Abstract Background: The tumor suppressor p53, encoded by the TP53 gene, is a transcription factor that regulates genes involved in DNA repair, cell cycle arrest, senescence, and apoptosis. TP53 is the most frequently altered tumor suppressor gene with mutations occurring in over 50% of human cancers. TP53 mutations result in a loss of function, rendering cells incapable of responding to a variety of cellular stresses, making them susceptible to tumorigenesis. The Y220C hotspot mutation accounts for 1.8% of all p53 mutations, occurring in ∼1% of all solid tumors. p53 Y220C is a structural mutation that causes destabilization of the p53 protein. Small molecules that bind to a pocket formed by the Y220C mutation, but absent in the p53 wild-type protein, can stabilize the protein to restore normal function. Rezatapopt (PC14586) is the first p53 Y220C reactivator to enter clinical trials, where it is showing clinical benefit. However, because this molecule has modest potency, its efficacy may be limited by insufficient p53 reactivation despite a high RP2D (2000 mg QD), particularly in KRAS mutant patients. Materials and Methods: Antares’s p53 Y220C reactivators were evaluated biochemically in SPR binding and thermal shift assays. Cellular activity was assessed in target engagement, target gene expression and cell proliferation assays across a panel of p53 Y220C mutant cell lines. Additionally, chromatin binding and target gene expression conferred by p53 Y220C reactivation were analyzed by ChIP-seq and RNA-seq. Finally, the in vivo efficacy was studied in several human CDX and PDX p53 Y220C models. Results: Antares’s orally bioavailable p53 Y220C reactivators demonstrated a ≥10-fold improvement in in vitro potency over rezatapopt in target engagement, ChIP-seq and RNA-seq assays, with corresponding potency improvements in cell proliferation assays across a panel of p53 Y220C mutant cell lines. In vivo, these compounds were effective in inhibiting tumor growth in multiple p53 Y220C CDX models at a significantly lower dose than rezatapopt. Importantly, these p53 reactivators exhibit a marked improvement in activity in a KRAS mutant model, both in vitro and in vivo. Conclusions: The significantly improved potency of the novel p53 reactivators described here offers the opportunity to restore p53 function in less sensitive patient populations and meaningfully improve upon the clinical response profile of rezatapopt. Citation Format: Chiou-Hong Lin, Benjamin C. Milgram, Brendon Ladd, Weixue Wang, John P. Vu, Jun Jacob Hu, Yemin Lan, Keyur Gada, Heidi Koldsoe, Stephanie M. Reeve, Robert Hicklin, Mint Sirisawad, Brendan J. Hilbert, Jack A. Henderson, Simon A. Roberts, Gregory Kryukov, Hsu-Ping Kuo, Natasja Brooijmans, Angel Guzman-Perez, Darrin D. Stuart, Erica L. Jackson. Highly potent and mutant-selective p53 Y220C reactivators with best-in-class potential [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 7092.

After L858R and ex19del epidermal growth factor receptor (EGFR) mutations, ex20ins mutations are the third most common class of driver-mutations in non-small cell lung cancer (NSCLC). Unfortunately, first-, second-, and third-generation EGFR tyrosine kinase inhibitors (TKIs) are generally ineffective for ex20ins patients due to insufficient mutant activity and selectivity over wild-type EGFR, leading to dose-limiting toxicities. While significant advances in recent years have been made toward identifying potent EGFR ex20ins mutant inhibitors, mutant vs wild-type EGFR selectivity remains a significant challenge. STX-721 (53) is a potent, irreversible inhibitor of the majority of EGFR/HER2 ex20ins mutants and demonstrates excellent mutant vs wild-type selectivity both in vitro and in vivo. STX-721 is currently in phase 1/2 clinical trials for EGFR/HER2 ex20ins-driven NSCLC.

