Inhibition Of Kinase Activity Research Articles

Discovery of LC-MI-3: A Potent and Orally Bioavailable Degrader of Interleukin-1 Receptor-Associated Kinase 4 for the Treatment of Inflammatory Diseases.

Interleukin-1 receptor-associated kinase 4 (IRAK4) is a promising therapeutic target in inflammation-related diseases. However, the inhibition of IRAK4 kinase activity may lead to moderate anti-inflammatory efficacy owing to the dual role of IRAK4 as an active kinase and a scaffolding protein. Herein, we report the design, synthesis, and biological evaluation of an efficient and selective IRAK4 proteolysis-targeting chimeric molecule that eliminates IRAK4 scaffolding functions. The most potent compound, LC-MI-3, effectively degraded cellular IRAK4, with a half-maximal degradation concentration of 47.3 nM. LC-MI-3 effectively inhibited the activation of downstream nuclear factor-κB signaling and exerted more potent pharmacological effects than traditional kinase inhibitors. Furthermore, LC-MI-3 exerted significant therapeutic effects in lipopolysaccharide- and Escherichia coli-induced acute and chronic inflammatory skin models compared with kinase inhibitors in vivo. Therefore, LC-MI-3 is a candidate IRAK4 degrader in alternative targeting strategies and advanced drug development.

Regnase-1 D141N mutation induces CD4+ T cell-mediated lung granuloma formation via upregulation of Pim2.

Regnase-1 is an RNase that plays a critical role in negatively regulating immune responses by destabilizing inflammatory mRNAs. Dysfunction of Regnase-1 can be a major cause of various inflammatory diseases with tissue injury and immune cell infiltration into organs. This study focuses on the role of RNase activity of Regnase-1 in developing inflammatory diseases. We have constructed mice with a single point mutation at the catalytic center of Regnase-1 RNase domain, which lacks endonuclease activity. D141N mutant mice demonstrated systemic inflammation, immune cell infiltration into various organs and progressive development of lung granuloma. CD4+ T cells, mainly affected by this mutation, upregulated mTORC1 pathway and facilitated the autoimmune trait in D141N mutation. Moreover, serine/threonine kinase Pim2 contributed to lung inflammation in this mutation. Inhibition of Pim2 kinase activity ameliorated granulomatous inflammation, immune cell infiltration and proliferation in the lungs. Additionally, Pim2 inhibition reduced the expression of adhesion molecules on CD4+ T cells, suggesting a role for Pim2 in facilitating leukocyte adhesion and migration to inflamed tissues. Our findings provide new insights into the role of Regnase-1 RNase activity in controlling immune function and underscore the therapeutic relevance of targeting Pim2 to modulate abnormal immune responses.

Abstract PO3-27-02: A novel PTK6 PROTAC degrader induces apoptosis of drug resistant breast cancer cells

Abstract Background. PTK6/Brk, a non-receptor tyrosine kinase, is an oncogenic driver for several tumor types, including breast, prostate, liver, and pancreatic cancers. PTK6 is expressed in all breast cancer subtypes and higher levels of expression are associated with worse patient outcomes. The optimal approach to targeting PTK6 for therapeutic benefit has not been established. Although several inhibitors of PTK6 kinase activity have been developed, none have thus far been successfully translated to clinical trials. This may be in part due to kinase activity-independent functions of PTK6, for which there is growing evidence, that are not effectively targeted by traditional small molecule kinase inhibitors. Downregulation of PTK6 using shRNA inhibits growth, invasion/migration, survival and metastasis of breast cancer cells, including those that are resistant to standard therapies. PTK6 kinase inhibitor treatment phenocopies effects of PTK6 downregulation with respect to some, but not all PTK6-driven oncogenic activities. Moreover, growth of PTK6 CRISPR-targeted cells can be rescued either with wild type or catalytically inactive PTK6, supporting kinase activity-independent activities of PTK6 in regulating growth and viability of breast cancer cells. Therefore, PROTAC degraders may be a better way to chemically target and downregulate PTK6 expression in cancer, thereby phenocopying the effects of PTK6 shRNA. Methods. We developed a first-in-class PTK6 degrader using PROTAC (proteolysis-targeting chimeric) technology to target PTK6 expression in breast cancer cells of diverse subtypes. Degrader controls were also synthesized to assess specificity of MS105’s effects. Results. PTK6 degrader, but not degrader controls, potently downregulates PTK6 expression in multiple breast cancer cell types. Proteomic and kinome profiling confirm the specificity of our PTK6 degrader. PTK6 PROTAC degrader inhibits viability and induces apoptosis of PTK6-expressing breast cancer cells, whereas treatment with parent kinase inhibitor or degrader controls that do not engage PTK6 or E3 ligase have minimal effect. Treatment with PTK6 degrader, but not kinase inhibitor or degrader controls, induces expression of pro-apoptotic Bim, phenocopying the effects observed with PTK6 shRNA. In contrast, PTK6 degrader and kinase inhibitor are comparably effective in inhibiting cell migration, supporting the differential kinase dependency of PTK6-dependent oncogenic functions. Therefore, our PTK6 PROTAC degrader effectively inhibits both kinase-dependent and independent oncogenic activities of PTK6. Our studies support the development of PTK6 PROTAC degraders as a preferred approach to clinically targeting PTK6 in cancer. Citation Format: Hanna Irie, Criseyda Martinez, Yan Xiong, Alison Bartkowski, Jessica Byerly, Jian Jin. A novel PTK6 PROTAC degrader induces apoptosis of drug resistant breast cancer cells [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-27-02.

