Significance Existing targeted therapies for solid tumors harboring FGFR2 alterations include pan-FGFR inhibitors, which often cannot be dosed to maximum efficacy due to FGFR1- and FGFR4-mediated toxicities. The structural similarity among FGFR family members has thwarted conventional approaches to structure-based design of FGFR2-selective inhibitors, so we used long-timescale molecular dynamics simulations to identify differential motions of FGFR2 and FGFR1 that could be leveraged to design FGFR2-selective inhibitors. Our efforts led to lirafugratinib (RLY-4008), an FGFR2 inhibitor exhibiting substantial selectivity over other FGFRs. Lirafugratinib was reported to have a 73% objective response rate in early clinical studies in FGFR-inhibitor naive, FGFR2 fusion-positive intrahepatic cholangiocarcinoma patients treated orally (once daily doses ≥70 mg) without inducing clinically significant adverse effects by inhibiting off-targets.

Unlike pan-FGFR inhibitors, RLY-4008 was designed to be selective for FGFR2 and induces clinical responses in FGFR2-altered solid tumors without clinically significant FGFR1-mediated hyperphosphatemia and FGFR4-mediated diarrhea.

FGFR2 fusions, amplifications, and mutations are oncogenic drivers that occur across multiple tumor types. Clinical efficacy observed with pan-FGFR inhibitors has validated the driver status of FGFR2 in FGFR2 fusion-positive intrahepatic cholangiocarcinoma (ICC), however, FGFR1-mediated toxicities (hyperphosphatemia, tissue mineralization) and the emergence of on-target FGFR2 resistance mutations limit the efficacy of pan-FGFR inhibitors. To overcome these limitations, we designed RLY-4008, a potent and highly selective, FGFR2 inhibitor. Despite significant investment in traditional structure-based drug design, selective targeting of FGFR2 has not been achieved. We leveraged differences in conformational dynamics between FGFR2 and other FGFR isoforms observed through molecular dynamics simulations to enable the design of RLY-4008. RLY-4008 inhibits FGFR2 with low nanomolar potency and demonstrates > 200-fold selectivity over FGFR1, and > 80- and > 5000-fold selectivity over FGFR3 and FGFR4, respectively, in biochemical assays. Additionally, RLY-4008 demonstrates high kinome selectivity for FGFR2 against a panel of > 400 human kinases. RLY-4008 has strong activity against primary and acquired FGFR2 resistance mutations in cellular assays, and potent antiproliferative effects on FGFR2-altered human tumor cell lines. In vivo, RLY-4008 demonstrates dose-dependent FGFR2 inhibition and induces regression in multiple human xenograft tumor models, including FGFR2 fusion-positive ICC, gastric, and lung cancers, FGFR2-amplified gastric cancer, and FGFR2-mutant endometrial cancer. Strikingly, RLY-4008 induces regression in an FGFR2 fusion-positive ICC model harboring the FGFR2V564F gatekeeper mutation and an endometrial cancer model harboring the FGFR2N549K mutation, two mutations that drive clinical progression on current pan-FGFR inhibitors. In the FGFR2V564F model, pan-FGFR inhibitors are ineffective, even at maximally tolerated doses. Notably, treatment of these tumors with RLY-4008 induces rapid regression and restores body weight. In rat and dog toxicology studies, RLY-4008 is well tolerated and is not associated with hyperphosphatemia or tissue mineralization at exposures significantly above those required to induce regression in all models. In contrast to pan-FGFR inhibitors, RLY-4008 is highly selective for FGFR2 and demonstrates strong activity against FGFR2 resistance mutations, suggesting that RLY-4008 may have broader therapeutic potential via preventing and overcoming therapeutic resistance. Together, these data and the favorable pharmaceutical properties of RLY-4008 strongly support its clinical development in FGFR2-altered tumors. Citation Format: Jessica Casaletto, Dejan Maglic, B. Barry Toure, Alex Taylor, Heike Schoenherr, Brandi Hudson, Kamil Bruderek, Songping Zhao, Patrick O9Hearn, Nastaran Gerami-Moayed, Demetri Moustakas, Roberto Valverde, Lindsey Foster, Hakan Gunaydin, Pelin Ayaz, Dina Sharon, Donald Bergstrom, James Watters. RLY-4008, a novel precision therapy for FGFR2-driven cancers designed to potently and selectively inhibit FGFR2 and FGFR2 resistance mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1455.

Although the Hippo transcriptional coactivator YAP is considered oncogenic in many tissues, its roles in intestinal homeostasis and colorectal cancer (CRC) remain controversial. Here, we demonstrate that the Hippo kinases LATS1/2 and MST1/2, which inhibit YAP activity, are required for maintaining Wnt signaling and canonical stem cell function. Hippo inhibition induces a distinct epithelial cell state marked by low Wnt signaling, a wound-healing response, and transcription factor Klf6 expression. Notably, loss of LATS1/2 or overexpression of YAP is sufficient to reprogram Lgr5+ cancer stem cells to this state and thereby suppress tumor growth in organoids, patient-derived xenografts, and mouse models of primary and metastatic CRC. Finally, we demonstrate that genetic deletion of YAP and its paralog TAZ promotes the growth of these tumors. Collectively, our results establish the role of YAP as a tumor suppressor in the adult colon and implicate Hippo kinases as therapeutic vulnerabilities in colorectal malignancies.

