SUMMARY Therapeutic blocking of the PD1 pathway results in significant tumor responses but resistance is common. We demonstrate that prolonged interferon signaling orchestrates PDL1-dependent and PDL1-independent resistance to immune checkpoint blockade (ICB), and to combinations such as radiation plus anti-CTLA4. Persistent type II interferon signaling allows tumors to acquire STAT1-related epigenomic changes and augments expression of interferon-stimulated genes and ligands for multiple T cell inhibitory receptors. Both type I and II interferons maintain this resistance program. Crippling the program genetically or pharmacologically interferes with multiple inhibitory pathways, and expands distinct T cell populations with improved function despite expressing markers of severe exhaustion. Consequently, tumors resistant to multi-agent ICB are rendered responsive to ICB monotherapy. Finally, we observe that biomarkers for interferon-driven resistance associate with clinical progression after anti-PD1 therapy. Thus, the duration of tumor interferon signaling augments adaptive resistance and inhibition of the interferon response bypasses requirements for combinatorial ICB therapies.

SUMMARY Stromal communication with cancer cells can influence treatment response. We show that stromal and breast cancer (BrCa) cells utilize paracrine and juxtacrine signaling to drive chemotherapy and radiation resistance. Upon heterotypic interaction, exosomes are transferred from stromal to BrCa cells. RNA within exosomes, which are largely non-coding transcripts and transposable elements, stimulates the pattern recognition receptor RIG-I to activate STAT1-dependent anti-viral signaling. In parallel, stromal cells also activate NOTCH3 on BrCa cells. The paracrine anti-viral and juxtacrine NOTCH3 pathways converge as STAT1 facilitates transcriptional responses to NOTCH3 and expands therapy resistant tumor-initiating cells. Primary human and/or mouse BrCa analysis support the role of anti-viral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance, which is abrogated by combination therapy with gamma secretase inhibitors. Thus, stromal cells orchestrate an intricate cross-talk with BrCa cells by utilizing exosomes to instigate anti-viral signaling. This expands BrCa subpopulations adept at resisting therapy and re-initiating tumor growth.

Interactions between stromal fibroblasts and cancer cells generate signals for cancer progression, therapy resistance, and inflammatory responses. Although endogenous RNAs acting as damage-associated molecular patterns (DAMPs) for pattern recognition receptors (PRRs) may represent one such signal, these RNAs must remain unrecognized under non-pathological conditions. We show that triggering of stromal NOTCH-MYC by breast cancer cells results in a POL3-driven increase in RN7SL1, an endogenous RNA normally shielded by RNA binding proteins SRP9/14. This increase in RN7SL1 alters its stoichiometry with SRP9/14 and generates unshielded RN7SL1 in stromal exosomes. After exosome transfer to immune cells, unshielded RN7SL1 drives an inflammatory response. Upon transfer to breast cancer cells, unshielded RN7SL1 activates the PRR RIG-I to enhance tumor growth, metastasis, and therapy resistance. Corroborated by evidence from patient tumors and blood, these results demonstrate that regulation of RNA unshielding couples stromal activation with deployment of RNA DAMPs that promote aggressive features of cancer. VIDEO ABSTRACT.