Abstract PO2-18-06: Targeting fibroblast growth factor receptor (FGFR1) expression through G-quadruplex stabilization inhibits metastatic breast cancer

Abstract Metastatic breast cancer (MBC) is the most advanced stage of breast cancer. Our understanding of the molecular mechanisms which drive MBC remain incomplete. Epithelial to mesenchymal transition (EMT) and mesenchymal to epithelial transition (MET) promote drug resistance and metastasis. It has been reported that fibroblast growth factor receptor 1 (FGFR1) plays a key role during the EMT:MET cycle. Furthermore, FGFR1 is amplified in 13% of primary and 20% of metastatic breast cancer patients. Therefore, optimizing inhibition of FGFR1 is crucial for the therapeutic targeting of the late stage breast cancer. First, we examined the efficacies of FGFR kinase inhibitors in the murine based dormant 4T07 tumor model. Inhibition of FGFR kinase activity leads to tumor growth inhibition but fail to eradicate dormant breast cancer cells. Therefore, we explored broader approaches to inhibit FGFR1 expression in addition to blockade of its kinase activity. G-quadruplex (G4) structures are secondary DNA structures commonly found upstream of transcriptional start sites (TSS) of oncogenes restricting their expression. Consequently, pharmacological stabilization of G4 structures within the promoters of cancer-related genes via use of small molecules has emerged as a promising therapeutic approach in cancer. Results herein demonstrate that the proximal promoter of FGFR1 contains sequences that form G4. Circular dichroism was used to verify formation of G4 in the FGFR1 proximal promoter. Importantly, use of the G4-binding compound CX-5461 stabilized the FGFR1 G4 structure, blocked the transcriptional activity of the FGFR1 proximal promoter and decreased FGFR1 expression. Therefore, we implemented the G4 stabilizers in FGFR1 expressing and metastatic drug-resistant BC cell lines. This approach results in dramatic downregulation of FGFR1 at the protein level after treatment with the G4 stabilizer. G4 stabilizing agents also interfere with ectopic FGFR1 expression and EMT-driven FGFR1 expression. Importantly, use of the G4-targeting compound CX5461 effectively blocked FGFR1 expression and inhibited FGFR1 downstream signaling, resulting in eradication of dormant breast cancer cells. Finally, in vivo application of CX5461 reduced FGFR1 expression, blocked pulmonary tumor formation and prolonged animal survival. In conclusion, consistent with the clinical observations our evaluation of FGFR kinase inhibitors validates the resistance to FGFR kinase inhibitors in MBC. Our findings indicate that targeting FGFR1 expression through G4 stabilization may be a potential strategy for MBC. Citation Format: Muhammad Safdar, Hang Lin, Sarah Dagher, Jonathan Dickerhoff, Mitchell Ayers, Luis Solorio, Danzhou Yang, Michael Wendt, Saeed Akhand. Targeting fibroblast growth factor receptor (FGFR1) expression through G-quadruplex stabilization inhibits metastatic breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO2-18-06.

Abstract RF02-02: BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism

Abstract PI3Kα is the most mutated kinase and the second most mutated oncogene in human cancer. Activation of PI3Ka can be achieved by receptor tyrosine kinases such as insulin receptor and insulin-like growth factor receptor 1 and/or by directly interacting with RAS family members. Previous elegant preclinical studies have established that RAS-driven PI3Ka activation is important in tumor cells but may not be involved in cell types controlling glucose metabolism. Alpelisib, a small molecule inhibitor of the kinase activity of PI3Ka, has been approved for the treatment of ER+ PIK3CA mutant breast cancer in combination with fulvestrant after endocrine therapy in advanced or metastatic breast cancer based on an improvement in PFS versus fulvestrant alone. Inhibition of PI3Ka activity by alpelisib in normal tissues resulted in a severe (G3/4) hyperglycemia rate of 37% with frequent dose interruptions and discontinuations. Additionally, preclinical studies have demonstrated that the dysregulation of glucose homeostasis resulting from PI3Ka kinase inhibition leads to hyperinsulinemia that increases pathway flux, rendering kinase inhibitors less effective. Here, we report on a novel covalent small molecule designed to inhibit RAS-mediated activation of the AKT pathway via PI3Ka without the resultant hyperglycemia associated with direct inhibition of PI3Ka kinase activity. BBO-10203 disrupts the physical interaction between RAS and PI3Ka in tumor cells resulting in potent signaling pathway inhibition. This agent selectively binds to PI3Ka and disrupts its interaction with K-,H-, and N-RAS with low single digit nanomolar potency (~5 nM). Breaking the interaction between these two oncogenes inhibits basal pAKT cellular levels (BT-474/KYSE-410 IC50: ~5 nM) in HER2 amplified (HER2amp) and wild-type or mutant PI3Ka cell lines. Even though BBO-10203 does not inhibit the kinase activity of PI3Ka, its effects on cancer cell signaling inhibition and transcriptional regulation highly resemble those of alpelisib. BBO-10203 displays excellent drug-like properties and oral bioavailability. Single dose treatment of KYSE-410 (HER2amp/KRASG12C) tumor bearing mice with increasing doses (1-100 mg/kg, PO) of BBO-10203 results in dose and time dependent inhibition of pAKT in vivo. Maximal inhibition (~80%) is achieved at 30 mg/kg and lasts for 24 hours. Repeated dose treatment of tumor bearing mice with BBO-10203 is well tolerated and results in significant efficacy in PIK3CA mutant as well as HER2amp human xenograft models. In the KYSE-410 xenograft model, BBO-10203 daily oral dosing of 30 mg/kg results in significant tumor regressions. Importantly, treatment with BBO-10203 does not affect insulin signaling in differentiated adipocytes in vitro, nor does it impact glucose metabolism in vivo at 3-times the maximal efficacious dose level in xenograft studies. In conclusion, we have identified a novel approach to inhibit the PI3Ka signaling pathway by blocking its interaction with, and activation by RAS. This approach can achieve strong pAKT inhibition in tumor cells without changes in glucose metabolism. Clinical investigation of BBO-10203 for the treatment of both ER+/PIK3CA mutant and HER2amp breast cancer is warranted. Citation Format: Pedro Beltran, Simanshu Dhirendra, Rui Xu, Ming Chen, Daniel Czyzyk, Sofia Donovan, Siyu Feng, Cindy Feng, Lijuan Fu, Felice Lightstone, Ken Lin, Anna Maciag, Dwight Nissley, Erin Riegler, Kerstin Sinkevicius, Andrew Stephen, James Stice, David Turner, Bin Wang, Keshi Wang, Yue Yang, Cathy Zhang, Frank McCormick, Eli Wallace. BBO-10203, a first-in-class, orally bioavailable, selective covalent small molecule that inhibits RAS-driven PI3Kalpha activity without affecting glucose metabolism [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr RF02-02.

