Lorenzo Tomassoni

Abstract Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations 1,2 . RMC-7977 is a highly selective inhibitor of the active GTP-bound forms of KRAS, HRAS and NRAS, with affinity for both mutant and wild-type variants 3 . More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS 4 . Here we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models. We observed broad and pronounced anti-tumour activity across models following direct RAS inhibition at exposures that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumour versus normal tissues. Treated tumours exhibited waves of apoptosis along with sustained proliferative arrest, whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. In the autochthonous KPC mouse model, RMC-7977 treatment resulted in a profound extension of survival followed by on-treatment relapse. Analysis of relapsed tumours identified Myc copy number gain as a prevalent candidate resistance mechanism, which could be overcome by combinatorial TEAD inhibition in vitro. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS-GTP inhibition in the setting of PDAC and identify a promising candidate combination therapeutic regimen to overcome monotherapy resistance.

PG-M3 is a new monoclonal antibody (MoAb) specifically directed against a peptide sequence located in the aminoterminal region of the human PML protein. PML gene fuses with the retinoic acid receptor alpha (RAR alpha) gene during the t(15; 17) chromosomal translocation of acute promyelocytic leukemia (APL). The epitope recognized by PG-M3 is species-specific and fixative-resistant and is shared by most PML isoforms and PML/RAR alpha fusion proteins. PML is consistently located within the nucleus, although a minority of cells (about 20%), both in vitro and in vivo, show positivity for PML also in the cytoplasm. The nuclear staining pattern of PG-M3 varies from speckled (cells other than APL) to micropunctate (APL cells). Although two physiologically expressed PML isoforms are detectable by immunocytochemistry only or predominantly in the cytoplasm of transfected cells, the cytoplasmic localization of PML is a property also shared by the PML isoforms that predominantly localize to the nuclei. Immunohistologic analysis of normal human tissues with the PG-M3 MoAb showed variable PML expression, with the highest levels of the protein in postmitotic, differentiated cell types, such as endothelial cells, epithelia, and tissue macrophages, especially activated ones. In keeping with this in vivo finding, PML appears strongly upregulated in the U937 promonocyte cell line after exposure to agents that induce monocyte/macrophage activation (interferon gamma) or maturation (vitamin D3 and transforming growth factor beta 1).

The sparse vascularity of pancreatic ductal adenocarcinoma (PDAC) presents a mystery: What prevents this aggressive malignancy from undergoing neoangiogenesis to counteract hypoxia and better support growth? An incidental finding from prior work on paracrine communication between malignant PDAC cells and fibroblasts revealed that inhibition of the Hedgehog (HH) pathway partially relieved angiosuppression, increasing tumor vascularity through unknown mechanisms. Initial efforts to study this phenotype were hindered by difficulties replicating the complex interactions of multiple cell types in vitro. Here we identify a cascade of paracrine signals between multiple cell types that act sequentially to suppress angiogenesis in PDAC. Malignant epithelial cells promote HH signaling in fibroblasts, leading to inhibition of noncanonical WNT signaling in fibroblasts and epithelial cells, thereby limiting VEGFR2-dependent activation of endothelial hypersprouting. This cascade was elucidated using human and murine PDAC explant models, which effectively retain the complex cellular interactions of native tumor tissues. SIGNIFICANCE: We present a key mechanism of tumor angiosuppression, a process that sculpts the physiologic, cellular, and metabolic environment of PDAC. We further present a computational and experimental framework for the dissection of complex signaling cascades that propagate among multiple cell types in the tissue environment. This article is featured in Selected Articles from This Issue, p. 201.

ABSTRACT Despite extensive efforts, reproducible assessment of pancreatic ductal adenocarcinoma (PDA) heterogeneity and plasticity at the single cell level remains elusive. Systematic, network-based analysis of regulatory protein activity in single cells identified three PDA Developmental Lineages (PDLs), coexisting in virtually all tumors, whose transcriptional states are mechanistically driven by aberrant activation of Master Regulator (MR) proteins associated with gastrointestinal lineages (GLS state), morphogen and EMT pathways (MOS state), and acinar-to-ductal metaplasia (ALS state), respectively. Each PDL is further subdivided into sub-states characterized by low vs. high MAPK pathway activity. This taxonomy was remarkably conserved across multiple cohorts, cell lines, and PDX models, and harmonized with bulk profile analyses. Cross-state plasticity and MR essentiality was confirmed by barcode-based lineage tracing and CRISPR/Cas9 assays, respectively, while MR ectopic expression induced PDL transdifferentiation. Together these data provide a mechanistic foundation for PDA heterogeneity and a roadmap for targeting PDA cellular subtypes.