Breast cancer cells can achieve protection from DNA damage both through cell-autonomous mechanisms and intercellular communication with the tumor microenvironment. Previously, we described an Interferon-Related DNA Damage Resistance Signature comprising of a network of interferon-stimulated genes (ISGs) that promotes and clinically predicts chemotherapy and radiation resistance in breast cancer. Here, we examine the heterotypic tumor-stroma interactions that regulate ISGs. We show that STAT1 and other ISGs are induced in breast cancer cells following interaction with stroma. STAT1 induces NOTCH3 expression, explaining a requirement for juxtacrine signaling. Moreover, we report that stroma upregulates ISG expression in breast cancer cells through exosomes. Exosomes transferred from the stroma and ISG induction are both dependent on RAB27B. In mice, targeting these pathways abrogate stroma-mediated resistance and results in long-term tumor-free survival. Analysis of primary human tumors supports the role of anti-viral/NOTCH3 pathways in NOTCH signaling and stroma-mediated resistance. To determine whether these observations can further predict clinical efficacy, cell lines derived from a genetically engineered mouse model for p53-induced breast cancer, K14cre;Brca1F/F;p53F/F, were tested. We show that stroma induces breast cancer ISGs through exosomes, which in turn can induce signaling to activate NOTCH3 and regulate DNA damage resistance. Citation Format: Tony J. Wu, Barzin Y. Nabet, Bihui Xu, Mirjam C. Boelens, Jos Jonkers, Andy J. Minn. Exosome transfer from stromal to breast cancer cells regulates therapy resistance pathways in triple-negative breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A45.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplasia, with macrophages as one of the most abundant, multifunctional immune cell populations in the tumor microenvironment (TME) and a major component of the immune infiltrate. To best improve clinical outcomes, consideration must be placed on targeted therapeutics that blunt heterotypic interactions between tumor cells and supporting cells within the TME. Here, we provide new insight into the dichotomous relationship between epithelial and mesenchymal phenotypes of PDAC cells in 3D culture. We report the ability of PDAC mesenchymal cells to form vascular mimicry-like structures in a 3D in vitro assay of invasion. Additionally, we demonstrate that macrophages have the ability to impart a proinvasive phenotype to PDAC cells when co-cultured in 3D, irrespective of EMT (epithelial-to-mesenchymal transition) status. To elucidate a mechanism for this macrophage-mediated proinvasive phenotype, we employed an unbiased, multi-omics approach. First, PDAC cell lines and primary macrophages were CTAP (cell-type specific labeling using amino acid precursors) labeled and admixed together for a prolonged period of time. To identify cell of origin of novel RNA and proteins, these mixed co-cultures were FACS sorted for downstream RNA-sequencing analysis or harvested in bulk for downstream proteome and secretome analysis. Preliminary integration of cell culture transcriptomes with CTAP-TMT (tandem mass tag) proteomes and secretomes implicates several key epithelial- and macrophage-derived signaling molecules as principal instructing signals for mediating the observed proinvasive phenotype. Blockade of this signaling axis, by inhibition of the signaling molecules or their receptors, disrupted the crosstalk between the two cell types and impaired the ability of macrophages to impart a proinvasive phenotype to PDAC cells. These preliminary preclinical data, recapitulated in vivo, suggest that targeting the TME of patients by using a staging regimen of chemokine inhibitor to disrupt tumor-macrophage cell interactions, followed with therapeutic modalities such as gemcitabine (and/or immunotherapy), would improve upon standard of care and inform future treatment options for PDAC in the clinic. Citation Format: Tony J. Wu, Oliver Cast, Danish Memon, Alejandro Jimenez-Sanchez, Anne Machel, Sebastian Kehrloesser, Michael B. Gill, Martin L. Miller. Identifying mechanisms of macrophage-mediated metastasis and therapy resistance in PDAC [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C65.

Background: The Circulating Cell-free Genome Atlas study (NCT02889978) is a multi-center, case-control, observational study with longitudinal follow-up (n=15,254; 56% cancer, 44% non-cancer) to support development of a cell-free DNA (cfDNA) multi-cancer early detection test. Previously, we reported that a targeted methylation assay detected and localized >20 cancer types at >99% specificity in individuals with cancer.1,2 Here, we report prediction of cancer (presence/absence) and tissue of origin (TOO) in individuals enrolled with clinical suspicion of cancer but without pathologic diagnosis or treatment at time of enrollment. Methods: Plasma cfDNA from blood samples collected prior to clinical diagnosis was subjected to targeted methylation sequencing. Samples were divided into a training set and an independent validation set to train and validate a machine learning classifier to assess cancer and predict TOO. Performance was assessed in a subset of participants enrolled with suspicion of cancer; subsequently, cancer was confirmed by evaluating a pathologic specimen. Results: Participants being evaluated for suspicion of cancer were classified as confirmed cancer (>20 cancer types; n=164 in training, n=75 in validation) or confirmed non-cancer (n=49 training, n=15 validation). In the confirmed non-cancer group, all training and validation samples were correctly predicted as non-cancer (100% specificity). In the confirmed cancer group, cancer detection across all stages was 40.2% (66/164; 95% confidence interval [CI], 32.7-48.2%) in training and 46.7% (35/75; 95% CI, 35.1-58.6%) in validation. Excluding stage I renal cancers (where detection/tumor fraction is low in plasma and which comprised 20% of participants in this subset) detection across stages was 50.4% (66/131; 95% CI, 41.5-59.2%) and 59.3% (35/59; 95% CI, 45.7-71.9%), respectively. In stages II and above, detection was 70.7% (58/82; 95% CI, 59.6-80.3%) and 78.9% (30/38; 95% CI, 62.7-90.4%), respectively. For detected cancers, TOO was predicted in 93.9% (62/66) samples in training and 100% (35/35) in validation. Of those with a TOO call, accuracy was 85.5% (53/62; 95% CI, 74.2-93.1%) and 97.1% (34/35; 95% CI, 85.1-99.9%), respectively. Conclusion: A cfDNA multi-cancer detection test has shown the potential to predict cancer and TOO in individuals with suspicion of cancer ahead of histologic diagnosis with performance comparable to those with confirmed cancer at the time of blood collection. This was achieved with high specificity and TOO accuracy. The high specificity suggests that the false positive rate could be comparable in populations with average versus higher risk (suspicion) of cancer. These findings suggest that a cfDNA multi-cancer detection test could accelerate the diagnostic resolution of suspicion of cancer. References: 1. Oxnard GR, et al. ASCO Breakthrough Meeting 2019; Abstract 44. 2. Oxnard GR, et al. ESMO Annual Meeting 2019; Abstract 5639. Citation Format: David D. Thiel, Xiaoji Chen, Kathryn N. Kurtzman, Jessica Yecies, Tony Wu, Quan Zhang, Hai Liu, Nan Zhang, Eric T. Fung, Michael V. Seiden, Minetta C. Liu, Geoffrey R. Oxnard, Earl Hubbell, Alexander M. Aravanis, Anne-Renee Hartman, Eric A. Klein. Prediction of cancer and tissue of origin in individuals with suspicion of cancer using a cell-free DNA multi-cancer early detection test [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr CT021.

