Abstract BBO-10203 is a first-in-class clinical stage small molecule that disrupts the interaction between RAS and PI3Kα resulting in blockade of RAS-driven PI3Kα pathway activation. Unlike PI3Kα kinase inhibitors, BBO-10203 inhibits PI3Kα signaling without directly targeting the kinase domain, thereby preventing hyperglycemia, as insulin receptor signaling does not rely on RAS proteins. BBO-10203 suppresses PI3Kα activation in preclinical models harboring oncogenic KRAS and/or PIK3CA mutations and exhibits complete suppression of AKT phosphorylation (pAKT) in most HER2 amplified or overexpressing cell lines (HER2+). Importantly, this potent inhibitory effect on pAKT drives robust efficacy in HER2+ xenograft models in vivo. Although much is known about HER2 and RAS signaling in tumor cells, it is not clear how inhibiting the interaction of RAS and PI3Kα results in pAKT inhibition in HER2+ cells. Using the HER2+ KYSE-410 esophageal cancer cell line we demonstrate that BBO-10203 inhibits PI3Kα /AKT signaling in HER2+ cells via a mechanism that is mainly driven through non-canonical RAS proteins. CRISPR-mediated knock-in of the PIK3CA RAS-binding domain (RBD) mutations (T208D/K227A), which disrupt the interaction of RAS with PI3Kα, significantly reduced pAKT in the KYSE-410 model, strongly supporting the role of RAS proteins in driving pAKT signaling. Treatment with the panRAS inhibitor RMC-6236 showed little effect on pAKT, suggesting that canonical RAS (K-, H- and N-RAS) may not be important players in pAKT signaling. Among individual RAS isoforms screened via siRNA knockdown, only RRAS/RRAS2 knockdown significantly reduced pAKT, whereas knockdown of other RAS members either elevated pAKT (consistent with compensatory expression) or had no effect. Through co-immunoprecipitation experiments, we identified that RRAS2 is present in the same protein complex as HER2 and HER3, providing mechanistic evidence that this association may contribute to pAKT activation in HER2+ cells. Current efforts are focused on elucidating how BBO-10203 affects the interaction of HER2/3, p85/110α and RRAS2 in HER2+ and other RTK-overexpressing cell line models. Given its orthogonal method of pAKT inhibition and potent activity in HER2+ models, we hypothesized that BBO-10203 would show combination activity with standard-of-care HER2-targeted therapies in HER2+ xenograft models. Indeed, BBO-10203 enhanced the anti-tumor activity of HER2-targeted therapies (tucatinib, trastuzumab, or Enhertu) in vivo, leading to tumor regression, even in the trastuzumab-resistant JIMT-1 model. BBO-10203 has entered phase 1 clinical trials (NCT06625775) and is being evaluated in HER2+ breast cancers, both as a monotherapy and in combination with trastuzumab. Citation Format: Siyu Feng, Cindy Feng, Miranda Cabanski-Dunning, Cathy Zhang, Ming Chen, Erin Riegler, Daniel J. Czyzyk, Yue Yang, Rui Xu, Eli M. Wallace, Dhirendra K. Simanshu, Dwight V. Nissley, Frank McCormick, Kerstin W. Sinkevicius, James P. Stice, Pedro J. Beltran. The RAS: PI3Kα breaker BBO-10203 inhibits PI3Kα/AKT activity in HER2+ models through non-canonical RAS signaling blockade [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 6780.

