 cooperativity as a model for high-risk biology, we now identify cell-autonomous Cxcl1 as a key mediator of spatial T-cell restriction via interactions with CXCR2+ neutrophilic myeloid-derived suppressor cells in human PDAC using imaging mass cytometry. Silencing of cell-intrinsic Cxcl1 in LSL-KrasG12D/+;Trp53R172H/+;Pdx-1Cre/+(KPC) cells reprograms the trafficking and functional dynamics of neutrophils to overcome T-cell exclusion and controls tumor growth in a T cell–dependent manner. Mechanistically, neutrophil-derived TNF is a central regulator of this immunologic rewiring, instigating feed-forward Cxcl1 overproduction from tumor cells and cancer-associated fibroblasts (CAF), T-cell dysfunction, and inflammatory CAF polarization via transmembrane TNF–TNFR2 interactions. TNFR2 inhibition disrupts this circuitry and improves sensitivity to chemotherapy in vivo. Our results uncover cancer cell–neutrophil cross-talk in which context-dependent TNF signaling amplifies stromal inflammation and immune tolerance to promote therapeutic resistance in PDAC.Significance:By decoding connections between high-risk tumor genotypes, cell-autonomous inflammatory programs, and myeloid-enriched/T cell–excluded contexts, we identify a novel role for neutrophil-derived TNF in sustaining immunosuppression and stromal inflammation in pancreatic tumor microenvironments. This work offers a conceptual framework by which targeting context-dependent TNF signaling may overcome hallmarks of chemoresistance in pancreatic cancer.

Differentially expressed pathways comparing transcriptomes in KRAS-TP53 co-altered (n=23) and KRAS-altered/TP53WT (n=5) derived from Cancer Cell Line Encyclopedia

Purpose: Dasatinib is a dual Src/Abl inhibitor recently approved for Bcr-Abl+ leukemias with resistance or intolerance to prior therapy. Because Src kinases contribute to multiple blood cell functions by triggering a variety of signaling pathways, we hypothesized that their molecular targeting might lead to growth inhibition in acute myeloid leukemia (AML).Experimental Design: We studied growth factor–dependent and growth factor–independent leukemic cell lines, including three cell lines expressing mutants of receptor tyrosine kinases (Flt3 or c-Kit) as well as primary AML blasts for responsiveness to dasatinib.Results: Dasatinib resulted in the inhibition of Src family kinases in all cell lines and blast cells at ∼1 × 10−9 mol/L. It also inhibited mutant Flt3 or Kit tyrosine phosphorylation at ∼1 × 10−6 mol/L. Mo7e cells expressing the activating mutation (codon 816) of c-Kit were most sensitive to growth inhibition with a GI50 of 5 × 10−9 mol/L. Primary AML blast cells exhibited a growth inhibition of −6 mol/L. Cell lines that showed growth inhibition at ∼1 × 10−6 mol/L showed a G1 cell cycle arrest and correlated with accumulation of p21 and p27 protein. The addition of rapamycin or cytotoxic agents enhanced growth inhibition. Dasatinib also caused the apoptosis of Mo7e cells expressing oncogenic Kit.Conclusions: Although all of the precise targets for dasatinib are not known, this multikinase inhibitor causes either growth arrest or apoptosis in molecularly heterogeneous AML. The addition of cytotoxic or targeted agents can enhance its effects. Clin Cancer Res; 16(4); 1149–58

Supplementary Data from Dasatinib Inhibits the Growth of Molecularly Heterogeneous Myeloid Leukemias

Background: The oncolytic vaccinia virus (VV) is a promising biotherapy undergoing clinical development in human cancer, including renal cell carcinoma (RCC). While VV induces potent tumor oncolysis and immunomodulation, clinical experience has shown that OV therapy alone leads to limited benefit. Moreover, OVs and checkpoint inhibitor combinations, do not significantly improve clinical responses, underscoring the need to identify new tumor microenvironment targets to overcome OV resistance in vivo. The adenosine pathway is a biologically and clinically relevant target in RCC and is associated with adaptive resistance to immunotherapy. The objectives of this study are to characterize the role of the adenosine axis in tumor escape mechanisms to vaccinia virus in RCC, and to determine the efficacy of novel OV-immunotherapy combinations targeting adenosine. Methods: The in vitro oncolytic effects of JX-594 and mJX-594 (oncolytic VVs expressing human or murine GMCSF, respectively) in human (786-0, ACHN, CAKi-1, UOK262) and murine (RENCA) renal cancer cell lines were assessed. Proteomic analysis of 786-0 lysates treated with JX-594 was performed for mechanistic studies. The effects of JX-594 and mJX 594 on CD73 and CD39 expression were determined in human and murine RCC. In vivo antitumor effects of mJX-594 alone, and in combination with A2AR, A2BR and Dual Adenosine receptor inhibition was determined in the RENCA model in immunocompetent mice. Results: Potent in vitro oncolysis and efficient viral replication were observed in human and murine RCC cells after infection with vaccinia virus. VV oncolysis was associated with significant modulation of proliferation, survival, and ER stress pathways, as determined by functional proteomics. JX-594 and mJX-594 treatment induced increased expression of CD39 and CD73 in RCC cell lines and in renal tumors in vivo. While no significant enhancement of vaccinia oncolysis was observed with combinations with A2AR and A2BR inhibitors in vitro, a significant improvement on in vivo tumor control and survival were observed when mJX594 given intravenously was combined with A2AR and A2BR inhibitors (triplet), at a higher degree than single agents or doublets (mJX-594 in combination with single A2AR or A2BR inhibitors). Characterization of treatment mediated changes in immune signatures (nanostring) is underway and will be presented at the meeting. Conclusion: Vaccinia virus potently induces in vitro oncolysis in RCC cell lines, while also increasing expression of adenosine rate limiting enzymes in vitro and in vivo, which could explain tumor escape to oncolytic VV. MJX-594 combination with A2AR and A2BR inhibition safely and significantly improves renal cancer oncolysis and tumor control in vivo. Our studies uncover a novel, translationally relevant strategy to improve OV efficacy in vivo, in RCC and other cancers. Citation Format: Valery A. Chavez, Floritza Bustamante, Carolina Merchan-Mendes, Shannon Saigh, Patricia Guevara, Eric Wieder, Jaime Merchan. Targeting immunosuppressive adenosine to enhance vaccinia virus renal cancer oncolysis in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6792.

Adoptive T-cell therapy holds great promise for the treatment of metastatic melanoma. However, prohibitive costs associated with current technology required for culture and expansion of tumor-reactive T-cells, the need for intense preconditioning regimens to induce lymphopenia, and the unpredictable anti-tumor effect of adoptively transferred T-cells remain significant impediments for its clinical implementation. Here we report a simplified combinatorial approach that involves short activation of CD8(+) T cells in the presence of IL-12 followed by adoptive transfer into tumor bearing animals after a single injection of cyclophosphamide. This approach resulted in complete eradication of B16 melanoma, and the establishment of long term immunological memory capable of fully protecting mice after a second B16 melanoma challenge. The activated donor cells were unique because they simultaneously exhibited traits for cytotoxic effector function, central memory-like, homing, and senescence. After tumor eradication and within three months after transfer, CD8+ cells exhibited a conventional memory CTL phenotype. Moreover, these memory CTLs acquired functional attributes characteristic of memory stem cells, including the ability to resist chemotherapy-induced toxicity. Our results suggest that short-term T-cell receptor signaling in the presence of IL-12 promotes promiscuous qualities in naïve CTL which - upon transfer into lymphopenic hosts- are sufficient to eradicate tumors and generate life-long tumor-specific memory.