Kinase Inhibition via Small Molecule-Induced Intramolecular Protein Cross-Linking.

Remarkable progress has been made in the development of cysteine-targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) has emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K-E/D salt bridge within the enzyme active-site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D-targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide-based small-molecule inhibitors capable of inducing intramolecular cross-linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide-facilitated, ligand-induced mechanism leading to intramolecular kinase cross-linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative.

Kinase Inhibition via Small Molecule‐Induced Intramolecular Protein Cross‐Linking

Remarkable progress has been made in the development of cysteine‐targeted covalent inhibitors. In kinase drug discovery, covalent inhibitors capable of targeting other nucleophilic residues (i.e. lysine, or K) has emerged in recent years. Besides a highly conserved catalytic lysine, almost all human protein kinases possess an equally conserved glutamate/aspartate (e.g. E/D) that forms a K‐E/D salt bridge within the enzyme active‐site. Electrophilic ynamides were previously used as effective peptide coupling reagents and to develop E/D‐targeting covalent protein inhibitors/probes. In the present study, we report the first ynamide‐based small‐molecule inhibitors capable of inducing intramolecular cross‐linking of various protein kinases, leading to subsequent irreversible inhibition of kinase activity. Our strategy took advantage of the close distance between the highly conserved catalytic K and E/D residues in a targeted kinase, thus providing a conceptually general approach to achieve irreversible kinase inhibition with high specificity and desirable cellular potency. Finally, this ynamide‐facilitated, ligand‐induced mechanism leading to intramolecular kinase cross‐linking and inhibition was unequivocally established by using recombinant ABL kinase as a representative.

Abstract ND12: Discovery of PF-07220060, a potent and selective CDK4 inhibitor

Abstract PF-07220060 was developed based on the clinical need to improve efficacy and safety outcomes in patients treated with currently approved dual CDK4/6 inhibitors, including Palbociclib. Although all dual CDK4/6 inhibitors increase response rates and prolong disease control, they show common dose-limiting hematologic toxicities, foremost neutropenia. This poses challenges to provide these agents at doses necessary to extinguish cell cycling in tumors. Human ER+ breast cancer cells are highly dependent on CDK4 but not CDK6. By contrast, CDK4 is dispensable for human bone marrow derived cells, or cells enriched for neutrophil precursors or hematopoietic stem cells. This is primarily due to the compensatory role of CDK6 in these compartments. These observations prompted us to develop PF-07220060, an aminopyrimidine-based inhibitor, endowed with 20-fold and 4-fold increased selectivity for CDK4 over CDK6 when compared to Palbociclib and Abemaciclib/Ribociclib, respectively. CDK4-over-CDK6 selectivity is explained by G-loop conformational differences between the two kinases, with the G-loop of CDK4 being more flexible, assuming a more open conformation when bound to CDK4 selective versus non-selective inhibitors. PF-07220060 reduces dose-limiting neutropenia in beagle dogs by 15-fold when compared with Palbociclib, consistent with early clinical data obtained with PF-07220060. Higher tolerated plasma concentrations of PF-07220060 versus dual CDK4/6 inhibitors enable increased inhibition of CDK4 kinase activity in tumors. This translates to improved tumor growth inhibition across CDK4-driven models of human breast and prostate cancer at clinically relevant doses. Our work establishes proof-of-concept for CDK4 selective inhibition and introduces a best-in-class CDK4 inhibitor for treatment of HR+ HER2- breast cancer and other CDK4 dependent tumor types. Citation Format: Lars Anders, Gary Gallego. Discovery of PF-07220060, a potent and selective CDK4 inhibitor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr ND12.

Abstract LB181: First disclosure of XRD-0394, a novel dual ATM/DNA-PK inhibitor, that potently radiosensitizes and potentiates PARP and topoisomerase inhibitors