Aberrant activation of the PI3Kα pathway is one of the most frequent oncogenic events across human cancers and leads to promotion of tumor cell growth, survival, glucose metabolism, and acute resistance to numerous standard of care cancer therapies. While small molecule inhibitors of the kinase activity of PI3Kα have been approved for the treatment of HR+ HER2- breast cancer patients with PI3Kα mutations, a large medical need remains to increase their safety profile due to dose-limiting on-target hyperglycemia. This toxicity may limit target coverage, the number of eligible patients, and the duration of treatment which could result in suboptimal efficacy. An alternative novel strategy is to block RAS-mediated activation of PI3Kα, a signaling event prevalent mostly in malignant cells. Previous elegant preclinical studies have established that RAS activation of PI3Kα is important in tumor cells but may not be involved in normal cell types controlling glucose metabolism because insulin activation of PI3Kα does not depend on RAS. Here, we report on a novel first-in-class covalent small molecule designed to block the PI3Kα:RAS protein-protein interaction and inhibit RAS-mediated activation of the AKT pathway via PI3Kα without the resultant hyperglycemia associated with direct inhibition of PI3Kα kinase activity. BBO-10203 covalently and selectively binds PI3Kα on cysteine 242 in the RAS binding domain, which prevents the interaction of PI3Kα with KRAS, HRAS, and NRAS. BBO-10203 shows potent cellular target engagement with an IC50 of 1.4 nM and full target engagement achieved at 10 nM in ER+ HER2amp breast cancer BT-474 cells, which harbor a PIK3CAK111N mutation. BBO-10203 potently inhibits phosphorylated AKT (pAKT) across a diverse panel of 18 human breast cancer cell lines with amplification of HER2 or mutations in PI3Kα with a mean EC50 of 3.2 nM. Transcriptional and post-translational cellular changes driven by treatment with BBO-10203 are consistent with PI3Kα-specific inhibition. BBO-10203 displays excellent drug-like properties and oral bioavailability. Single dose treatment of BT-474 tumor bearing mice with increasing doses (10 to 100 mg/kg) of BBO-10203 results in dose and time dependent inhibition of pAKT. In the BT-474 xenograft model, BBO-10203 daily oral dosing of 100 mg/kg results in 88% tumor growth inhibition. Importantly, BBO-10203 does not induce hyperglycemia or hyperinsulinemia during an oral glucose tolerance test in fasted male C57BL/6 mice, demonstrating independence of insulin receptor signaling from RAS. Since activation of AKT provides acute resistance to multiple cancer therapies, BBO-10203 has the potential to enhance the long-term responses in combination with targeted and conventional anti-cancer agents in multiple settings. In vitro and in vivo studies show that BBO-10203 significantly enhances the anti-tumor activity of the HER2-targeted antibody trastuzumab in the BT-474 (ER+, HER2amp, and PIK3CAK111N) and MDA-MB-453 (ER-, HER2+, and PIK3CAH1047R) breast cancer models. In addition, BBO-10203 also significantly enhances the anti-tumor activity of the SERD fulvestrant or the CDK4/6 inhibitor palbociclib in the MCF7 (ER+, HER2-, and PIK3CAE545K) breast cancer model. All of these combinations induce tumor stasis or regression through direct effects on tumor cells and are well tolerated. In conclusion, BBO-10203 blocks RAS-mediated activation of PI3Kα and strongly inhibits pAKT signaling in tumor cells without affecting glucose metabolism. BBO-10203 has entered phase 1 clinical trials and may provide clinical benefit without the limiting toxicities that have restricted the use of PI3Kα inhibitors. Citation Format: Kerstin Sinkevicius, James Stice, Erin Riegler, Siyu Feng, Cathy Zhang, Daniel J. Czyzyk, John-Paul Denson, Yue Yang, Sofia Donovan, Ming Chen, Cindy Feng, Brian P. Smith, Lijuan Fu, Ken Lin, Felice C. Lightstone, Anna E. Maciag, Keshi Wang, Dwight V. Nissley, Dhirendra K. Simanshu, Eli M. Wallace, Rui Xu, Frank McCormick, Pedro J. Beltran. BBO-10203, a first-in-class, orally bioavailable, selective blocker of the PI3Kα:RAS interaction inhibits tumor growth alone and in combination with standard of care therapies in breast cancer models without inducing hyperglycemia [abstract]. In: Proceedings of the San Antonio Breast Cancer Symposium 2024; 2024 Dec 10-13; San Antonio, TX. Philadelphia (PA): AACR; Clin Cancer Res 2025;31(12 Suppl):Abstract nr PS7-04.