PURPOSE:Commonly occurring oncogenic mutations in EGFR render non-small cell lung cancers sensitive to approved EGFR-targeted drugs. EGFR in-frame exon 20 insertion (ex20ins) mutants are, however, less sensitive to such drugs. The efficacy of existing medicines may in part be limited by their selectivity for ex20ins mutations relative to wild-type EGFR, which is important for epithelial tissue homeostasis. EXPERIMENTAL DESIGN:We solved high-resolution crystal structures of clinically frequent ex20ins mutants to better understand how to achieve mutant-selective inhibition. Analyses of these structures demonstrate targetable ex20ins-mutant-biased dynamic protein states. To exploit these findings, we designed STX-721, an irreversible EGFR/ERBB2 ex20ins-mutant-selective inhibitor, and characterized its activity relative to clinical phase benchmarks across multiple preclinical assays. RESULTS:Structural analyses predict that STX-721 takes advantage of ex20ins-mutant-selective dynamic states. In carefully benchmarked biochemical, biophysical, and cellular assays, STX-721 demonstrated superior ex20ins-mutant selectivity relative to other tested benchmark clinical phase compounds and achieved ex20ins-mutant-selective tumor regression in vivo. CONCLUSIONS:These data highlight that STX-721 shows a high level of mutant EGFR selectivity across human preclinical cancer models and may provide an improved clinical efficacy versus adverse event profile relative to existing drugs.

Abstract Activating mutations in PI3Kα are among the most prevalent genetic aberrations in breast cancer. Constitutive signaling of PI3Kα is an established oncogenic driver in these cancers, as these mutations are predictors of response to alpelisib, an approved PI3Kα inhibitor, which inhibits both wild type and mutant PI3Kα. While inhibiting PI3Kα has proven to be efficacious, the concomitant inhibition of wild type enzyme in normal tissue results in metabolic dysfunction and ultimately dose-limiting toxicities. Selectively targeting the mutant PI3Kα enzyme is expected to inhibit tumor growth while sparing normal tissues, thus increasing the therapeutic index compared to non-mutant selective PI3Kα inhibitors. Therefore, we have developed STX-478, an allosteric, CNS penetrant, mutant-selective PI3Kα inhibitor. In a high-throughput viability screen, STX-478 selectively inhibited growth of cell lines with kinase domain and helical domain mutations in PI3Kα. In a panel of PI3Kα mutant CDX and PDX models, STX-478 provided efficacy that was similar or superior to higher than clinically achievable doses of alpelisib. Robust suppression of pharmacodynamic (PD) markers, pAKT, was observed in tumors, but not in skeletal muscle. Importantly, the efficacy achieved with STX-478 occurred at doses that did not cause metabolic dysfunction, in contrast to alpelisib which caused insulin release and suppressed glucose uptake in skeletal muscle in a 13C glucose tolerance test. Collectively, these data indicate that STX-478 has an expanded therapeutic window to selectively target mutant PI3Kα in vivo. In efficacy studies using human tumor xenograft models, STX-478 caused tumor regressions as a single agent. In multiple advanced ER+ breast cancer PDX models including models that are insensitive to the approved CDK4/6 inhibitor palbociclib, STX-478 combined with standards of care was highly efficacious and well tolerated. In a representative PDX model, STX-478 combined with the ER degrader fulvestrant and palbociclib was well tolerated for > 90 days of dosing in mice and resulted in durable tumor regressions that were superior to STX-478 alone. In conclusion, these data demonstrate STX-478 is a highly efficacious, well tolerated, mutant-specific PI3Kα inhibitor that can be combined safely with standards of care in breast cancer and provide the therapeutic benefits of PI3Kα inhibition without the side effects associated with inhibiting WT PI3Kα. These properties combined with the potential for CNS penetration and excellent pharmacokinetic profile in higher species make STX-478 a potentially best-in-class mutant-selective PI3Kα inhibitor. STX-478 is currently being evaluated in a Phase I clinical trial (NCT05768139). Citation Format: Trang Tieu, Leonard Buckbinder, David St. Jean, Samantha Manimala, Gregory Dowdell, Michael Huff, Jacob Alltucker, Erica Jackson, Angel Guzman-Perez, Darrin Stuart. STX-478 is a potentially best-in-class mutant-selective PI3Kα inhibitor that demonstrates robust efficacy in ER+ breast cancer models as monotherapy and in combination with standard of care agents [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 PO2-18-04.