Background: Pharmacodynamic (PD) biomarkers are an increasingly valuable tool for decision-making and prioritization of lead compounds during preclinical and clinical studies as they link drug-target inhibition in cells with biological activity. They are of particular importance for novel, first-in-class mechanisms, where the ability of a targeted therapeutic to impact disease outcome is unknown. We recently discovered CB-5083, a novel small molecule inhibitor of p97, a protein involved in several facets of protein homeostasis, including ubiquitin-dependent protein degradation, endoplasmic reticulum-associated degradation (ERAD) and autophagy. Accumulation of K48 poly-ubiquitinated proteins is a hallmark of protein degradation inhibition, and we used this proximal biomarker to follow CB-5083 target engagement on p97 in various pre-clinical models. Results: CB-5083 is a potent inhibitor of p97, with a biochemical IC50 of 11 nM. When cancer cells are exposed to CB-5083, biological consequences linked to p97 inhibition are detected, including ERAD inhibition, ER (endoplasmic reticulum) stress, ER stress-mediated cell death and accumulation of poly-ubiquitinated proteins. In mouse models, CB-5083 is orally bio-available and causes rapid and sustained accumulation of K48 poly-ubiquitin in tumor xenografts after a single administration. Concurrent with increases in K48 poly-ubiquitin levels, activation of ER stress response pathways and induction of apoptosis markers are also observed. Accumulation of K48 poly-ubiquitin also occurs in other tissues in the body and we developed a quantitative method to detect accumulation of K48 poly-ubiquitin as a marker of target engagement in whole blood. With this method, we were able to compare the kinetics and level of K48 poly ubiquitin accumulation following CB-5083 administration in both tumor and whole blood. We also performed a dose escalation of CB-5083 in cynomolgus monkeys and defined the minimal plasma AUC and Cmax required to see accumulation of K48 poly-ubiquitin in monkey whole blood. This approach allowed us to predict human exposures that should lead to target engagement and consequent biological activity in our ongoing phase 1 studies where a flow cytometry based assay is being used to monitor K48 poly-ubiquitin levels in patient blood samples. Conclusion: K48 poly-ubiquitin is a target engagement biomarker of p97 inhibition. CB-5083, a p97 inhibitor, can induce sustained induction of K48 poly-ubiquitin, not only in tumor but also in surrogate tissues such as whole blood. K48 poly-ubiquitin accumulation is currently being assessed to follow target engagement in the blood of patients in our ongoing phase 1 dose escalation studies of CB-5083. Citation Format: Mary-Kamala MENON, Ferdie SORIANO, Steve WONG, Eduardo VALLE, Stevan Djakovic, Brajesh KUMAR, Bing YAO, Antonett MADRIAGA, Tony WU, Julie RICE, Jinhai WANG, Alessandra CESANO, Laura SHAWVER, Han-Jie ZHOU, David WUSTROW, Daniel ANDERSON, Mark ROLFE, Ronan LE MOIGNE. Utilization of K48 poly-ubiquitin modulation as a biomarker of target engagement for a protein homeostasis inhibitor in the clinic: Preclinical validation with CB-5083, a first-in-class inhibitor of the AAA ATPase p97. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4541.

e15705 Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal, incurable disease. Macrophages are one of the most abundant, multifunctional immune cell populations in the tumor microenvironment and a major component of the immune infiltrate in many solid tumors. Methods: Employing a multi-omics approach, PDAC cell lines and primary macrophages were CTAP (cell-type specific labelling using amino acid precursors)-labelled and admixed together for a prolonged period of time. To identify cell-of-origin of novel RNA and proteins, these mixed co-cultures were FACS sorted for downstream RNA-sequencing analysis or harvested in bulk for downstream proteome and secretome analysis. Results: Here, we provide new insight into the dichotomous relationship between epithelial and mesenchymal phenotypes of PDAC cells in 3D culture. We report the ability of PDAC mesenchymal cells to form vascular mimicry-like structures in a 3D in vitro assay of invasion. Additionally, we demonstrate that macrophages have the ability to impart a pro-invasive phenotype to PDAC cells when co-cultured in 3D, regardless of EMT (epithelial-to-mesenchymal transition) status. Preliminary integration of cell culture transcriptomes with CTAP-TMT proteomes and secretomes implicates several key epithelial- and macrophage-derived signalling molecules as principal instructing signals for mediating the observed pro-invasive phenotype. Conclusions: Blockade of these signalling molecules or their receptors disrupted the crosstalk between PDAC cells and macrophages within the tumor microenvironment and impaired the ability of macrophages to induce a pro-invasive phenotype to PDAC cells.