Approved inhibitors of KRASG12C prevent oncogenic activation by sequestering the inactive, GDP-bound (OFF) form rather than directly binding and inhibiting the active, GTP-bound (ON) form. This approach provides no direct target coverage of the active protein. Expectedly, adaptive resistance to KRASG12C (OFF)-only inhibitors is observed in association with increased expression and activity of KRASG12C(ON). To provide optimal KRASG12C target coverage, we have developed BBO-8520, a first-in-class, direct dual inhibitor of KRASG12C(ON) and (OFF) forms. BBO-8520 binds in the Switch-II/Helix3 pocket, covalently modifies the target cysteine, and disables effector binding to KRASG12C(ON). BBO-8520 exhibits potent signaling inhibition in growth factor-activated states, in which current (OFF)-only inhibitors demonstrate little measurable activity. In vivo, BBO-8520 demonstrates rapid target engagement and inhibition of signaling, resulting in durable tumor regression in multiple models, including those resistant to KRASG12C(OFF)-only inhibitors. BBO-8520 is in phase 1 clinical trials in patients with KRASG12C non-small cell lung cancer. Significance: BBO-8520 is a first-in-class direct, small molecule covalent dual inhibitor that engages KRASG12C in the active (ON) and inactive (OFF) conformations. BBO-8520 represents a novel mechanism of action that allows for optimal target coverage and delays the emergence of adaptive resistance seen with (OFF)-only inhibitors in the clinic. See related commentary by Zhou and Westover, p. 455.

BBO-10203 is an orally available drug that covalently and specifically binds to the rat sarcoma (RAS)-binding domain of phosphoinositide 3-kinase α (PI3Kα), preventing its activation by HRAS, NRAS, and KRAS. It inhibited PI3Kα activation in tumors with oncogenic mutations in KRAS or PIK3CA and in tumors with human epidermal growth factor receptor 2 (HER2) amplification or overexpression. In preclinical models, BBO-10203 caused significant tumor growth inhibition across multiple tumor types and showed enhanced efficacy in combination with inhibitors of cyclin-dependent kinase 4/6 (CDK4/6), estrogen receptor (ER), HER2, and KRAS-G12C mutant, including in tumors harboring mutations in Kelch-like ECH-associated protein 1 (KEAP1) and serine/threonine kinase 11 (STK11). Notably, these antitumor effects occurred without inducing hyperglycemia, because insulin signaling does not depend on RAS-mediated PI3Kα activation to promote glucose uptake.

