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.

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.

Our recent work has indicated that the DMP1 locus on 7q21, encoding a haplo‐insufficient tumour suppressor, is hemizygously deleted at a high frequency in breast cancer. The locus encodes DMP1α protein, an activator of the p53 pathway leading to cell cycle arrest and senescence, and two other functionally undefined isoforms, DMP1β and DMP1γ. In this study, we show that the DMP1 locus is alternatively spliced in ∼30% of breast cancer cases with relatively decreased DMP1α and increased DMP1β expression. RNA‐seq analyses of a publicly available database showed significantly increased DMP1β mRNA in 43–55% of human breast cancers, dependent on histological subtypes. Similarly, DMP1β protein was found to be overexpressed in ∼60% of tumours relative to their surrounding normal tissue. Importantly, alteration of DMP1 splicing and DMP1β overexpression were associated with poor clinical outcomes of the breast cancer patients, indicating that DMP1β may have a biological function. Indeed, DMP1β increased proliferation of non‐tumourigenic mammary epithelial cells and knockdown of endogenous DMP1 inhibited breast cancer cell growth. To determine DMP1β's role in vivo, we established MMTV‐DMP1β transgenic mouse lines. DMP1β overexpression was sufficient to induce mammary gland hyperplasia and multifocal tumour lesions in mice at 7–18 months of age. The tumours formed were adenosquamous carcinomas with evidence of transdifferentiation and keratinized deposits. Overall, we identify alternative splicing as a mechanism utilized by cancer cells to modulate the DMP1 locus through diminishing DMP1α tumour suppressor expression, while simultaneously up‐regulating the tumour‐promoting DMP1β isoform. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

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.

Despite recent advances in therapeutic approaches and early detection, breast cancer remains significant health care burden in many developing countries. In fact, it has been suggested that up to ~30% of breast cancer cases are overdiagnosed as a result of early detection mammography screening. This finding poses a hypothesis that better patient stratification using prognostic/predictive indicators, especially for the patients with early diagnosis, would provide significant improvement in clinical management and reduce health care cost. Our recent work identified Dmp1 as a critical tumor suppressor in breast cancer. Dmp1 is a transcription factor that induces cell cycle arrest and senescence by activating the p14ARF-p53 pathway. Overexpression of Her2/neu activates the Dmp1 promoter via PI3K-Akt-NFκB pathway leading to increase of p53 target genes. Loss of Dmp1 accelerates mammary tumor development in MMTV-neu mouse model without difference between Dmp1+/- and Dmp1-/- genotypes. Human DMP1 locus on 7q21 is hemizygously deleted in ~42% of breast carcinomas, which is mutually exclusive of INK4a/ARF or p53 inactivation. In the cases with hemizygous DMP1 deletion, the other DMP1 allele remains wild-type without mutation or promoter hypermethylation, which suggests that DMP1 is haploinsufficient tumor suppressor. The h DMP1 locus encodes three distinct transcripts via alternative splicing of pre-mRNA at Exon 10. The bona fide tumor suppressor protein is named DMP1α, while the two other transcripts without known biological function were named DMP1β and DMP1γ. The qPCR analysis of 46 matched breast cancer samples revealed that 30% of tumor samples have splicing alteration of DMP1 to increase DMP1β isoform. Importantly, the patients with high DMP1β/α ratio in tumor samples had shorter relapse-free survival compared to those patients without splicing alteration. Furthermore, DMP1β/α ratio was increased in MMTV-neu mouse mammary tumors. Immunohistochemistry of 50 breast tumor samples showed that DMP1β protein is overexpressed which was also associated with shorter relapse-free survival. Expression of DMP1β in non-tumorigenic breast epithelial cell line, MCF10A, significantly increased the rate of cell proliferation and size of the mammospheres in Matrigel©. Knockdown of the endogenous DMP1β inhibits proliferation of breast cancer cell lines. To ascertain role of DMP1β in development of breast cancer in vivo, we developed a MMTV-DMP1β mouse model. The mammary glands from MMTV-DMP1β female mice show dysplastic morphological changes in the epithelium with multifocal tumors at 18 months of age. The tumor tissue and surrounding mammary glands were hyperproliferative as they expressed high levels of Ki67 and Cyclin D1. Overall, we provide evidence that alternative splicing to increase DMP1β expression leads to proliferation of human cells and mouse mammary gland and offers poor prognosis for breast cancer patients. Citation Format: Dejan Maglic, Robert Kendig, Mark Cline, Guangchao Sui. DMP1β, an alternative splice isoform of tumor suppressor h DMP1 locus, has oncogenic properties in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr A013.

