Imlunestrant, elacestrant, and fulvestrant mediated ER degradation, PGR gene inhibition and cell proliferation inhibition in T-47D parental and T-47D ESR1-Y537N cells.

In vivo single agent imlunestrant efficacy in ESR1-mutant and wild type xenograft and PDX breast cancer models.

Statistical analysis of imlunestrant and fulvestrant efficacy alone and in combination with abemaciclib, alpelisib, and everolimus in ESR1-wild type and mutant xenograft and PDX breast cancer models

Imlunestrant PK/PD and uterotrophic activity.

Statistical survival analysis and body weight change in MCF-7 intracranial model treated with imlunestrant or other SERDs.

The impact of first-generation covalent KRASG12C inhibitors has been reduced due to the development of drug resistance, tolerability and challenges combining with immunotherapy. We designed olomorasib, a next-generation GDP-binding KRASG12C inhibitor, for nanomolar potency as well as selectivity over wild-type inhibition. In both in vitro and in vivo models of KRASG12C -mutant cancers, olomorasib reduces RAS activity and pERK levels, leading to substantial and significant tumor growth inhibition. Additionally, olomorasib combined with immune checkpoint inhibitors demonstrates greater anti-tumor activity compared to monotherapy. Furthermore, we demonstrate that olomorasib binds tightly to KRASG12C even in the presence of clinically relevant second site mutations, a known mechanism of resistance and limitation to currently approved KRASG12C inhibitors. These findings suggest that olomorasib could be effective for patients with KRASG12C mutant cancers either as monotherapy or in combination with immunotherapy. Olomorasib monotherapy and combination treatments are currently being investigated clinically.

In vivo imlunestrant efficacy alone and in combination with abemaciclib, alpelisib, and everolimus in CDX breast cancer models.

Biochemical and in vitro characterization of imlunestrant.

SMARCA4 alterations occur in approximately 10% of NSCLC and are associated with worse overall survival [1]. SMARCA4 mutations, which also occur in multiple cancer types, create a dependency on SMARCA2, resulting in synthetic lethality when SMARCA2 is selectively inhibited [2]. LY4050784 (FHD-909) is a selective SMARCA2 inhibitor that has demonstrated robust synthetic lethality with single-agent tumor regressions across multiple murine xenograft models of SMARCA4 mutated NSCLC [3]. A phase 1 study of LY4050784 is ongoing (NCT06561685). Of note, SMARCA4 deficiency frequently co-occurs with KRAS mutations in NSCLC and has been shown to result in poor clinical outcomes upon treatment with KRAS inhibitors or with immune checkpoint inhibitors [1, 4]. Here, we report the synergistic activity of LY4050784 in combination with standard-of-care agents and several novel KRAS inhibitors that are currently in development. To assess the combination benefits of LY4050784 in NSCLC, we evaluated combination treatments with chemotherapies, pembrolizumab, and KRAS inhibitors in vitro and in vivo using preclinical SMARCA4-mutant human cancer models. LY4050784 demonstrated robust activity in vitro and in vivo when combined with standard lung cancer chemotherapies (paclitaxel, cisplatin, and pemetrexed). Additionally, we evaluated LY4050784 in combination with pembrolizumab in a humanized KRAS- and SMARCA4-mutant lung cancer xenograft model. Here, LY4050784 sensitized the tumors to pembrolizumab resulting in synergistic combination benefit, while pembrolizumab alone had no effect on tumor growth as compared to the vehicle control. Finally, we explored the activity of LY4050784 in combination with both mutant-selective KRAS G12C and G12D inhibitors (olomorasib and LY3962673, respectively) and the isoform-selective pan-KRAS inhibitor LY4066434. In SMARCA4 and KRAS co-mutated lung, pancreatic, and colon cancer models, the KRAS inhibitors resulted in synergistic increases in activity both in vitro and in vivo when combined with LY4050784. These results demonstrate that LY4050784 exhibits robust antitumor activity in combination with chemotherapy, pembrolizumab, and multiple KRAS inhibitors in preclinical models of SMARCA4-mutant cancers. Investigation of these combinations clinically is warranted. Alessi et al., 2021, doi: 10.1016/j.jtho.2021.03.024 Hoffman et al., 2014, doi: 10.1073/pnas.1316793111 Lee, J.Y. et al. Poster presented at the AACR Annual Meeting; April 8, 2024; San Diego, CA Negrao et al., 2023, doi: 10.1158/2159-8290.CD-22-1420. Nathan A. Brooks, Shuang Chen, Candace Langan, Bryan Perria, Bonita D. Jones, Hai Hu, Mia Lei, Renan Escalante Chong, Wei Zheng, Rowena Almonte-Baldonado, Xiaodong Huang, Robert Stephen Flack, Janice Y. Lee, Kevin Wilson, Anke Klippel, Laurie LeBrun. LY4050784, a selective inhibitor of SMARCA2, demonstrates synergistic activity in combinations with pembrolizumab or KRAS inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3779.