Abstract The glycine to cysteine mutation on codon 12 of KRAS (KRASG12C) is found in ~15% of non-small cell lung cancers and in a low percentage of colorectal and pancreatic adenocarcinomas. This activating mutation pushes the balance of cellular KRAS towards its active, GTP-bound (ON) state that signals downstream and drives cellular proliferation. Recently approved inhibitors of KRASG12C that bind and sequester the oncogenic protein in its inactive GDP-bound (OFF) state, have demonstrated clinical efficacy; however, median duration of response has been < 9 months. Rapid tumor cell adaptation to KRASG12C (OFF)-only inhibitors like sotorasib and adagrasib has been attributed to reactivation of MAPK signaling through increased RTK flux and KRASG12C gene amplification that result in increased KRASG12C (ON) presence and activity. To block rapid KRASG12C (ON)-driven adaptation to KRASG12C (OFF) inhibitors, we have developed a first-in-class, direct, small molecule covalent inhibitor of both KRASG12C (ON) and (OFF) states. BBO-8520 binds in the switch II pocket and covalently modifies both the (ON) and (OFF) forms of KRASG12C independently of any other partner proteins. Mass spectrometry analysis of KRASG12C covalent engagement by BBO-8520 shows complete modification of both KRASG12C states within 15 minutes, while sotorasib and adagrasib, only modify the (OFF) state. 31P NMR studies demonstrate that BBO-8520 inhibits KRASG12C (ON) by locking the GTP-bound protein in state 1, a conformation where it is unable to bind effectors. The ability to directly bind KRASG12C (ON) leads to potent (~30 nM) inhibition in an effector (Raf1) binding assay where inactive (OFF) inhibitors demonstrate no measurable potency. BBO-8520 displays highly significant binding to KRASG12C in a global cysteine proteome analysis and is 100x more selective for KRASG12C than for WT KRAS and other mutant isoforms, with no measurable activity against N- or H-RAS. Cellular signaling and viability assays show that BBO-8520 has sub-nanomolar potency against KRASG12C mutant cell lines. In effector binding assays, BBO-8520 rapidly and completely blocks the RAS-RAF1 interaction, clearly differentiating it from inactive (OFF) inhibitors that require longer times to allow for cycling to the inactive (OFF) state. Additionally, long-term clonogenic assays that detect the emergence of resistance in the presence of inhibitors show that BBO-8520 is at least 30x more potent than sotorasib and adagrasib at preventing outgrowth. Importantly, the presence of growth factors (e.g., EGF) that push KRASG12C into its (ON) state, and significantly lower the potency of inactive (OFF) inhibitors (>20x), have only minor effects on BBO-8520’s potency. Drug-like pharmacokinetic properties allow BBO-8520 to achieve strong dose- and time-dependent pharmacodynamic effects (>80% inhibition of pERK) following a single, oral dose in KRASG12C mutant tumor bearing mice. In vivo target engagement and pERK inhibition in the MIAPaCa-2 and H358 KRASG12C mutant tumor models resulted in durable tumor regressions at 10 mg/kg. Similarly, daily dosing of 10 mg/kg of BBO-8520 in the KrasG12C-p53 driven GEMM model for 6 weeks resulted in better than 50% lung tumor volume regression. BBO-8520’s potent activity against KRASG12C (ON) presents, for the first time, the potential opportunity to directly target all mutant KRASG12C in cells, including the active form of KRASG12C, enabling efficacy following complete or near-complete target inhibition. Citation Format: Anna E. Maciag, James Stice, Bin Wang, Alok Sharma, Albert Chan, Ken Lin, Devansh Singh, Marcin Dyba, Yue Yang, Saman Setoodeh, Brian P. Smith, Dana Rabara, Zuhui Zhang, Erik K. Larsen, Dom Esposito, John Paul Denson, Michela Ranieri, Mary Meynardie, Sadaf Mehdizadeh, Patrick Alexander, Maria Abreu Blanco, David Turner, Rui Xu, Felice C. Lightstone, Kwok Kin Wong, Dhirendra Simanshu, Keshi Wang, Andrew G. Stephen, Kerstin Sinkevicius, Dwight V. Nissley, Eli Wallace, Frank McCormick, Pedro J. Beltran. BBO-8520, a first-in-class, direct inhibitor of KRASG12C (ON), locks GTP-bound KRASG12C in the state 1 conformation resulting in rapid and complete blockade of effector binding [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 ND07.

Individual oncogenic KRAS mutants confer distinct differences in biochemical properties and signaling for reasons that are not well understood. KRAS activity is closely coupled to protein dynamics and is regulated through two interconverting conformations: state 1 (inactive, effector binding deficient), and state 2 (active, effector binding enabled). Here we use 31P NMR to delineate the differences in state 1 and state 2 populations present in wild-type (WT) and common KRAS oncogenic mutants (G12C, G12D, G12V, G13D, and Q61L) bound to its natural substrate GTP or a commonly used nonhydrolyzable analogue GppNHp. Our results show that GppNHp-bound proteins exhibit significant state 1 population, whereas GTP-bound KRAS is primarily (90% or more) in the state 2 conformation. This observation suggests that the predominance of state 1 shown here and in other studies is related to GppNHp and is most likely nonexistent in cells. We characterize the impact of this differential conformational equilibrium of oncogenic KRAS on RAF1 kinase effector RBD (RAS Binding Domain) binding and intrinsic hydrolysis. Through a KRAS G12C drug discovery, we have identified a novel small molecule inhibitor, BBO-8956, which is effective against both GDP and GTP-bound KRAS G12C. We show that binding of this inhibitor significantly perturbs the state 1 - state 2 equilibrium and induces an inactive state 1 conformation in GTP-bound KRAS G12C. In the presence of BBO-8956, RAF1 RBD is unable to induce a signaling competent state 2 conformation within the ternary complex, demonstrating the mechanism of action (MOA) for this novel, active-conformation inhibitor.