Abstract A majority of cancer patients receive radiation therapy (RT) as part of their treatment regimens, whether using external beam therapy or locally-delivered radioisotopes. While often effective, RT may not be efficacious for some tumors while exerting significant short-term and long-term toxicities. Thus, enhancing therapeutic effectiveness of radiation therapy remains an important goal. ATM (ataxia-telangiectasia, mutated) and DNA-PKcs (deoxyribonucleic acid [DNA]-dependent protein kinase, catalytic subunit) are related protein kinases and both are critical regulators of cellular responses to ionizing radiation. We report here the identification and initial characterization of XRD-0394, a potent, specific dual inhibitor of both ATM and DNA-PKcs, whose chemical structure will be disclosed. A dual ATM/DNA-PK inhibitor has practical and theoretical advantages over compounds which inhibit just one of these kinases: 1) blunting both DNA damage response (DDR) pathways enhances radiation-induced tumor cell kill compared to single target inhibition; and 2) simultaneous inhibition of two DDR pathways should overcome intrinsic or acquired resistance of tumor cells to either target alone. Notably, transient inhibition of both kinases by XRD-0394 has little adverse effects on cells in the absence of irradiation. XRD-0394 is orally bioavailable, with excellent pharmacological profiles, demonstrating significant in vitro and in vivo sensitizing activity in combination with ionizing radiation and in combination with immunotherapy and radiation. In addition, a CRISPR screen identified alterations in the Fanconi A/BRCA pathway as an opportunity for synthetic lethality in tumor cells exposed to XRD-0394, an insight that was validated in multiple tumor cell types. In vitro studies demonstrated synergy of XRD-0394 in combination with PARP inhibitors, particularly in BRCA-mutant cells. XRD-0394 also potentiates the effectiveness of topoisomerase inhibitors in vitro. Single doses of XRD-0394 were well-tolerated in combination with palliative RT in subjects with advanced cancer in a Phase Ia clinical trial (NCT05002140). No DLTs were observed. At a dose of 160 mg, plasma concentrations were well above the preclinical target plasma level of 530 ng/mL (1 µM) for up to 15 h and inhibition of ATM kinase activity was demonstrated by inhibition of radiation-induced pKAP1 in tumor tissue. Importantly, this concentration of drug is sufficient to induce potent radiosensitization in numerous preclinical models. The median tmax (2.3 h) and mean terminal t½ (11.1 h) at the 160 mg dose in subjects support coadministration of XRD-0394 and RT within a clinically feasible time frame. These results provide a rationale for future clinical trials with XRD-0394 in combination with RT, RT and immunotherapy, PARP inhibitors and targeted delivery of topoisomerase inhibitors. Citation Format: Tona M. Gilmer, Chun-Hsiang Lai, Kexiao Guo, Katherine Deland, Kathleen A. Ashcraft, Amy E. Stewart, Yaode Wang, Jianmin Fu, Kris C. Wood, Deborah A. Smith, Jonathan T. Yang, Christopher T. Chen, Michael F. Gensheimer, Paul B. Romesser, Steven H. Lin, David G. Kirsch, Michael B. Kastan. First disclosure of XRD-0394, a novel dual ATM/DNA-PK inhibitor, that potently radiosensitizes and potentiates PARP and topoisomerase inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB181.

LRRK2 G2019S Promotes Colon Cancer Potentially via LRRK2-GSDMD Axis-Mediated Gut Inflammation.

Leucine-rich repeat kinase 2 (LRRK2) is a serine-threonine protein kinase belonging to the ROCO protein family. Within the kinase domain of LRRK2, a point mutation known as LRRK2 G2019S has emerged as the most prevalent variant associated with Parkinson's disease. Recent clinical studies have indicated that G2019S carriers have an elevated risk of cancers, including colon cancer. Despite this observation, the underlying mechanisms linking LRRK2 G2019S to colon cancer remain elusive. In this study, employing a colitis-associated cancer (CAC) model and LRRK2 G2019S knock-in (KI) mouse model, we demonstrate that LRRK2 G2019S promotes the pathogenesis of colon cancer, characterized by increased tumor number and size in KI mice. Furthermore, LRRK2 G2019S enhances intestinal epithelial cell proliferation and inflammation within the tumor microenvironment. Mechanistically, KI mice exhibit heightened susceptibility to DSS-induced colitis, with inhibition of LRRK2 kinase activity ameliorating colitis severity and CAC progression. Our investigation also reveals that LRRK2 G2019S promotes inflammasome activation and exacerbates gut epithelium necrosis in the colitis model. Notably, GSDMD inhibitors attenuate colitis in LRRK2 G2019S KI mice. Taken together, our findings offer experimental evidence indicating that the gain-of-kinase activity in LRRK2 promotes colorectal tumorigenesis, suggesting LRRK2 as a potential therapeutic target in colon cancer patients exhibiting hyper LRRK2 kinase activity.

Abstract 4669: Differential preclinical activity of Bruton tyrosine kinase inhibitors (BTKis) against BTK resistance-associated mutations

Abstract Background: Differences in profiles of acquired mutations of resistance to BTKis might have implications for treatment sequencing. We evaluated preclinical activity of BTKis (ibrutinib [Ibr], acalabrutinib [Acala], zanubrutinib [Zanu], and pirtobrutinib [Pirto]) against multiple clinically relevant BTK mutations and report on the clinical impact of BTK C481S in patients (pts) treated with Ibr. Methods: Inhibition of kinase activity by BTKis was evaluated in a cell-free kinase activity assay (Nanosyn). Cell-killing activity on CRISPR-generated BTK mutant knock-in TMD8 cells was assessed after 3 days of BTKi treatment. Time from first detection of C481S mutation to progressive disease (PD) was evaluated in pts treated with Ibr with previously untreated (n=247) or relapsed/refractory (R/R; n=172) CLL using pooled data from 5 clinical trials. Results: In vitro kinase activity was highest for Ibr compared with Acala or Zanu against C481S (Ibr IC50: 26.3 nM; Zanu: 84.8 nM; Acala: 338 nM) and T474I (Ibr: 1.2 nM; Zanu: 4.4 nM; Acala: 43.6 nM). Ibr maintained cell-killing activity against all tested BTK mutants, whereas limited activity was seen with Acala against C481S and A428D; with Zanu against C481S, L528W, and A428D; and with Pirto against T474I, L528W, V416L, and A428D (Table). In pts treated with Ibr, C481S was detected prior to clinical PD in 3 previously untreated pts and in 26 pts with R/R CLL; median (range) time from first detection of C481S to PD in these pts was 17.0 mo (11.2-34.3) and 10.0 mo (2.6-35.7), respectively. Conclusion: This study demonstrates activity of Ibr against all evaluated BTK variants, which are associated with resistance to covalent and noncovalent BTKis. Clinical data shows that pts who develop C481S mutations continue to derive benefit from Ibr for several months before PD. Together, these data suggest a role for Ibr in the treatment of pts with a broad spectrum of BTK variants. Table. Cell-killing activity of BTKis against wild type and mutant BTK EC50 (nM) BTK Wild type C481S T474I L528W V416L A428D Ibr 0.6 414.0 1.9 181.4 0.5 205.3 Acala 3.9 2973.8 43.3 3.5 106.3 3000.0 Zanu 0.7 2322.0 11.7 3000.0 0.6 1726.0 Pirto 8.2 12.6 2727.5 3000.0 1898.0 3000.0 Citation Format: Matthew S. Davids, Inhye E. Ahn, Jacob J. Riehm, Melih Acar, Vincent Girardi, Sima Patel, Tarikere Gururaja, Jennifer Woyach, Lloyd T. Lam, Edith Szafer-Glusman. Differential preclinical activity of Bruton tyrosine kinase inhibitors (BTKis) against BTK resistance-associated mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4669.