Introduction: PI3Kα mutations are oncogenic drivers found in approximately 40% of HR+ breast cancers as well as multiple other solid tumors. Approved treatments for PI3Kα mutant-driven HR+ breast cancers include direct inhibitors of either PIK3CA or downstream AKT. However, these inhibitors block PI3K pathway signaling in unmutated host tissues resulting in hyperglycemia, rash, and GI toxicity. Herein we describe the preclinical profile of LY4045004, a next-generation, mutant-selective inhibitor of PI3Kα. The compound exhibits potent inhibition across the most common PI3Kα mutations in breast cancer (H1047R, E545K, and E542K) while sparing wild type (WT) PI3Kα. Strong efficacy in multiple PI3Kα-mutant breast cancer models is demonstrated while avoiding the hyperglycemia and insulin increases characteristic of non-selective inhibitors. Methods: PI3Kα biochemical potency was measured using ADP Glo kinase assay. PI3Kα on-rate and off-rate were measured using Transcreener FI assay. PI3Kα cell potency was measured using cell viability and signal transduction assays. Tumor growth inhibition, pharmacokinetic (PK), and pharmacodynamic effects were assessed in in vivo studies using PI3Kα mutant cell-derived xenograft (CDX) and patient-derived xenograft (PDX)-models. Plasma insulin and C-peptide levels were measured using ELISA. Results: LY4045004 is a potent, allosteric inhibitor of both kinase and helical PI3Kα mutations in enzyme and cell-based assays, inhibiting growth and signaling responses in multiple mutant-driven breast cancer cell lines. The compound is selective for mutant PI3Kα over WT PI3Kα in both enzyme and cell-based assays and exhibits a very slow off-rate from PI3Kα. LY4045004 demonstrates favorable in vitro ADME properties and excellent PK with high oral bioavailability across preclinical species. In vivo, orally administered LY4045004 demonstrated dose-dependent tumor regressions in PI3Kα H1047R-driven and E545K-driven breast cancer models both as monotherapy and in combination with fulvestrant without inducing hyperglycemia (no significant increase in insulin or C-peptide) or body weight change. Tumor pharmacodynamic analyses confirmed strong pathway inhibition at doses that caused regressions. Additionally, LY4045004 demonstrated tumor regressions in PI3Kα H1047R and E545K PDX models. LY4045004 was well-tolerated at efficacious doses in mice both as a single agent and in combination, and well-tolerated in rat, dog, and cynomolgus monkey toxicology studies. Conclusions: The favorable potency, selectivity, and PK properties of LY4045004 are predicted to result in efficacy with improved tolerability in patients with prevalent PI3Kα helical- and kinase-domain mutant HR+ breast cancers. Global regulatory submissions are planned in the first half of 2025. Citation Format: Raymond Gilmour, Andrew L. Faber, Weihua Shen, Harold B. Brooks, Lisa J. Kindler, Sarah M. Bogner, Loredana Puca, Viviana Volta, Michele Dowless, Jennifer R. Stephens, Anke Klippel, Rui Wang, Divya Ramchandani, Parisa Zolfaghari, Kannan Karukurichi, Alex Gousie, Robert Bondi, Gereint Sis, Ross Wallace, Ronee Baracani, Nathan Wright, Gabrielle Kolakowski, Laurie LeBrun, Steven W. Andrews. Preclinical characterization of LY4045004, a next-generation, mutant-selective PI3Kα inhibitor [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 P4-12-24.