Small-molecule drugs have enabled the practice of precision oncology for genetically defined patient populations since the first approval of imatinib in 2001. Scientific and technology advances over this 20-year period have driven the evolution of cancer biology, medicinal chemistry, and data science. Collectively, these advances provide tools to more consistently design best-in-class small-molecule drugs against known, previously undruggable, and novel cancer targets. The integration of these tools and their customization in the hands of skilled drug hunters will be necessary to enable the discovery of transformational therapies for patients across a wider spectrum of cancers.SIGNIFICANCE:Target-centric small-molecule drug discovery necessitates the consideration of multiple approaches to identify chemical matter that can be optimized into drug candidates. To do this successfully and consistently, drug hunters require a comprehensive toolbox to avoid following the "law of instrument" or Maslow's hammer concept where only one tool is applied regardless of the requirements of the task. Combining our ever-increasing understanding of cancer and cancer targets with the technological advances in drug discovery described below will accelerate the next generation of small-molecule drugs in oncology.

Supplementary Table 3: A total of 246 cell lines were evaluated for both SHP2 knockdown and SHP099 sensitivity. Data displays PTPN11 shRNA ATARIS Quantile Score and SHP099 IC50 and Amax for each cell line tested.

p-ERK MSD results in RAS mutant cells ectopically expressed ARAF variants

Proliferation IC50 values for various cell lines and cell line derivatives

Supplementary Table 4: List of cell lines evaluated for sensitivity to SHP099 (Tab 1) or an RTK-inhibitor (Tab 2) in 2D or 3D. Included is the lineage and the KRAS mutation status. Where data are blank, no viable data was available, or the cell line did not grow appropriately as a tumor spheroid.

(A) Immunoblot of p-RSK3 and qPCR for DUSP6 from MIA PaCa-2 xenografts collected 3 hours after the last dose from Fig 5C. (B) Immunoblot for the designated proteins from MIA PaCa-2 cells grown in 2D, 3D and from in vivo xenografts without compound treatment. Protein loading amount was normalized and verified by tubulin loading control. Each separate column represents an individual treated tumor. (C) Dependency of MET by DRIVE pooled shRNA screen (y axis, ATARIS Quantile score of less than -0.5 indicates a significant effect) and expression of HGF (x-axis) by RNAseq in pancreatic cancer cell lines in CCLE (n=21). (D) Immunoblot of p-RSK3 and qPCR for DUSP6 from KP4 xenografts collected 3 hours after the last dose from Fig 5E. Protein loading amount was normalized and verified by tubulin loading control. Each separate column represents an individual treated tumor (E) Immunoblot for the designated proteins from KP4 cells grown in 2D, 3D and from in vivo xenografts without compound treatment (F) Schematic of RTK-SHP2 signaling highlighting that SHP2 acts downstream of one or more activated RTKs to elicit downstream signaling in KRAS mutant and also additional SHP2-specific, non-MAPK signaling. SHP2 inhibition by SHP099 can serve as a surrogate for cancers where KRAS mutant cancers are dependent on upstream RTKs.

Response to encorafenib and binimetinib of BRAFV600 mutant melanoma PDX models.