Abstract 5902: Targeting MAPK-driven tumors via inhibition of MAP2K4

Abstract MAPK pathway activation is a feature of multiple tumor types. Drugging KRAS and BRAF, the two main oncogenic drivers in the MAPK pathway, has proven successful in the clinic. Inhibition of the downstream effectors, MEK and ERK, can also induce tumor regression. Despite this, many tumors are intrinsically resistant to MAPK pathway inhibitors, or acquire resistance under selective pressure to drug treatment. This creates a need for combination treatments to improve clinical responses. A synthetic lethal (SL) interaction between inhibition of the MAPK pathway and blockade of JNK-JUN signaling has recently been described1. Specifically, data from yeast genetics and CRISPR knockout experiments in human cells have identified MAP2K4 as a potential therapeutic target that could be combined with MAPK inhibitors. To date, however, no potent MAP2K4 inhibitors with in vitro and cellular selectivity against key anti-targets have been reported. Here, we describe the development of potent covalent inhibitors of MAP2K4 kinase activity. Biochemical and cell-based assays show that the compound(s) are selective for MAP2K4 versus anti-targets including MAP2K7 and ERK kinases. The combination of MAP2K4 and MEK/ERK inhibitors was effective in cell lines driven by MAPK signaling. These data provide the rationale for further development of MAP2K4 inhibitors to advance our understanding of this novel drug combination.

Abstract 3913: Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy

Abstract Prednisone is an anti-inflammatory glucocorticoid (GC) that is cytotoxic for normal and malignant B cells and, on this basis, has long been included in combination chemotherapies to treat aggressive B-cell lymphomas. However, the mechanisms by which GCs kill malignant lymphoma cells still largely remains elusive due to the pleiotropic consequences of GC binding to the glucocorticoid receptor (GR; NR3C1), a ligand-induced transcription factor. This prompted us to search for critical direct targets of GC action with the hypothesis that GCs may inhibit key survival pathways in lymphomas. To determine effector genes that increase glucocorticoid toxicity, we performed genome-wide CRISPR-Cas9 screens in the presence and absence of prednisolone. Screens in cell line models of Burkitt lymphoma (BL) and diffuse large B cell lymphoma (DLBCL) revealed strong synergy between GC treatment and inactivation of genes encoding components of the B cell receptor (BCR) signaling pathway (≥2 SD). To identify direct transcriptional GR targets and binding sites genome-wide, we performed the cleavage under targets and release using nuclease (CUT&RUN) assay and integrated this dataset with RNA-seq upon GC treatment. The pairwise comparison analysis (p<0.05) for GR-bound genes across BL and DLBCL cell lines revealed a significant enrichment of negative regulators of BCR/PI3K signaling and BCR-related tumor suppressors (LAPTM5, KLHL14, ARID5B, DDIT4, DAPP1, INPP5D, and CSK). Interestingly, expression of CSK, an inhibitor of all Src-family kinases (SFKs), was repressed while that of other BCR negative regulators were directly activated by binding of GR. Combinatorial deletion of activated genes counteracted the toxicity of GC up to 83.9%. In contrast, depletion of CSK paradoxically increased the cytotoxic effects of GC in BCR-dependent aggressive lymphoma with decreased proximal BCR signaling. To gain further insights on CSK function in aggressive lymphomas, we performed phospho-proteome and ubiquitinome profiling by treating lymphoma models with a small molecule inhibitor of CSK kinase activity (CSKi). The CSKi treatment initially increased constitutive BCR signaling, as expected, but then triggered exuberant ubiquitination of the LYN, HCK and BLK, leading to their proteasomal degradation. Consequently, the CSKi blocked BCR-dependent NF-κB activation in ABC DLBCL models and BCR-dependent PI3 kinase activation in models of GCB DLBCL and BL. In summary, inhibition of oncogenic BCR signaling is a major mode of action for GCs. Furthermore, small molecule inhibition of CSK kinase activity potentiated the effect of GCs on oncogenic BCR signaling and strongly synergized with GCs in killing ABC and GCB DLBCL models in vitro and preventing the growth of ABC and GCB DLBCL and patient-derived xenografts, warranting the development of clinical-grade CSK inhibitors for the treatment of these aggressive cancers. Citation Format: Jaewoo Choi, Michele Ceribelli, James D. Phelan, Björn Häupl, Da Wei Huang, George W. Wright, Tony Hsiao, Vivian Morris, Francesco Ciccarese, Boya Wang, Sean Corcoran, Sebastian Scheich, Xin Yu, Weihong Xu, Yandan Yang, Hong Zhao, Joyce Zhou, Grace Zhang, Jagan Muppidi, Giorgio Ga Inghirami, Thomas Oellerich, Craig J. Thomas, Wyndham H. Wilson, Louis M. Staudt. Molecular mechanism of action and targets of glucocorticoids in lymphoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3913.