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.

Pharmacokinetic properties of our first-generation HIF-2α antagonist PT2385, including modest solubility, resulted in a high recommended phase 2 dose (RP2D) of 800 mg BID and motivated the pursuit of novel scaffolds which could improve solubility and formulation parameters with the goal of improved pharmacokinetics. Herein we disclose our successful efforts to identify such HIF-2α antagonists through an optimization strategy characterized by: (1) increasing the fraction of sp 3 hybridized carbons (Fsp 3 ), (2) replacing the aromatic portion of the indane core with pyridine heterocycles, and (3) improving a putative O lp →π* Ar interaction, an underutilized electrostatic contact in medicinal chemistry. These efforts emphasize the importance of employing multiple strategies in parameter optimization. In isolation, modifications to areas (1) and (2) improved solubility, but with the compromise of reduced potency. In area (3), understanding the importance of an O lp →π* Ar interaction, as documented through a wealth of crystal structures and retrospective calculations, proved essential in guiding SAR and identifying the trifluoromethyl group as a suitable replacement of the sulfone. Only by combining these three strategies could inhibitors with substantially improved solubility and comparable potency be discovered. Finally, the overall improvement in pharmacokinetic properties of the newly identified inhibitors is highlighted through a battery of ADME and in vivo data, including use of pharmacodynamic biomarkers indicative of HIF-2α antagonism.

Supplementary Data, Yeh, et al. from Biological Characterization of ARRY-142886 (AZD6244), a Potent, Highly Selective Mitogen-Activated Protein Kinase Kinase 1/2 Inhibitor

Supplementary method includes detailed experimental procedure for the synthesis of PT2385. Supplementary Figures 1-7 - Lack of cytotoxicity of PT2385 in cultured 786-O and A498 cells (1); PT2385 pharmacokinetics in CD-1 mice (2); Inhibition of HIF-2ï¡ gene expression and circulating hVEGFa in A498 mouse xenograft model after treatment with PT2385 (3); HIF-2ï¡ mRNA and protein levels in 786-O cells in vitro after treatment with PT2385 (4); Levels of HIF-1ï¡ and HIF-2ï¡ in patient-derived xenograft tumors (5); Body weight of mice in efficacy studies treated with either vehicle, PT2385 or sunitinib (6); Binding of PT2385 to rat HIF-2ï¡ as determined by ITC and inhibition of mouse kidney EPO gene expression with PT2385 treatment (7).