PI3Kα is highly mutated in cancer resulting in hyperactivation of lipid kinase activity and downstream AKT signaling. H1047 is the most common site of oncogenic mutation and occurs in ~14% of all breast cancers. Initial therapeutic benefit of targeting PI3Kα was established with alpelisib, an alpha-selective PI3K inhibitor that is equipotent against wild-type and mutant forms. However, wild-type PI3Kα inhibition results in frequent dose-limiting toxicities including hyperglycemia, restricting the full potential of this drug. Selective targeting of H1047X-mutant PI3Kα is expected to both improve anti-tumor activity and reduce toxicity. STX-478 is an allosteric, CNS-penetrant, selective PI3Kα H1047X inhibitor, having excellent drug-like properties and exceptional kinome selectivity. STX-478 demonstrated minimal inhibition of CYP enzymes in vitro, supporting the potential for combinations with a wide range of therapeutics in breast cancer and a variety of other tumor types. STX-478 selectivity extended to the inhibition of other activating kinase domain mutations in biochemical assays. In a diverse panel of PI3Kα H1047X mutant cell lines, STX-478 selectively reduced the cellular levels of pAKT (S473) with a strong correlation between pAKT inhibition and cell viability (R = 0.8). In a high-throughput viability screen of 467 cancer cell lines, the presence of PIK3CA H1047X and other kinase domain mutations were the single strongest predictor of STX-478 sensitivity with potency superior to alpelisib. STX-478 also selectively inhibited the proliferation of cell lines with PI3Kα helical domain mutations, potentially due to the selective dependency of these cells on mutant PI3Kα. When combined with fulvestrant, lapatinib, or abemaciclib, STX-478 demonstrated synergistic anti-proliferative activity in cell lines with relevant ER/HER2 status. Unlike alpelisib, STX-478 did not impair glucose metabolism or cause insulin resistance at efficacious doses. In the T47D (PI3Kα H1047R) breast cancer model, STX-478 (100 mg/kg) monotherapy caused tumor regression whereas alpelisib caused only stasis. STX-478 combination with fulvestrant was well-tolerated, with more consistent and deeper tumor regression. Similar results were observed in a PI3Kα H1047R mutant ER+/HER2- PDX model, where fulvestrant monotherapy showed minimal activity, while combination with STX-478 yielded tumor regressions. In an ER+/HER2+ PDX model (PI3Kα H1047R/R108H), palbociclib and STX-478 (100 mg/kg) monotherapy resulted in similar efficacy while the combination was well tolerated and yielded tumor regression. Together these data indicate robust STX-478 monotherapy activity that was well tolerated and improved when dosed in combination with fulvestrant or CDK4/6 inhibitors. Finally, we investigated the effect of STX-478 treatment in an ER+ PDX model carrying a helical domain mutation. STX-478 treatment resulted in tumor growth inhibition at doses that did not result in metabolic dysfunction, suggesting that STX-478 may also be efficacious in treating PIK3CA mutant tumors with helical domain mutations. In summary, STX-478 efficacy was superior to alpelisib at a dose level that exceeds the clinically relevant exposure in mice without causing metabolic dysfunction. STX- 478 has a predicted low human dose, CNS exposure, low risk of DDI, and a predicted long half-life with minimal variation in peak-to-trough plasma concentrations which further supports a favorable therapeutic index. STX-478 has the potential to provide a best-in-class profile to improve outcomes in patients harboring tumors with prevalent PI3Kα H1047X mutations as well as other kinase and helical domain mutant tumors. The significant CNS exposure of STX-478 is expected to enable this treatment for patients with brain tumors and brain metastases not afforded by existing options. STX-478 is currently in IND enabling studies and is expected to enter human clinical trials in 2023. Citation Format: Leonard Buckbinder, David J. St. Jean, Brendon Ladd, Trang Tieu, Philip Jonsson, Jacob Alltucker, Samantha Manimala, Weixue Wang, Angel Guzman-Perez, Darrin D. Stuart, Gregory Dowdell. STX-478, a mutant-selective PI3Kα H1047X inhibitor clinical candidate with a best-in-class profile: Pharmacology and therapeutic activity as monotherapy and in combination in breast cancer xenograft models [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P4-07-04.

(A) Average body weight of mice treated with each BRAF inhibitor in the experiment depicted in Figure 1C; (B) Encorafenib plasma exposure in mice following the last dose from the experiment depicted in Figure 1C. To estimate daily exposure on the BID regimen, multiply the AUC x 2. Encorafenib is 98.6% protein bound in mouse plasma; (C) Gastric hyperplasia and hyperkeratosis observed in the forestomach of mice treated with BRAFi. Hematoxylin and eosin stains of forestomach sections from mice following 14 days of treatment with vehicle, encorafenib, dabrafenib, or vemurafenib from the study depicted in Figure 1C.

