Cellular pathways controlling chemotaxis, growth, survival, and oncogenesis are activated by receptor tyrosine kinases and small G-proteins of the Ras superfamily that stimulate specific isoforms of phosphatidylinositol-3-kinase (PI3K). These PI3K lipid kinases phosphorylate the constitutive lipid phosphatidylinositol-4,5-bisphosphate (PIP2) to produce the signaling lipid phosphatidylinositol-3,4,5-trisphosphate (PIP3). Progress has been made in understanding direct, moderate PI3K activation by receptors. In contrast, the mechanism by which receptors and Ras synergistically activate PI3K to much higher levels remains unclear, and two competing models have been proposed: membrane recruitment versus activation of the membrane-bound enzyme. To resolve this central mechanistic question, this study employs single-molecule imaging to investigate PI3K activation in a six-component pathway reconstituted on a supported lipid bilayer. The findings reveal that simultaneous activation by a receptor activation loop (from platelet-derived growth factor receptor, a receptor tyrosine kinase) and H-Ras generates strong, synergistic activation of PI3Kα, yielding a large increase in net kinase activity via the membrane recruitment mechanism. Synergy requires receptor phospho-Tyr and two anionic lipids (phosphatidylserine and PIP2) to make PI3Kα competent for bilayer docking, as well as for subsequent binding and phosphorylation of substrate PIP2 to generate product PIP3. Synergy also requires recruitment to membrane-bound H-Ras, which greatly speeds the formation of a stable, membrane-bound PI3Kα complex, modestly slows its off rate, and dramatically increases its equilibrium surface density. Surprisingly, H-Ras binding significantly inhibits the specific kinase activity of the membrane-bound PI3Kα molecule, but this minor enzyme inhibition is overwhelmed by the marked enhancement of membrane recruitment. The findings have direct impacts for the fields of chemotaxis, innate immunity, inflammation, carcinogenesis, and drug design.

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Alterations in the fibroblast growth factor receptors (FGFRs) have been identified as oncogenic drivers in many human cancers. Specifically, activating FGFR3 gene alterations are found in ~15% of metastatic bladder cancers. One pan-FGFR inhibitor has been approved for patients with FGFR3-altered bladder cancer and others are in clinical development. Importantly, all of these agents inhibit FGFR1-3 with approximate equal potency. Consequently, these agents are associated with toxicities driven by off-target inhibition of FGFR1 and FGFR2, potentially limiting efficacy. Additionally, existing drugs lose potency in the setting of FGFR3 gatekeeper mutations and acquired resistance due to gatekeeper mutations has been described. LOX-24350 is a highly potent and isoform-selective FGFR3 inhibitor with activity against wild-type FGFR3, FGFR3 activating mutations such as S249C, and FGFR3 gatekeeper (V555M) mutations. Here, we describe the preclinical profile of LOX-24350. Compound potency and selectivity were measured using enzyme fluorescent activity assays, and cell-based assays using in-cell western and cell-titer Glo methods. Tumor growth inhibition and PK/PD studies were performed in mice. LOX-24350 showed greater than 56-fold selectivity for FGFR3 S249C over wild-type FGFR1 in mechanistic cellular inhibition assays, while maintaining potency for the V555M gatekeeper mutation. In HEK293 cells stably expressing FGFR3 S249C and FGFR3 S249C/V555M, LOX-24350 inhibited FGFR3 phosphorylation with IC50 values of 3.1 and 5.0 nM, respectively, as compared to FGFR1 and FGFR2 IC50 values of 174.5 and 90.7 nM, respectively. Similarly, in NIH3T3 cells engineered to express FGFR3 S249C or FGFR3 S249C/V555M, LOX-24350 inhibited cell growth with IC50 values of 12.2 and 22.9 nM, respectively. LOX-24350’s isoform-selectivity was best exemplified in cancer cell line models, with IC50 values of 15.1 and 12.6 nM in RT112 (FGFR3-TACC3) and UMUC14 (FGFR3 S249C) cell lines, respectively, as compared to 4712.6 nM in DMS114 (FGFR1 amp). LOX-24350 demonstrated high oral bioavailability in preclinical species as well as favorable in vitro ADME properties. In vivo, LOX-24350 demonstrated tumor regressions in FGFR3-driven tumor models on par with pan-FGFR inhibitors, without body weight loss or hyperphosphatemia seen with pan-FGFR inhibitors. This wider therapeutic index is predicted to allow for greater efficacy in patients. These data demonstrate that LOX-24350 potently and selectively inhibits FGFR3, the S249C activating mutation, and its gatekeeper mutation, V555M, while sparing FGFR1, FGFR2, and other problematic off-targets. We hypothesize that this profile will lead to differentiated efficacy and tolerability for patients with FGFR3-driven cancers. An IND submission is planned for 2022. Citation Format: Joshua A. Ballard, Timothy Kercher, David Abraham, Ryan Brecht, Nathan A. Brooks, Thomas Buckles, Desta Bume, David Busha, Ernst Peder Cedervall, Kevin Condroski, Kevin Ebata, Severine Isabelle Gharbi, Robert Hazlitt, Tony Morales, Nisha Patel, Jessica Podoll, Kaveri Urkalan, Sandra Gomez Villalain, Shane Walls, Faith Watson, Peiyi Yang, Barbara J. Brandhuber, Steven W. Andrews. Preclinical characterization of LOX-24350, a highly potent and isoform-selective FGFR3 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P141.