RAS mutations occur in approximately 20% of human cancers including the majority of pancreatic ductal adenocarcinoma (PDAC), half of colorectal cancers, and a third of all lung cancers. In cases of oncogenic activating RAS mutations, GTP hydrolysis is impaired, and the protein is preferentially held in the active GTP-bound state. When activated, RAS triggers multiple proliferative signaling cascades, including the MAPK/ERK and PI3K pathways, to induce cell growth, division, and differentiation. In solution, RAS exists in two interconverting GTP-bound conformational states, State 1 (inactive) and State 2 (active). State 2 is the active conformation since it binds to RAS Binding Domain (RBD) of effector proteins. Though the State 1 conformation is GTP-bound, it is unable to bind effectors and represents an inactive KRAS conformation. This State 1/State 2 equilibrium is closely coupled to the conformations (γ1 and γ2) exhibited by the γ-phosphate of GTP and its analogues. Solution-state 31P NMR spectroscopy captures and quantifies the γ1 and γ2 peaks representing State 1 and State 2 (Spoerner M et al., JBC 2010). We have initiated solution-state NMR investigation to determine the structural differences between wild-type (WT) and oncogenic mutant KRAS proteins (G12C, G12D, G12V, Q61L, and G13D) when bound to GTP and GppNHp. We quantified the State 1/State 2 equilibrium using 31P NMR. Our results show that GppNHp-bound KRAS proteins harboring oncogenic mutations differentially modulate the State 1/State 2 conformational equilibria resulting in remarkable divergence in conformational populations compared to WT protein; a notion for presence of mutation induced structural plasticity. Much work on the structural biology, biophysics and biochemistry of KRAS proteins is done when bound to the non-hydrolysable GTP analogue, GppNHp. Strikingly, our 31P NMR results show that GTP-bound KRAS elicits significant variations in State 1 and State 2 populations in comparison to GppNHp-bound proteins. These data suggest GppNHp binding induces a significant increase in the State 1 population; an observation that does not translate into GTP-bound KRAS. Our results provide additional insights into the differential binding affinities seen between WT/mutant KRAS and effector proteins (e.g., Hunter JC et al., Mol Cancer Res 2015) as well as interpretation for State 1 conformations in KRAS crystal structures when bound to mostly GppNHp and not GTP. We have also applied this 31P NMR approach to the evaluation of KRAS small molecule inhibitors that shift the population equilibrium to the State 1 (inactive) conformation in the protein-ligand complex. This demonstrates an intriguing therapeutic opportunity and novel mechanism of action that we are further exploring as part of our drug discovery pipeline. Citation Format: Alok K. Sharma, Marcin Dyba, Dominic Esposito, Bin Wang, Pedro J. Beltran, Eli Wallace, Dwight Nissley, Frank McCormick, Anna E. Maciag. Structural plasticity of KRAS oncogenic mutants – A case of misleading conclusions from GTP analogues [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr PR01.

Abstract Background: Mutations on codon 12 of KRAS are observed in many human cancers. KRASG12C mutations are found in ~15% of non-small cell lung cancers and in a low percentage of colorectal and pancreatic adenocarcinomas. These activating mutations in KRAS push cellular balance towards its active, GTP-bound state that signals downstream and drives cellular transformation. Recently approved inhibitors of KRASG12C that bind and sequester the oncogenic protein in its inactive, GDP-bound state, have demonstrated clinical efficacy in patients with KRASG12C cancers, including NSCLC, CRC and pancreatic adenocarcinoma. However, duration of response has been shorter than expected from the potent inhibition of a driver oncogene. This quick emergence of acquired resistance has been attributed to reactivation of MAPK signaling through multiple mechanisms, including RTK signaling and KRASG12C gene amplification, resulting in increased active, GTP-bound KRASG12C. Materials and Methods: Mass spectrometry was used to measure covalent modification of KRASG12C. Inhibition of the active state of KRASG12C was measured biochemically using a protein:protein interaction or RAS:RAF1 ELISA assays. Downstream consequences of inhibiting active-state KRASG12C was performed by western blotting. Results: In order to overcome active KRASG12C-driven resistance, we have developed direct, small molecule inhibitors of KRASG12C that inhibit both the active, GTP-bound and inactive, GDP-bound forms of KRASG12C through interactions with the switch II pocket, and independently of any other partner proteins. Mass spectrometry analysis of KRASG12C covalent engagement shows complete modification of both KRASG12C states, while sotorasib, adagrasib, and divarasib only modify the inactive, GDP-bound state. As expected, our active state inhibitors also show potent inhibitory activity in an effector (Raf1) binding assay where inactive, GDP-bound inhibitors demonstrate no measurable potency. Interestingly, during our work assessing the potency of these direct KRASG12C inhibitors of the active state, we discovered that employing the broadly used non-hydrolysable GTP nucleotide analogue GppNHP as a surrogate for the natural nucleotide results in overestimation of potency. We found that potency against the GppNHP-bound form of KRASG12C was consistently higher (3-10x) than when using GTP. These differences in potency between GppNHP and GTP were biologically meaningful as only compounds with strong activity against GTP-bound KRASG12C were able to demonstrate cellular activity consistent with inhibition of the active, GTP-bound state. Conclusion: We demonstrate here that inhibiting the active, GTP-bound state of KRASG12C is possible with switch II pocket binders and that using the natural, physiological nucleotide, GTP, in biochemical assays is indispensable to identify compounds with promising cellular activity that is differentiated from the inactive, GDP-bound molecules. Citation Format: Bin Wang, Alok Sharma, James Stice, Brian Smith, Marcin Dyba, Devansh Singh, John-Paul Denson, Dana Rabara, Erik Larsen, Yue Yang, Felice C Lightstone, Andrew Stephen, Dwight Nissley, Frank McCormick, Eli Wallace, Anna E Maciag, Pedro J Beltran. Use of the natural nucleotide, GTP, is essential for the identification of potent, active-state KRASG12C inhibitors that bind in the switch II pocket [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 A084.

