Tumour-selective activity of RAS-GTP inhibition in pancreatic cancer.

Broad-spectrum RAS inhibition has the potential to benefit roughly a quarter of human patients with cancer whose tumours are driven by RAS mutations1,2. 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 variants3. More than 90% of cases of human pancreatic ductal adenocarcinoma (PDAC) are driven by activating mutations in KRAS4. 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.

Pharmacological reactivation of inactive X-linked Mecp2 in cerebral cortical neurons of living mice.

Rett syndrome (RTT) is a genetic disorder resulting from a loss-of-function mutation in one copy of the X-linked gene methyl-CpG-binding protein 2 (MECP2). Typical RTT patients are females and, due to random X chromosome inactivation (XCI), ∼50% of cells express mutant MECP2 and the other ∼50% express wild-type MECP2. Cells expressing mutant MECP2 retain a wild-type copy of MECP2 on the inactive X chromosome (Xi), the reactivation of which represents a potential therapeutic approach for RTT. Previous studies have demonstrated reactivation of Xi-linked MECP2 in cultured cells by biological or pharmacological inhibition of factors that promote XCI (called "XCI factors" or "XCIFs"). Whether XCIF inhibitors in living animals can reactivate Xi-linked MECP2 in cerebral cortical neurons, the cell type most therapeutically relevant to RTT, remains to be determined. Here, we show that pharmacological inhibitors targeting XCIFs in the PI3K/AKT and bone morphogenetic protein signaling pathways reactivate Xi-linked MECP2 in cultured mouse fibroblasts and human induced pluripotent stem cell-derived postmitotic RTT neurons. Notably, reactivation of Xi-linked MECP2 corrects characteristic defects of human RTT neurons including reduced soma size and branch points. Most importantly, we show that intracerebroventricular injection of the XCIF inhibitors reactivates Xi-linked Mecp2 in cerebral cortical neurons of adult living mice. In support of these pharmacological results, we also demonstrate genetic reactivation of Xi-linked Mecp2 in cerebral cortical neurons of living mice bearing a homozygous XCIF deletion. Collectively, our results further establish the feasibility of pharmacological reactivation of Xi-linked MECP2 as a therapeutic approach for RTT.

Tumor Explants Elucidate a Cascade of Paracrine SHH, WNT, and VEGF Signals Driving Pancreatic Cancer Angiosuppression.

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.

Tumor-selective effects of active RAS inhibition in pancreatic ductal adenocarcinoma.

Broad-spectrum RAS inhibition holds the potential to benefit roughly a quarter of human cancer patients whose tumors are driven by RAS mutations. However, the impact of inhibiting RAS functions in normal tissues is not known. 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 (WT) variants. As >90% of human pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, we assessed the therapeutic potential of RMC-7977 in a comprehensive range of PDAC models, including human and murine cell lines, human patient-derived organoids, human PDAC explants, subcutaneous and orthotopic cell-line or patient derived xenografts, syngeneic allografts, and genetically engineered mouse models. We observed broad and pronounced anti-tumor activity across these models following direct RAS inhibition at doses and concentrations that were well-tolerated in vivo. Pharmacological analyses revealed divergent responses to RMC-7977 in tumor versus normal tissues. Treated tumors exhibited waves of apoptosis along with sustained proliferative arrest whereas normal tissues underwent only transient decreases in proliferation, with no evidence of apoptosis. Together, these data establish a strong preclinical rationale for the use of broad-spectrum RAS inhibition in the setting of PDAC.

Visualization of Xist Long Noncoding RNA with a Fluorescent CRISPR/Cas9 System.

X-inactive specific transcript (Xist) is a long noncoding RNA that is essential for initiating and maintaining epigenetic silencing of one copy of the X chromosome in mammalian females. But the mechanism by which Xist localizes and spreads on the X chromosome and facilitates transcriptional silencing remains largely unknown. This limited understanding, at least in part, is due to the technical difficulties in the visualization and functional characterization of Xist. Development of a successful method for Xist tracking is a key to better understanding of the X chromosome silencing, as well as to gain insight into the regulatory role of other long noncoding RNAs. Here, we describe an alternative method for visualization of Xist lncRNA in cells using a CRISPR/Cas9-based approach. This strategy is relatively simple approach to track Xist at different stages of cell differentiation, providing mechanistic insights into the initiation, maintenance, and establishment of X inactivation.

A Non-random Mouse Model for Pharmacological Reactivation of Mecp2 on the Inactive X Chromosome.

X chromosome inactivation (XCI) is the random silencing of one X chromosome in females to achieve gene dosage balance between the sexes. As a result, all females are heterozygous for X-linked gene expression. One of the key regulators of XCI is Xist, which is essential for the initiation and maintenance of XCI. Previous studies have identified 13 trans acting X chromosome inactivation factors (XCIFs) using a large-scale, loss-of-function genetic screen. Inhibition of XCIFs, such as ACVR1 and PDPK1, using short-hairpin RNA or small molecule inhibitors, reactivates X chromosome-linked genes in cultured cells. But the feasibility and tolerability of reactivating the inactive X chromosome in vivo remains to be determined. Towards this goal, a XistΔ:Mecp2/Xist:Mecp2-Gfp mouse model has been generated with non-random XCI due to deletion of Xist on one X chromosome. Using this model, the extent of inactive X reactivation was quantitated in the mouse brain following treatment with XCIF inhibitors. Recently published results show, for the first time, that pharmacological inhibition of XCIFs reactivates Mecp2 from the inactive X chromosome in cortical neurons of the living mouse brain.

Anti-FGFR1 aptamer-tagged superparamagnetic conjugates for anticancer hyperthermia therapy.

Compounds that recognize and strongly bind to molecular targets are one of the cornerstones of modern pharmaceutics. Work has been ongoing for the past 25 years on the therapeutic use of aptamers, nucleic acid molecules, whose three-dimensional structure is the result of interactions between complementary base pairs. The aptamers selection methods allow the oligonucleotides which bind the molecular target in its native environment to be quickly isolated from a large library of random oligonucleotides. The possibilities presented for aptamers in the field of targeted therapy require the application of effective carriers to counter the renal clearance effect and/or functional cargo to exert therapeutic action if the aptamer is only used as a targeting moiety. Lately, a material gaining ground in biomedical research is iron oxide particles, which exhibit a superparamagnetic characteristic at nanoscale levels. This allows the iron oxide nanoparticles to convert external magnetic energy into heat, a mechanism known as hyperthermy, and efficiently supports conventional oncological treatment. In this study, we describe an experimentally confirmed functional model of targeted anticancer hyperthermia therapy. Using the systematic evolution of ligands by exponential enrichment technique, we selected a DNA aptamer that specifically binds to the extracellular domain of recombinant fibroblast growth factor receptor type-1 (FGFR1) with a nanomolar dissociation constant. The chosen target plays an important role in many crucial cellular processes and is also considered a candidate protein that is involved in tumor initiation, survival and progression. Next, we combined the selected aptamer with iron oxide nanoparticles to produce aptamer superparamagnetic conjugates (ASCs). Finally, we found that targeted ASCs selectively destroy FGFR1-overexpressing human osteosarcoma cells U2OS upon magnetic field irradiation.