SUMMARY Hippo signaling has been recognized as a key tumor suppressor pathway. Here, we perform a comprehensive molecular characterization of 19 Hippo core genes in 9,125 tumor samples across 33 cancer types using multidimensional “omic” data from The Cancer Genome Atlas. We identify somatic drivers among Hippo genes and the related microRNA (miRNA) regulators, and using functional genomic approaches, we experimentally characterize YAP and TAZ mutation effects and miR-590 and miR-200a regulation for TAZ. Hippo pathway activity is best characterized by a YAP/TAZ transcriptional target signature of 22 genes, which shows robust prognostic power across cancer types. Our elastic-net integrated modeling further reveals cancer-type-specific pathway regulators and associated cancer drivers. Our results highlight the importance of Hippo signaling in squamous cell cancers, characterized by frequent amplification of YAP/TAZ, high expression heterogeneity, and significant prognostic patterns. This study represents a systems-biology approach to characterizing key cancer signaling pathways in the post-genomic era.

The mammalian Hippo signaling pathway, through its effectors YAP and TAZ, coerces epithelial progenitor cell expansion for appropriate tissue development or regeneration upon damage. Its ability to drive rapid tissue growth explains why many oncogenic events frequently exploit this pathway to promote cancer phenotypes. Indeed, several tumor types including basal cell carcinoma (BCC) show genetic aberrations in the Hippo (or YAP/TAZ) regulators. Here, we uncover that while YAP is dispensable for homeostatic epidermal regeneration, it is required for BCC development. Our clonal analyses further demonstrate that the few emerging Yap‐null dysplasia have lower fitness and thus are diminished as they progress to invasive BCC. Mechanistically, YAP depletion in BCC tumors leads to effective impairment of the JNK‐JUN signaling, a well‐established tumor‐driving cascade. Importantly, in this context, YAP does not influence canonical Wnt or Hedgehog signaling. Overall, we reveal Hippo signaling as an independent promoter of BCC pathogenesis and thereby a viable target for drug‐resistant BCC.

Both epigenetic silencing and genetic deletion of tumor suppressors contribute to the development and progression of breast cancer. SOX7 is a transcription factor important to development, and its down-regulation has been reported in tumor tissues and cell lines of prostate, colon, and lung cancers. However, the regulation of SOX7 expression and its functional role in breast cancer have not been reported. The current study demonstrates that SOX7 mRNA and protein expression are down-regulated in breast cancer tissues and cell lines compared with adjacent normal tissues and nontumorigenic cells, respectively. The SOX7 promoter is hypermethylated in breast cancer cell lines compared with nontumorigenic cells, and the inhibition of DNA methylation increases SOX7 mRNA levels. With shRNA-mediated SOX7 silencing, nontumorigenic immortal breast cells display increased proliferation, migration, and invasion and form structures that resemble that of breast cancer cells in a three-dimensional culture system. Conversely, ectopic SOX7 expression inhibits proliferation, migration, and invasion of breast cancer cells in vitro and tumor growth in vivo. Importantly, we discovered that SOX7 transcript levels positively correlated with clinical outcome of 674 breast cancer patients. Overall, our data suggest that SOX7 acts as a tumor suppressor in breast cancer. SOX7 expression is likely regulated by multiple mechanisms and potentially serves as a prognostic marker for breast cancer patients.

Our recent study shows a pivotal role of Dmp1 in quenching hyperproliferative signals from HER2 to the Arf-p53 pathway as a safety mechanism to prevent breast carcinogenesis. To directly demonstrate the role of Dmp1 in preventing HER2/neu-driven oncogenic transformation, we established Flag-Dmp1α transgenic mice (MDTG) under the control of the mouse mammary tumor virus (MMTV) promoter. The mice were viable but exhibited poorly developed mammary glands with markedly reduced milk production; thus more than half of parous females were unable to support the lives of new born pups. The mammary glands of the MDTG mice had very low Ki-67 expression but high levels of Arf, Ink4a, p53, and p21Cip1, markers of senescence and accelerated aging. In all strains of generated MDTG;neu mice, tumor development was significantly delayed with decreased tumor weight. Tumors from MDTG;neu mice expressed Flag-Dmp1α and Ki-67 in a mutually exclusive fashion indicating that transgenic Dmp1α prevented tumor growth in vivo. Genomic DNA analyses showed that the Dmp1α transgene was partially lost in half of the MDTG;neu tumors, and Western blot analyses showed Dmp1α protein downregulation in 80% of the cases. Our data demonstrate critical roles of Dmp1 in preventing mammary tumorigenesis and raise the possibility of treating breast cancer by restoring Dmp1α expression.

Lung cancer is the leading cause of cancer deaths in the world, which is a cause for more solid tumor-related deaths than all other carcinomas combined. More than 170,000 new cas‐ es are diagnosed each year in the United States alone, of whom ~160,000 will eventually die, accounting for nearly 30% of all cancer deaths (Siegel et al., 2012). The annual incidence for lung cancer per 100,000 population is highest among African Americans (76.1), followed by whites (69.7), American Indians/Alaska Natives (48.4), and Asian/Pacific Islanders (38.4). Hispanic people have much lower lung cancer incidence (37.3) than non-Hispanics (71.9) (CDC, 2010). These results identify the racial/ethnic populations and geographic regions that would benefit from enhanced efforts in lung cancer prevention, specifically by reducing cig‐ arette smoking and exposure to environmental carcinogens.