Abstract Src homology 2 domain-containing phosphatase (SHP2), a ubiquitously expressed non-receptor tyrosine phosphatase, plays a critical role in the regulation of the MAPK signaling pathway and cellular proliferation. Activating mutations in SHP2 are associated with the development of multiple malignancies including leukemia, lung cancer and neuroblastoma. In addition, SHP2 promotes the conversion of oncogenic KRAS to its active GTP-bound state and it’s inhibition can enhance efficacy of GDP-KRASG12C inhibitors as well as other MAPK pathway inhibitors (RAF, MEK and ERK) which have suboptimal clinical efficacy as single agents. As a result, inhibition of SHP2 through genetic manipulation or pharmacological means has been shown to suppress tumor growth and presents an attractive potential avenue for the treatment of malignancies as monotherapy or in combination with other MAPK/PI3K inhibitors. Here we describe BBP-398, a potent, orally bioavailable allosteric small molecule inhibitor of SHP2. BBP-398 displays high selectivity against other phosphatases, kinases, GPCRs, transporters and hERG. Predicted human PK properties show good oral bioavailability with half-life of ~12-16 hours enabling continuous daily dosing and optimal therapeutic index in combination with other targeted therapeutics. In cellular assays, BBP-398 demonstrates potent pERK/DUSP6 inhibition and loss of viability across a panel of cell lines with active MAPK signaling, such as mutant EGFR and KRASG12C. In vivo, BBP-398 strongly suppresses RAS-ERK signaling in RTK- or RAS-driven xenografts. In the EGFR-dependent non-small cell lung cancer (NSCLC) HCC827 and esophageal squamous cell carcinoma KYSE-520 xenograft models, BBP-398 drives dose dependent efficacy consistent with the level of target inhibition. Detailed analysis of tumor response shows that efficacy is driven by maintaining better than 50% inhibition of pERK for most of the dosing interval. In addition to its strong single agent activity, BBP-398 also leads to enhanced efficacy in vitro and in vivo when used in combination with targeted therapeutics against driver MAPK genetic alterations, such as KRAS, EGFR or MET. Combination targeting, such as with the GDP-KRASG12C inhibitor sotorasib in the NSCLC NCI-H358 xenograft model, or with the mutant EGFR inhibitor osimertinib in the HCC827 erlotinib resistant (ER) xenograft model, drives strong suppression of MAPK activity and results in tumor regressions. Collectively, these findings highlight that SHP2 inhibition is a promising molecular therapeutic strategy in cancer which can potentially strongly suppress tumor growth as a single agent or in combination with other MAPK pathway inhibitors. Given its preclinical properties and projected favorable clinical pharmacokinetic profile, BBP-398 is currently being evaluated in a Phase 1/1b trial in patients with advanced solid tumors (NCT04528836). Citation Format: James P. Stice, Sofia Donovan, Yuting Sun, Nancy Kohl, Barbara Czako, Faika Mseeh, Paul Leonard, Anna Wade, Justin Lim, Phil Jones, Eli Wallace, Kerstin Sinkevicius, Pedro Beltran. BBP-398, a potent, small molecule inhibitor of SHP2, enhances the response of established NSCLC xenografts to KRASG12C and mutEGFR inhibitors [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 P207.