KRAS is altered in ∼16% of all cancers and is an oncogenic driver in NSCLC, pancreatic, colorectal, and other cancers. Next generation KRAS inhibitors, designed to target multiple oncogenic KRAS mutations, have the potential to improve outcomes for the large burden of KRAS-mutated disease. Importantly, preclinical data suggest that a pan-KRAS approach that spares wildtype HRAS and NRAS may avoid the skin toxicities associated with pan-RAS inhibition.1 This is particularly important in the context of treating KRAS-mutated colorectal cancer in combination with EGFR inhibition, which also carries skin toxicity. Next generation KRAS inhibitors could treat a wide range of patients including the approximately 35% of those with NSCLC who are at increased risk of developing CNS metastases. LY4066434 is an orally bioavailable, highly potent pan-KRAS inhibitor that has high selectivity over HRAS and NRAS.2 Here, we report the antitumor activity of LY4066434 in patient-derived xenograft (PDX) and intracranial cancer models. We evaluated the activity of LY4066434 in PDX models harboring different KRAS mutations (both common and less frequent) and representing diverse histologies. LY4066434 showed robust anti-tumor activity, ranging from significant tumor growth inhibition to strong regression, in KRAS-driven PDX models of NSCLC, pancreatic, colorectal, and gastric cancers. Furthermore, we analyzed the activity of LY4066434 in combination with standard-of-care therapies. LY4066434 when combined with cetuximab showed enhanced antitumor activity in PDX models of NSCLC, pancreatic, and colorectal cancers with various KRAS mutations. Additionally, the combination of LY4066434 with chemotherapies also demonstrated greater efficacy in KRAS-mutant PDX models of endometrial and ovarian cancers. Finally, evaluation of LY4066434 activity in two KRAS-mutant orthotopic brain tumor models of NSCLC showed that LY4066434 inhibited tumor growth and promoted survival in these brain tumor models. Taken together, these results demonstrate the potential of LY4066434 as a promising therapeutic option for a range of cancer types driven by KRAS mutations including NSCLC, pancreatic, and colorectal cancers. LY4066434 is currently being investigated clinically (NCT06607185). 1. Manousaridis I. et al. 2013. J Eur Acad Dermatol and Venereol, 27: 11-18. 2. 2. Prieto L. et al. AACR-NCI-EORTC. Oct 11-15, 2023. Boston, MA. Hong Gao, Youyan Zhang, Chun Ping Yu, Wei Guo Xu, Binghui Li, Huimin Bian, Manuj Tandon, Tao Wang, Trent R. Stewart, Mark H. Bender, Wenyu Ming, Megan A. Johnson, Lisa M. Kays, Madeleine Leonard, Mark A. Castanares, Andrew Capen, Arthur Xintian You, Wen Ting Bian, Lourdes Prieto, Timothy Kercher, Laurie LeBrun, Anke Klippel, Chandrasekar Iyer, Xueqian Gong. LY4066434, an oral small molecule pan-KRAS inhibitor, demonstrates robust anti-tumor activity in KRAS-mutant models, including in the CNS [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 4375.