Abstract 4321: The oncogenic DMTF1β splice variant promotes autophagy-dependent cancer cell motility

Abstract The oncogenic isoform DMTF1β, which is generated by alternative splicing of the DMTF1 tumor suppressor gene is associated with increased cell proliferation and tumorigenesis. Despite these correlative findings little is known about the DMTF1β mechanism of action. In order to decipher the oncogenic DMTF1β signaling pathway, we performed RNA sequencing of DMTF1β depleted prostate cancer cells and analyzed the DMTF1β-specific protein interactome in these cells. Interestingly, autophagy-related pathways belonged to the most downregulated networks in DMTF1β knockdown cells. Moreover, we found that predominantly DMTF1β, but not a protein lacking the β-specific domain interacts with the key autophagy ULK1-ATG13 protein complex. These interactions were confirmed by co-immunoprecipitation and proximity ligation assays. Importantly, DMTF1β binding to ULK1 stabilized this key autophagy protein. Accordingly, knocking down DMTF1β significantly lowered autophagic flux in breast and prostate cancer cells as assessed by LC3-dependent and independent autophagy assays. Since published data show an important role for autophagy in migration and invasion of cancer cells, we next investigated whether DMTF1β is regulating these cellular processes. Indeed, DMTF1β depleted breast and prostate cancer cells displayed significantly decreased wound closure, trans-well invasion and metastatic foci formation in a zebra fish xenograft model. Importantly, pharmacological inhibition of ULK1 kinase activity or knocking down ULK1 reversed DMTF1β-mediated cancer cell migration. In agreement, decreased cell motility in DMTF1β knockdown cancer cells could be rescued by ectopic expression of the VPS34 complex member Beclin-1. In summary, we identified a novel DMTF1β-ULK1 signaling pathway that activates aberrant cancer cell migration most probably by increasing basal cell autophagy. Targeting autophagy using ULK1 inhibitors may represent a promising strategy to lower metastatic activity of tumor cells. Citation Format: Mario P. Tschan, Jun Xu, Anna M. Bill. The oncogenic DMTF1β splice variant promotes autophagy-dependent cancer cell motility [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4321.

Abstract 592: A novel pMEK inhibitor ABM-4095 for the treatment of pancreatic cancer

Abstract The MEK-ERK pathway is often activated in tumors by mutations of upstream BRAF- or RAS-proteins. The RAS is frequently mutated in several cancers, especially in pancreatic cancer with an about 95% rate. Pancreatic cancer has the highest mortality rate with a 5-year survival rate of 8% and a median survival of 6 months. Targeted therapies have been extensively evaluated in pancreatic cancer, however, survival improvement of this aggressive disease using a targeted strategy has been minimal. The only currently approved targeted therapy is erlotinib in combination with the chemotherapy drug gemcitabine. To date, four MEK inhibitors that block RAF-activation have been approved by the FDA, but their ability to inhibit ERK signal activated by RAS-mutations is limited due to the induction of MEK phosphorylation by relief of ERK-dependent feedback inhibition of RAF. Here, we report a novel small molecule, ATP-uncompetitive, pMEK inhibitor ABM-4095 that potently prevents phosphorylation of MEK by RAF with moderate inhibition of MEK kinase activity. Most of MEK inhibitors approved by FDA or in clinical trials increase pMEK levels instead. ABM-4095 has a good cell permeability. Its anti-cancer efficacy has been demonstrated both in the in vitro cancer cell line proliferation assay and in vivo xenograft models. ABM-4095 showed a synergy effect with erlotinib in pancreatic cancer cells. In a Miapaca-2 xenograft model, ABM-4095 showed good dose-dependent tumor inhibition as a single agent or combining with the KRAS G12C inhibitor AMG-510, and tumor regressions were observed. In summary, ABM-4095 is a potent pMEK inhibitor with a high activity against the RAS-mutant pancreatic cancer in vitro and in vivo. Detailed preclinical results will be presented. Citation Format: Min Xu, Yong Hu, Lanjiao Zhao, Xiuquan Chen, Youqin Chen, Chen Yang, Jizhi Li, Chen Chen, Xiaobing Lv. A novel pMEK inhibitor ABM-4095 for the treatment of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 592.

Abstract 5147: Identification of pharmacodynamic biomarkers in PBMCs and cancer cells for SIK2 kinase inhibitor therapy

Abstract Salt-inducible kinase 2 (SIK2) is an AMPK family kinase involved in multiple signaling pathways including the LBK1-SIK-HDAC, LKB1-SIK-CRTC/CREB, PI3K-PKA-mTOR and Hippo pathways. The expression of SIK2 is significantly upregulated in fraction of ovarian and breast cancers, and associated with poor prognosis. Inhibition of SIK2 kinase activity suppresses growth of ovarian and breast cancers as well as progression of acute myeloid leukemia. SIK2 inhibitors synergistically enhance sensitivity of ovarian and breast cancer cells to paclitaxel, carboplatin and PARP inhibitors. Utilization of SIK2 inhibitors to treat cancers has been investigated in preclinical studies and in a Phase I clinical trial (NCT04711161). To enable pharmacodynamic (PD) analysis in clinical studies, we have developed biomarkers that measure the effect of GRN-300, a SIK2/3 inhibitor, on SIK2 signaling in ovarian cancer cells. Class IIa histone deacetylases (HDAC4/5/7) are SIK2 substrates in cancer cells; and are also expressed in peripheral blood mononuclear cells (PBMCs). We found that GRN-300 decreased phosphorylated HDAC4/5/7 levels in ovarian and breast cancer cells (Lu et al. J Clin Invest, 2022) and now report that this SIK2 inhibitor also decreases pHDAC4/5/7 in PBMCs ex vivo in a dose-dependent manner using Western blot analysis. We also identified three mRNA markers in PBMCs by RNAseq analysis - SEPP1, RNASE1 and MS4A6A - which are up-regulated by GRN-300 in PBMCs isolated from healthy donors. However, these mRNA markers were either expressed at very low levels or not dramatically changed in cancer cells. We identified an additional mRNA marker panel using RNAseq technology by screening seven ovarian and six breast cancer cell lines: A2780, Caov3, IGROV1, OC316, OVCAR3, OVCAR8, SKOv3ip, BT549, Cal51, HCC1937, MDA-MB-231, MDA-MB-436 and MDA-MB-468. mRNA expression of three genes - DDIT4, NUPR1 and IL6 - is upregulated by GRN-300 in more than 9 of 13 cell lines tested. Five genes - ID1, ID2, ID3, ID4 and TGFBI - are down-regulated in response to GRN-300 treatment in more than 10 of 13 cell lines. The decrease in HDAC4/5/7 phosphorylation, and alterations in mRNA markers we discovered could serve as PD biomarkers for clinical trials to evaluate the effects of SIK2 inhibitors and provide insights in the mechanisms of drug action. Citation Format: Hailing Yang, Weiqun Mao, Philip Rask, Markus Peter, Gayathri Swaminath, Stephan W. Morris, Zhen Lu, Robert C. Bast. Identification of pharmacodynamic biomarkers in PBMCs and cancer cells for SIK2 kinase inhibitor therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5147.

