Acquired cancer resistance to combination immunotherapy from transcriptional loss of class I HLA.

Understanding mechanisms of late/acquired cancer immunotherapy resistance is critical to improve outcomes; cellular immunotherapy trials offer a means to probe complex tumor-immune interfaces through defined T cell/antigen interactions. We treated two patients with metastatic Merkel cell carcinoma with autologous Merkel cell polyomavirus specific CD8+ T cells and immune-checkpoint inhibitors. In both cases, dramatic remissions were associated with dense infiltration of activated CD8+s into the regressing tumors. However, late relapses developed at 22 and 18 months, respectively. Here we report single cell RNA sequencing identified dynamic transcriptional suppression of the specific HLA genes presenting the targeted viral epitope in the resistant tumor as a consequence of intense CD8-mediated immunologic pressure; this is distinguished from genetic HLA-loss by its reversibility with drugs. Transcriptional suppression of Class I loci may underlie resistance to other immunotherapies, including checkpoint inhibitors, and have implications for the design of improved immunotherapy treatments.

WNT/ß-catenin signaling regulates mitochondrial activity to alter the oncogenic potential of melanoma in a PTEN-dependent manner.

Aberrant regulation of WNT/ß-catenin signaling has a crucial role in the onset and progression of cancers, where the effects are not always predictable depending on tumor context. In melanoma, for example, models of the disease predict differing effects of the WNT/ß-catenin pathway on metastatic progression. Understanding the processes that underpin the highly context-dependent nature of WNT/ß-catenin signaling in tumors is essential to achieve maximal therapeutic benefit from WNT inhibitory compounds. In this study, we have found that expression of the tumor suppressor, phosphatase and tensin homolog deleted on chromosome 10 (PTEN), alters the invasive potential of melanoma cells in response to WNT/ß-catenin signaling, correlating with differing metabolic profiles. This alters the bioenergetic potential and mitochondrial activity of melanoma cells, triggered through regulation of pro-survival autophagy. Thus, WNT/ß-catenin signaling is a regulator of catabolic processes in cancer cells, which varies depending on the metabolic requirements of tumors.

WNT7B mediates autocrine Wnt/ß-catenin signaling and anchorage-independent growth in pancreatic adenocarcinoma.

Developmental and cancer models show Wnt/ß-catenin-dependent signaling mediates diverse phenotypic outcomes in the pancreas that are dictated by context, duration and strength of activation. While generally assumed to be pro-tumorigenic, it is unclear to what extent dysregulation of Wnt/ß-catenin signaling impacts tumor progression in pancreatic adenocarcinoma (PDAC). In the present study, Wnt/ß-catenin activity was characterized across a spectrum of PDAC cell lines and primary tumors. Reporter and gene expression-based assays revealed wide heterogeneity in Wnt/ß-catenin transcriptional activity across PDAC cell lines and patient tumors, as well as variable responsiveness to exogenous Wnt ligand stimulation. An experimentally generated, pancreas-specific gene expression signature of Wnt/ß-catenin transcriptional activation was used to stratify pathway activation across a cohort of resected, early-stage PDAC tumors (N=41). In this cohort, higher Wnt/ß-catenin activation was found to significantly correlate with lymphvascular invasion and worse disease-specific survival (median survival time 20.3 versus 43.9 months, log-rank P=0.03). Supporting the importance of Wnt ligand in mediating autocrine Wnt signaling, Wnt/ß-catenin activity was significantly inhibited in PDAC cell lines by WLS gene silencing and the small-molecule inhibitor IWP-2, both of which functionally block Wnt ligand processing and secretion. Transcriptional profiling revealed elevated expression of WNT7B occurred in PDAC cell lines with high levels of cell autonomous Wnt/ß-catenin activity. Gene-knockdown studies in AsPC-1 and HPAF-2 cell lines confirmed WNT7B-mediated cell autonomous Wnt/ß-catenin activation, as well as an anchorage-independent growth phenotype. Our findings indicate WNT7B can serve as a primary determinant of differential Wnt/ß-catenin activation in PDAC. Disrupting the interaction between Wnt ligands and their receptors may be a particularly suitable approach for therapeutic modulation of Wnt/ß-catenin signaling in PDAC and other cancer contexts where Wnt activation is mediated by ligand expression rather than mutations in canonical pathway members.

The neurofibromatosis-2 homologue, Merlin, and the tumor suppressor expanded function together in Drosophila to regulate cell proliferation and differentiation.

Neurofibromatosis-2 is an inherited disorder characterized by the development of benign schwannomas and other Schwann-cell-derived tumors associated with the central nervous system. The Neurofibromatosis-2 tumor suppressor gene encodes Merlin, a member of the Protein 4.1 superfamily most closely related to Ezrin, Radixin and Moesin. This discovery suggested a novel function for Protein 4.1 family members in the regulation of cell proliferation; proteins in this family were previously thought to function primarily to link transmembrane proteins to underlying cortical actin. To understand the basic cellular functions of Merlin, we are investigating a Drosophila Neurofibromatosis-2 homologue, Merlin. Loss of Merlin function in Drosophila results in hyperplasia of the affected tissue without significant disruptions in differentiation. Similar phenotypes have been observed for mutations in another Protein 4.1 superfamily member in Drosophila, expanded. Because of the phenotypic and structural similarities between Merlin and expanded, we asked whether Merlin and Expanded function together to regulate cell proliferation. In this study, we demonstrate that recessive loss of function of either Merlin or expanded can dominantly enhance the phenotypes associated with mutations in the other. Consistent with this genetic interaction, we determined that Merlin and Expanded colocalize in Drosophila tissues and cells, and physically interact through a conserved N-terminal region of Expanded, characteristic of the Protein 4.1 family, and the C-terminal domain of Merlin. Loss of function of both Merlin and expanded in clones revealed that these proteins function to regulate differentiation in addition to proliferation in Drosophila. Further genetic analyses suggest a role for Merlin and Expanded specifically in Decapentaplegic-mediated differentiation events. These results indicate that Merlin and Expanded function together to regulate proliferation and differentiation, and have implications for understanding the functions of other Protein 4.1 superfamily members.

Identification of the tobacco and Arabidopsis homologues of the pollen-expressed LAT59 gene of tomato.

We describe the complete genomic sequences for the tobacco and Arabidopsis homologues of tomato LAT59, a previously described member of a family of pectate lyase-like genes. Translation of the tobacco gene, Nt59, predicts a protein with 93.5% overall amino acid similarity to LAT59. Nt59 has two introns whose positions are exactly conserved with the two introns of LAT59. Both LAT59 and Nt59 are specifically expressed in pollen and their promoter and 5'-UTR sequences are highly similar. Furthermore, two promoter elements shown to be important for pollen expression of LAT59 are conserved in the Nt59 promoter. The Arabidopsis homologue, At59, was found by examination of four candidates. At59 has 72.6% amino acid similarity to LAT59 and the position of one of its two introns is conserved with one of the LAT59 introns. At59 is also pollen-expressed and although its promoter sequence is quite different from the Nt59 and LAT59 promoters, the two promoter elements are somewhat conserved.