The efficacy of STX-478 is similar or superior to high-dose alpelisib across PI3Kα-mutant tumor xenografts while sparing insulin resistance (p.o., oral administration; q.d., daily). A, Final tumor volume percent change is represented for GP2d, Detroit 562, NCI-H1048, and HCC1954 CDX tumors. BrCa, breast cancer. B, Tumor volumes over time from PDX models (N = 3/group) harboring PI3Kα mutations in the kinase domain (ST1056: H1047R PI3Kα), kinase and helical domains (ST1799: E542K/H1065L PI3Kα), and helical domain (ST2652: E545K PI3Kα) treated with either vehicle, STX-478, or alpelisib. Statistical significance was calculated using two-way ANOVA and Dunnett multiple comparisons tests. End-of-study tumors were harvested 4 hours after the final dose, and pAKT (S473) was analyzed by Western blot. IsoSeq analysis of a tumor from model ST1799 showed that the E542K/H1065L PI3Kα mutations are in cis. AKT and pAKT (S473) were blotted separately, and a representative vinculin blot is shown.

Gene set enrichment results for the DEG clustering analysis using MSigDB and Metacore gene sets.

Supplementary Material and Methods. File contains the following: Transcriptome sequencing and analysis, Soft agar assay, 2D and 3D Cell proliferation screen and compound characterization information.

Abstract Background: EGFR mutations are well validated clinical targets in non-small cell lung cancer (NSCLC). Osimertinib, a highly-selective EGFR mutation-targeting covalent drug, is increasingly used in the first line setting for patients with NSCLC bearing EGFR L858R mutation or exon 19 deletions (ex19del). In a subset of these patients, co-occurring L858R/C797x or ex19del/C797x mutations (“double mutants”) are emerging as an on-target resistance mechanism, necessitating the need for new therapies, particularly in patients with CNS metastases. Materials and methods: STX-241 was tested across a panel of in vitro biochemical, cell signaling, and proliferation assays for potency against EGFR L858R and ex19del single mutants and the corresponding C797S double mutants. Additionally, STX-241 was tested for in vivo activity in mice bearing human NSCLC cell line xenografts NCI-H3255 (L858R) and PC-9 (ex19del), and in a PC-9-derived ex19del/C797S double mutant knock-in xenograft. Free CNS penetration (Kp,uu) was determined in non-tumor bearing mice. Osimertinib and gefitinib (an approved reversible EGFR inhibitor) were used as benchmark molecules across assays. Results: STX-241, an ATP-competitive reversible EGFR inhibitor representing novel chemical matter, demonstrated potent and selective inhibition of recombinant EGFR L858R/C797S mutant protein relative to wild-type, with increased mutant residence time relative to gefitinib. Potent (high picomolar to low nanomolar) inhibition of L858R, ex19del, L858R/C797S, and ex19del/C797S mutants was observed in proliferation assays using engineered Ba/F3 and human NSCLC cell lines. STX-241 demonstrated >150x selectivity for all tested EGFR mutants relative to wild-type EGFR in engineered Ba/F3 cells and human cancer cells. In these assays, STX-241 selectivity exceeded that of the benchmark reversible EGFR inhibitor gefitinib. Strong potency and double mutant selectivity was also observed for STX-241 in pharmacodynamic assays measuring EGFR pathway activation (pEGFR). STX-241 was well tolerated at doses of 15 mg/kg BID or 50 mg/kg QD in mice, where regression of EGFR exon 19 or 21 mutant NSCLC xenografts was observed, concomitant with EGFR pathway suppression. Notably, using an isogenic pair of PC-9 (EGFR ex19del) NSCLC xenografts that differ only by the presence of a C797S mutation, STX-241 demonstrates no drop off in antitumor activity in the presence of C797S. In mouse pharmacokinetic studies measuring free CNS penetration, Kp,uu measurements were comparable to Osimertinib, run in parallel as a benchmark. Conclusions: STX-241 demonstrates strong potency and selectivity against EGFR L858R/C797S and ex19del/C797S double mutants, as well as robust CNS penetrance. These data warrant further exploration of this potential best-in-class inhibitor in the clinic for patients progressing on Osimertinib via C797x mutation. Citation Format: Raymond A Pagliarini, Benjamin C Milgram, Deanna R Borrelli, Erin O'Hearn, Michael R. Huff, Brendon Ladd, Natasja Brooijmans, Weixue Wang, Petr Kuzmic, Angel Guzman-Perez, Darrin D Stuart. Identification of STX-241, a CNS-penetrant and mutant-selective EGFR inhibitor with activity on osimertinib-resistant C797x mutations [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr B168.