Abstract 603: BB5523, a multiple kinase inhibitor with HPK1, VEGFR2 and TNIK inhibition, enhanced antitumor immunity in tumor models

Abstract Introduction: Immuno-suppressive micro-environment and abnormal signaling pathways are crucial for the proliferation of tumor cells. Hematopoietic progenitor kinase 1 (HPK1) inhibitor plays important roles in activation of exhausted T cells and enhancement of anti-tumor immune responses. VEGF/VEGFR2 pathway is the central therapeutic target in anti-angiogenic treatment in multiple cancers. Traf2- and Nck-interacting kinase (TNIK) is a member of the germinal center kinase family, and is reported to be essential for Wnt signaling and colorectal cancer (CRC) proliferation and progression. Blockade of critical kinases in multiple signaling pathways inhibits the proliferation, invasion and metastasis of cancer cells. Therefore, intergradation of these functions in a small molecule serves as the next generation of immune-oncology small molecules for cancer therapy. Methods: BB5523 was developed through structure-based drug design, and optimized by SAR analysis and medicinal chemistry iteration. Biochemical assays and cell-base functional assays were applied in compound evaluation. Pharmacokinetic (PK) and preliminary toxicity studies were performed with mice. CDX and syngeneic tumor models in mice were conducted to evaluate the in vivo efficacy of BB5523 as a single agent. Results: BB5523 shows high inhibition potency on HPK1 with good selectivity against other TCR signaling-related kinases, such as FYN and ZAP70. Inhibition of HPK1 kinase activity (IC50 = 3.3 nM) by BB5523 strongly suppresses the phosphorylation of the downstream biomarker protein SLP-76 (IC50 = 30 nM); and boosts the IL-2 secretion in purified human T cells and human peripheral blood mononuclear cells. BB5523 also shows good activities on VEGFR2 (IC50 = 1.6 nM) and TNIK (IC50 = 0.7 nM) which are crucial for the proliferation of cancer cells. In in vitro experiments, BB5523 significantly inhibits the proliferation of various tumor cell lines at sub-micromolar concentrations. PK profile of BB5523 shows good oral bioavailability (F > 80%). In preliminary safety evaluation, no abnormal symptoms were observed in mice dosed up to 100 mpk for 14 days. BB5523 showed significant tumor-growth inhibition efficacy in TE-8, JIMT-1, B16-F10 and MC38 tumor models. Conclusion: As a potent and oral available small molecule kinase inhibitor, BB5523 boosts anti-tumor immunity through HPK1 inhibition, while also blocking crucial signaling pathways in tumor micro-environment. BB5523 shows promising in vitro and in vivo efficacy with a well-tolerated safety profile. The preclinical study of BB5523 is in progress. Citation Format: Gongping Duan, Min Li, Puyong Zhang, Wei Hua, Enlun Hao, Shipeng Wang, Xiaoyi Ma, Junheng Wang, Lijie Wei, Xingmin Zhang. BB5523, a multiple kinase inhibitor with HPK1, VEGFR2 and TNIK inhibition, enhanced antitumor immunity in tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 603.

Abstract 260: Depletion of Ezh2 in the intestinal adenoma induced by JAK2V617F mutation leads to the malignant neoplasm