Leukocyte migration is controlled by a membrane-based chemosensory pathway on the leading edge pseudopod that guides cell movement up attractant gradients during the innate immune and inflammatory responses. This study employed single cell and population imaging to investigate drug-induced perturbations of leading edge pseudopod morphology in cultured, polarized RAW macrophages. The drugs tested included representative therapeutics (acetylsalicylic acid, diclofenac, ibuprofen, acetaminophen) as well as control drugs (PDGF, Gö 6976, wortmannin). Notably, slow addition of any of the four therapeutics to cultured macrophages, mimicking the slowly increasing plasma concentration reported for standard oral dosage in patients, yielded no detectable change in pseudopod morphology. This finding is consistent with the well established clinical safety of these drugs. However, rapid drug addition to cultured macrophages revealed four distinct classes of effects on the leading edge pseudopod: (i) non-perturbing drug exposures yielded no detectable change in pseudopod morphology (acetylsalicylic acid, diclofenac); (ii) adaptive exposures yielded temporary collapse of the extended pseudopod and its signature PI(3,4,5)P 3 lipid signal followed by slow recovery of extended pseudopod morphology (ibuprofen, acetaminophen); (iii) disruptive exposures yielded long-term pseudopod collapse (Gö 6976, wortmannin); and (iv) activating exposures yielded pseudopod expansion (PDGF). The novel observation of adaptive exposures leads us to hypothesize that rapid addition of an adaptive drug overwhelms an intrinsic or extrinsic adaptation system yielding temporary collapse followed by adaptive recovery, while slow addition enables gradual adaptation to counteract the drug perturbation in real time. Overall, the results illustrate an approach that may help identify therapeutic drugs that temporarily inhibit the leading edge pseudopod during extreme inflammation events, and toxic drugs that yield long term inhibition of the pseudopod with negative consequences for innate immunity. Future studies are needed to elucidate the mechanisms of drug-induced pseudopod collapse, as well as the mechanisms of adaptation and recovery following some inhibitory drug exposures.

The lipid‐anchored GTPase Ras is well known for its role in oncogenesis, with around 25% of human tumors showing mutations in a Ras family member. One of the best characterized Ras effectors is the highly oncogenic lipid kinase phosphatidylinositol 3‐kinase (PI3K). PI3K phosphorylates the constitutive plasma membrane lipid PIP2, yielding the vital second messenger lipid PIP3. This PIP3 signal regulates many cell processes including migration and growth. Calmodulin also activates PI3K by tightly binding MARCKS, a PIP2 sequestering protein, freeing the lipid. The enhanced free PIP2 population serves as additional substrate for PI3K, increasing its activity. The findings presented here reveal that surprisingly, Ras activation of PI3K is blocked by the presence of calmodulin, with important implications for PIP3 signaling. We are observing these mechanisms using single molecule TIRF microscopy to probe the interactions and activities of single proteins on a supported target membrane surface. Preliminary evidence monitoring PI3K binding and lipid kinase activity at the single molecule level reveals that biologically relevant concentrations of calmodulin strongly inhibit Ras activation of PI3K. These results imply a new and additional level of control for an important and oncogenic signaling pathway.Support or Funding InformationFunding for this work was provided by the National Institutes of Health ( R01 GM063235 to J.J.F.), the Medical Research Council ( MC U105184308 to R.L.W.), the AstraZeneca/LMB Blue Skies Initiative ( MC A024‐5PF9G to R.L.W.), and St. Catharine's College (G.R.M.).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.