Pulmonary arterial hypertension (PAH) is a destructive disease of the pulmonary vasculature often leading to right heart failure and death. Current therapeutic intervention strategies only slow disease progression. The role of aberrant hypoxia-inducible factor (HIF)2α stability and function in the initiation and development of pulmonary hypertension (PH) has been an area of intense interest for nearly two decades.Here we determine the effect of a novel HIF2α inhibitor (PT2567) on PH disease initiation and progression, using two pre-clinical models of PH. Haemodynamic measurements were performed, followed by collection of heart, lung and blood for pathological, gene expression and biochemical analysis. Blood outgrowth endothelial cells from idiopathic PAH patients were used to determine the impact of HIF2α-inhibition on endothelial function.Global inhibition of HIF2a reduced pulmonary vascular haemodynamics and pulmonary vascular remodelling in both su5416/hypoxia prevention and intervention models. PT2567 intervention reduced the expression of PH-associated target genes in both lung and cardiac tissues and restored plasma nitrite concentration. Treatment of monocrotaline-exposed rodents with PT2567 reduced the impact on cardiovascular haemodynamics and promoted a survival advantage. In vitro, loss of HIF2α signalling in human pulmonary arterial endothelial cells suppresses target genes associated with inflammation, and PT2567 reduced the hyperproliferative phenotype and overactive arginase activity in blood outgrowth endothelial cells from idiopathic PAH patients. These data suggest that targeting HIF2α hetero-dimerisation with an orally bioavailable compound could offer a new therapeutic approach for PAH. Future studies are required to determine the role of HIF in the heterogeneous PAH population.

Background/AimExposure to polycyclic aromatic hydrocarbons (PAH) may increase risk of pediatric asthma. Effects of prenatal PAH exposure, specifically, are hypothesized but understudied. The ECHO PATHWAYS Consortium investigated these relationships in a large, diverse prospective cohort study.MethodsWe included 919 mother-child dyads from the CANDLE Study, a longitudinal pregnancy cohort set in Shelby County, TN. PAH metabolites were measured in second trimester urine and adjusted for specific gravity. Seven metabolites detected in >80% of women were included in analysis. When children were 4-6 years old, mothers completed the International Study on Allergies and Asthma in Childhood survey. Poisson regression with robust standard errors was used to estimate relative risk of current wheeze, current asthma, and ever asthma associated with each metabolite in separate models, adjusted for maternal age, race, education and other asthma risk factors. Effect modification by child sex and maternal asthma was assessed using interaction models. We did not adjust for multiple comparisons.ResultsParticipants were 66% Black, 44% White; 58% with high school education or less. On average, urinary PAH metabolite concentrations were higher than in other US cohorts. Mean (SD) child age at assessment was 4.3 (0.4) years. Prevalence of reported current wheeze and asthma and ever asthma was 19.4%, 16.1%, and 14.6%, respectively. In multivariable models, we observed no evidence that any metabolite was associated with any outcome. No differences by child sex were observed. Maternal asthma modified associations between 1/9-hydroxyphenanthrene and current asthma (RRmaternal asthma = 0.88; 95%CI: 0.78, 0.99 versus RRwithout maternal asthma = 1.09; 95%CI: 0.97, 1.23 per two-fold increase in exposure; pinteraction=0.012).ConclusionsResults of our analysis, the largest cohort study of prenatal PAH and childhood airway outcomes conducted to date, did not support the hypothesis that prenatal PAHs increases risk of early childhood asthma.

E7820 and indisulam are two examples of aryl sulfonamides that recruit RBM39 to Rbx-Cul4-DDA1-DDB1-DCAF15 E3 ligase complex, leading to its ubiquitination and degradation by the proteasome. To understand their mechanism of action, we performed kinetic analysis on the recruitment of RBM39 to DCAF15 and solved a crystal structure of DDA1-DDB1-DCAF15 in complex with E7820 and the RRM2 domain of RBM39. E7820 packs in a shallow pocket on the surface of DCAF15 and the resulting modified interface binds RBM39 through the α1 helix of the RRM2 domain. Our kinetic studies revealed that aryl sulfonamide and RBM39 bind to DCAF15 in a synergistic manner. The structural and kinetic studies confirm aryl sulfonamides as molecular glues in the recruitment of RBM39 and provide a framework for future efforts to utilize DCAF15 to degrade other proteins of interest.