Abstract Background The increased JAK2 and EZH2 plays a critical role in the colorectal or colon cancer (CRC) progression. Targeting aberrant JAK2 expression caused by JAK2 gain-of-function (GOF) mutation, JAK2V617F and deregulated EZH2 kinase activity, JAK2 and/or EZH2 inhibitors have been used to treat the advanced CRCs. However, it is unclear whether the aberrant JAK2 can directly induce CRC or intestinal epithelial (IEC) malignancy. We hypothesize that the overexpressed JAK2 driven by JAK2V617F GOF mutation increases IEC neoplasia in which depletion of EZH2 can lead to IEC malignant neoplasm. Materials and Methods Colorectal samples were collected from 20 cases of CRC patients. Immunohistochemistry staining (IH), confocal immunofluorescence staining (IF) and bulk RNAseq were used to determine LGR5, Ki67, JAK2, EZH2, mutant β-catenin and JAK2V617F GOF mutation. Crypt-derived organoids or tumoroids were used to determine the JAK2 and EZH2 in the proliferative LGR5 cells (Ki67hiLGR5+) and were treated with JAK2 and/or EZH2 inhibitors. The mice with JAK2 and/or EZH2 deficiency in IECs were generated by crossing VilCreER mice, floxed-Jak2 and/or floxed-Ezh2 mice to determine the intestinal and colonic neoplasia. Lgr5, Ki67, Cdx2 Jak2, Ezh2, and mutant β-catenin IH or IF, organoids culture and peritoneal xenograft were used to determine the malignant neoplasm. The JAK2V617F-transfected HT29 or Caco-2 cells were used to determine the migration induced by JAK2V617F GOF mutation, then treated with EZH2 inhibitors to determine the effects of EZH2 inhibition on JAK2V617F GOF mutation-induced migration. The spheres were formed with Caco-cells with JAK2V617F GOF mutation and were used to observe the effects of JAK2V617F GOF mutation on IEC neoplasia formation. Results JAK2 and/or EZH2 overexpression are positively correlated with the probability of CRC initiation. JAK2 and EZH2 are highly expressed in the CRC tumoroids. Inhibition of EZH2 increases the CRC tumoroids growth and spheroid formation, and reduces budding numbers while reducing the proliferation of organoids from healthy crypts. Jak2 over-expression in IECs increases intestinal microadenoma, and depletion of EZH2 in the adenoma increases mutant β-catenin and severity of intestinal adenoma, suggesting that depletion of EZH2 enhances the malignancy of overexpressed Jak2-derived IEC neoplasia. Consistently, JAK2V617F GOF mutation increases HT29 or Caco-2 cell migration. Conclusion JAK2V617F GOF mutation can drive IEC neoplastic proliferation and adenoma, depletion of EZH2 in the adenoma leads to IEC malignant neoplasm. Our research suggests that loss of Ezh2 may synergistically cooperate with the Jak2V617F mutation in the pathogenesis of IEC malignant neoplasm. Citation Format: Nardana Esmaeili, Ahmed Bakheet, Wen Gao, Haifeng Li, Dan Cai, Xiaonan Han. Depletion of Ezh2 in the intestinal adenoma induced by JAK2V617F mutation leads to the malignant neoplasm [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 260.

Abstract 648: Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays

Abstract Essential biological signaling pathways affecting e.g., proliferation, differentiation, migration, and apoptosis are regulated by protein kinases. Deregulation of protein kinases is observed in many tumor cells and frequently the development and progression of human cancers and other diseases is found to be causally connected to altered activity of specific protein kinases. Therefore, protein kinases have become a prime molecular target for therapeutic intervention. Multiple small molecule inhibitors targeting different kinases are currently in clinical use for treatment of various types of cancer and other diseases. A first step in preclinical development of new compounds is testing candidate substances in biochemical in-vitro assays, either based on binding of the compounds to their target or based on alterations of the in-vitro activity of the target kinase monitored in biochemical activity assays. Depending on the actual mode of action of a compound, the relevance of the results of such in-vitro assays may depend on the actual setup of the assay. Mainly for reasons of cost and technical feasibility, the use of generic substrates for in-vitro kinase activity assays is well established. While many of those substrates are highly artificial and can only be considered as generic phosphate-group acceptors, they have been successfully used in the past in the development of many approved small molecule kinase inhibitors currently in clinical use. However, even while many kinases may show activity with generic substrates, for some physiological substrates are required. Members of e.g. the RAF family and other members of the MAPK pathway are highly substrate specific and will not show kinase activity with generic substrates. Such kinases which have been established with generic substrates are rarely switched to more physiological substrates as most often this will result in increased complexity and associated cost of preclinical compound development. We have compared the in-vitro activity of WEE1 using different in-vitro activity assay readouts like autophosphorylation, phosphorylation of generic substrates and phosphorylation of its physiological substrate CDK1. In addition, we examined the substrate specificity of WEE1 towards CDK1 alone and in complex with Cyclin B1. We compared the potency of a selection of WEE1 inhibitors when different substrates were used. While in early preclinical development the use of rather artificial biochemical assays has been successfully been applied in the past, it should be taken into consideration that at least for the kinase target in focus, the identification, establishment and use of a more physiologically relevant substrate may increase the value of early-stage compound screening results significantly. Citation Format: Daniel G. Mueller, Andreas Gericke, Frank Totzke, Carolin Heidemann-Dinger, Constance Ketterer, Diane Kraemer, Thomas Weber, Michael H. Kubbutat. Relevance of substrate selection for the results of biochemical WEE1 in-vitro kinase activity inhibition assays [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 648.

Postnatal survival of phrenic motor neurons is promoted by BDNF/TrkB.FL signaling.

The number of motor neurons (MNs) declines precipitously during the final trimester before birth. Thereafter, the number of MNs remains relatively stable, with their connections to skeletal muscle dependent on neurotrophins, including brain-derived neurotrophic factor (BDNF) signaling through its high affinity full length tropomyosin related kinase receptor subtype B (TrkB.FL) receptor. As a genetic knockout of BDNF leads to extensive MN loss and postnatal death within 1-2 days after birth; we tested the hypothesis that postnatal inhibition of BDNF/TrkB.FL signaling is important for postnatal PhMN survival. In the present study, we used a 1NMPP1-sensitive TrkBF616A mutant mouse to evaluate the effects of inhibition of TrkB kinase activity on phrenic MNs (PhMNs) numbers and diaphragm muscle (DIAm) fiber cross-sectional area (CSA). Pups were exposed to 1NMPP1 or vehicle (DMSO) from birth to 21 days old (weaning) via the mother's ingestion in the drinking water. Following weaning, the right phrenic nerve was exposed in the neck and the proximal end dipped in a rhodamine solution to retrogradely label PhMNs. After 24 h, the cervical spinal cord and DIAm were excised. Labeled PhMNs were imaged using confocal microscopy, while DIAm strips were frozen at ~1.5x resting length, cryosectioned and stained with H&E to assess CSA. We observed an ~34% reduction in PhMN numbers and increased primary dendrite numbers in 1NMPP1 treated TrkBF616A mice. The distribution of PhMN size (somal surface area) DIAm fiber cross-sectional areas did not differ. We conclude that survival of PhMNs during early postnatal development is sensitive to BDNF/TrkB.FL signaling.