Excessive Wnt signaling is associated with 1) poor prognosis in triple-negative breast cancer (TNBC) and other cancers and 2) immune checkpoint inhibitor resistance, thus limiting its therapeutic application. From our previous work and Wnt signaling principles, we devised CHA1 as an inhibitor of Wnt signaling. CHA1 combines the green tea catechin EGCG (Epigallocatechin-3-gallate) and the DNA methyltransferase inhibitor decitabine, which have been used in numerous clinical trials and/or FDA approved for other cancers, respectively. Our investigations showed that CHA1 treatment (but not EGCG or decitabine alone) reduced primary tumors and metastases in TNBC xenograft and decreased Wnt signaling. Unexpectedly, CHA1 reprogrammed intrinsic tumor properties for antigen presentation and immune cell infiltration. The implications governing tumor-immune cell interactions are discussed in the context of increasing checkpoint inhibitor susceptibility. Both Immune-compromised and immune-competent TNBC preclinical models were used for mechanistic elaboration. In CHA1-treated human TNBC tumors in immune-compromised mice, biochemical and RNA seq analyses show that Wnt signaling was decreased due to induction of Wnt pathway inhibitors (e.g., SFRP1, DKK1, HBP1). The RNA seq analysis also revealed induction of >100 genes for antigen presentation and associated processes. MHC staining of CHA1-treated tumors verified the genotypic changes, which were also accompanied by robust gamma interferon signaling. All results were recapitulated in the immune-competent 4T1 syngeneic TNBC model. We observed large increases in tumor-infiltrating CD8+ T cells with CHA1 treatment. Lastly, recent reports collated the molecularly disparate properties of a “cold-to-hot” transition, in which “hot” tumors have increased immune cell infiltration and sensitivity to checkpoint inhibitors. Remarkably, the TNBC tumors (typically “cold”) that were treated with CHA1 now exhibited the unrelated list of “hot” tumor properties: 1) epigenetic reprogramming, 2) suppressed Wnt signaling, 3) E-cadherin and epithelial marker re-expression, 4) CD8+ T-cell enrichment, 5) increased tumor antigen presentation properties, and 6) increased tumor PD-L1 expression. The CHA1 mechanism is consistent with a global reprogramming of intrinsic tumor properties, triggered by differential Wnt and interferon signaling. CHA1 engages the fundamental processes that regulate tumor-immune cell dynamics, which, in turn, govern immune cell infiltration and determine checkpoint inhibitor sensitivity. Our work additionally establishes a molecular framework for assessing compounds that engage a fundamental “cold-to-hot” tumor reprogramming and that may predict new immune checkpoint inhibitor sensitivity. Thus, CHA1 treatment may reprogram tumor-immune cell dynamics to significantly expand the spectrum of TNBC and other tumors that can be efficaciously treated with immune checkpoint inhibitors (e.g., anti-PDL1 and/or anti-PD1). Citation Format: Mariam Alamoudi, Mollie Chipman, Francesca Deleso-Frechette, Eileen Liu, Rui Zhang, Zixu Wang, K. Eric Paulsopn, Amy S. Yee. A therapeutic strategy to inhibit Wnt signaling also reprograms breast tumor-immune cell interactions: Perspectives for conferring immune checkpoint inhibitor susceptibility [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr A39.

Triple negative breast cancers (TNBC) pose exceptional challenges with fatal brain metastases as a clear and unmet need. Immune checkpoint inhibitors (ICIs) are promising therapeutic strategies, but most TNBC are resistant, or cold tumors, due to lack of tumor-resident immune engagement. No FDA-approved therapies exist which promote a cold-to-hot transition or induce the important biomarker PD-L1, often used for ICI clinical decision-making. Maximal ICI susceptibility, or a full cold-to-hot transition, requires reciprocal Wnt signaling inhibition and Jak/STAT/interferon signaling activation. We report a new compound combination (CHA1) that fits the above criteria. CHA1 is comprised of EGCG (epigallocatechin-3-gallate; green-tea compound) and decitabine (DNA-methyltransferase (DNMT1) inhibitor; 5-deaza-cytidine; FDA-approved for hematologic malignancies). We used immune-compromised and syngeneic TNBC pre-clinical models to investigate tumor-intrinsic and tumor-resident T-cell effects, respectively. All results required CHA1 (but not EGCG or decitabine alone) and utilized attainable human dose equivalences with manageable safety profiles. CHA1 triggered efficient Wnt signaling inhibition by elevating Wnt pathway inhibitors (HBP1 and SFRP1) and traversed the blood-brain barrier to reduce both tumor and brain metastatic growth. Transcriptomic and expression analyses revealed that CHA1 treatment effectuated a robust tumor-intrinsic JAK/STAT/IFN response 1) to induce PDL1 and 2) to induce antigen presentation and processing genes, including MHC-1, MHC-2 and numerous genes attributed to professional antigen-presenting cells; 3) to induce CD8+-T-cell infiltration and activation. Additionally, CHA1 pre-treatment improved anti-PDL1 efficacy in a syngeneic setting. Lastly, we derived a composite gene signature emblematic of CHA1 treatment and of a favorable clinical prognosis in-silico. Together, our work supports a model in which CHA1 influences epigenetics, Wnt and Jak/STAT/IFN signaling mechanisms, all to reprogram an epithelial-mesenchymal TNBC tumor to express antigen-presenting properties and to recruit and activate tumor-resident CD8+-T-cells. We discuss our findings in the context of cancer biology and immunity with implications for improving ICI susceptibility for TNBC.