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.

Abstract : Breast cancer is the most common malignancy in women. Selection of patients for personalized therapy based on a risk-benefit assesment has not been improved. Dmp1 is a tumor suppressor that activates the p14ARF-p53 pathway to prevent breast cancer development. Regulation of Dmp1 at a transcription level by oncogenes is well understood; however, the role of Dmp1 phosphorylation is unknown. We identify a serine/threonine kinase of the MAPK pathway, MEKK1, which directly phosphorylates Dmp1. MEKK1 phosphorylates Dmp1 on multiple Serine/Threonines residues. Dmp1 and MEKK1 synergize on the Arf transactivation and the p53 pathway induction. Expression of MEKK1 in breast cancer cell lines leads to accumulation of ARF, p53, and p53 target genes. Analysis of MEKK1 locus in human breast tumor samples reveled that MEKK1 is frequently deleted and low expression of MEKK1 may be a new prognostic indicator of patient outcome.

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Breast cancer remains a significant public health issue in the industrialized countries. Physicians depend on surgery, cytotoxic adjuvant chemotherapy, or radiation for treatment of breast cancer patients. Few novel therapies are approved for clinical use. Although existing therapies are effective in controling the disease in many patients, the cytotoxic side-effects make them limited. This is especially the case in the patients with early stage breast cancer whose prognosis with surgery alone is good and avoiding adjuvant chemotherapy would spare them toxic side effects. On the other hand, significant fraction of patients relapses and develops metastasis. Therefore, it is critical to identify markers for better breast cancer patient stratification based on their prognosis that would guide physicians in selection and aggressiveness of therapies. Recently, we linked transcription factor DMP1 (cyclin D binding protein 1, Dmtf1) as a critical tumor suppressor that blocks proliferative signals from ErbB2 and oncogenic Ras by activating p14Arf-p53 pathway. hDMP1 is hemizygously deleted in ∼50% of human breast tumors that retain wild-type ARF and p53. In this study we identified an upstream kinase, MEKK1, which cooperates with Dmp1 to activate Arf transcription. In constitutively active form (C-terminal kinase domain or CA-MEKK1), MEKK1 increased transcriptional activity of Dmp1 via direct phosphorylation, while the full length (regulatory and kinase domain) form behaved as a feedback inhibitor by increasing Dmp1 ubiquitination. Phosphorylation sites on DMP1 that increase its transcriptional activity were mapped to the C-terminal transactivation domain. Expression of CA-MEKK1 in breast cancer cell lines activated endogenous Arf-p53 pathway. hMEKK1 is located on human 5q11 chromosome, a locus frequently deleted in breast and lung cancer. Since MEKK1 appeared to be an activator of p53 tumor suppressor pathway, we wondered if MEKK1 is involved in human cancer. Using DNA from breast cancer patient tumors and matched normal tissue, we analyzed hMEKK1 gene deletion using specific loss of heterozygosity (LOH) primers. MEKK1 was found hemizygously deleted in ∼20% of patients. Immunohistochemistry confirmed reduction of MEKK1 protein intensity to LOH(+) compared to LOH (-) patients. In the independent cohort of breast cancer patients, we show that low MEKK1 mRNA expression is associated with adverse outcome. Here we show that constitutively active MEKK1 is a novel activator of Dmp1-Arf-p53 pathway via Dmp1 phosphorylation. The MEKK1 gene was frequently deleted in breast tumor tissue and its expression was correlated with disease outcome. Overall, MEKK1 is a potential prognostic/predictive indicator for breast cancer and could be used by physicians to better stratify patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4158. doi:1538-7445.AM2012-4158