Abstract Targeting of the estrogen receptor (ER) by antiestrogens is the standard of care for patients with ER+ HER2− advanced/metastatic breast cancer. Although antiestrogens that degrade ERα (fulvestrant) or block estrogen production (aromatase inhibitors) have improved patient outcomes, clinically important challenges remain related to drug administration, limited bioavailability, lack of brain exposure, and acquired resistance due to ESR1 mutations. These limitations indicate a need for more robust ER-targeted therapies. Here, we discovered and characterized imlunestrant, a next-generation potent, brain-penetrant oral selective ER degrader. Imlunestrant degraded ERα and decreased ERα-mediated gene expression both in vitro and in vivo. Cell proliferation and tumor growth in ESR1 wild-type (WT) and mutant models were significantly inhibited by imlunestrant. Combining imlunestrant with abemaciclib (CDK4/6 inhibitor), alpelisib (PI3K inhibitor), or everolimus (mTOR inhibitor) further enhanced tumor growth inhibition, regardless of ESR1 mutational status. In an ER+ breast cancer intracranial tumor model, imlunestrant prolonged survival compared with vehicle or alternative selective ER degrader therapies. Together, these findings support the potential of imlunestrant to degrade ERα and suppress the growth of ESR1-WT and mutant breast cancer, including brain metastatic tumors. Significance: Imlunestrant, a next-generation, brain-penetrant oral ERα degrader, displays potent activity in ESR1 wild-type and mutant breast cancer, enhances combination activity with standard-of-care agents, and inhibits growth of ER+ intracranial tumors.

Abstract KRAS G12D mutations are activating oncogenic events that occur in approximately 35%, 13%, and 4% of pancreatic, colorectal, and non-small cell lung cancers, respectively, and less commonly in other cancers. We previously demonstrated that LY3962673 is a highly potent inhibitor of KRAS G12D and is selective against wild-type (WT) KRAS in mutant -cell lines and -in vivo models. Here, we describe the mechanism by which LY3962673 inhibits KRAS G12D and report a more comprehensive evaluation of LY3962673 activity across a panel of genetically and histologically diverse cancer cell lines, as well as in multiple patient-derived xenograft (PDX) models. LY3962673 is a non-covalent KRAS G12D inhibitor with high affinity binding to KRAS G12D-GDP (Kd 0.071 nM) compared to KRAS G12D-GTPγS (Kd 26.7 nM). In a panel of cancer cell lines with KRAS G12D mutations, non-G12D mutations or KRAS WT, LY3962673 selectively suppressed MAPK signaling and inhibited the growth of KRAS G12D mutant cancer cells while sparing KRAS WT and non-G12D mutant cells. Sensitivity to LY3962673 varied among the KRAS G12D-mutant cells tested, suggesting that not all cell lines share the same dependence on KRAS G12D for their growth and survival. Furthermore, in multiple KRAS G12D-mutant PDX models representing diverse tumor types, LY3962673 demonstrated anti-tumor activities, ranging from tumor growth inhibition to robust tumor regression. LY3962673 also showed enhanced efficacy when combined with other anti-cancer agents. Taken together, the findings underscore the potential of LY3962673 as a monotherapy or in combination with other anti-cancer agents, as a promising oral therapeutic option for a range of cancer types with KRAS G12D mutations. Citation Format: Xueqian Gong, Hong Gao, Mark H. Bender, Wenyu Ming, Youyan Zhang, Trent R. Stewart, Chun Ping Yu, Wei Guo Xu, Aurthur Xintian You, Wen Ting Bian, Binghui Li, Tao Wang, Huimin Bian, Manuj Tandon, Andrew Capen, Rachel N. Cavitt, Bryan D. Anderson, Wayne Bocchinfuso, Anke Klippel, Chandrasekar Iyer. LY3962673, an oral, highly potent, mutant-selective, and non-covalent KRAS G12D inhibitor demonstrates robust anti-tumor activity in KRAS G12D 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 3316.

Abstract Smad2 and Smad3 are intracellular mediators of transforming growth factor β (TGFβ) signaling that share various biochemical properties, but data emerging from functional analyses in several cell types indicate that these two Smad proteins may convey distinct cellular responses. Therefore, we have investigated the individual roles of Smad2 and Smad3 in mediating the cytostatic and proapoptotic effects of TGFβ as well as their function in epithelial-to-mesenchymal transition. For this purpose, we transiently depleted mouse mammary epithelial cells (Nme) of Smad2 and/or Smad3 mainly by a strategy relying on RNaseH-induced degradation of mRNA. The effect of such depletion on hallmark events of TGFβ-driven epithelial-to-mesenchymal transition was analyzed, including dissolution of epithelial junctions, formation of stress fibers and focal adhesions, activation of metalloproteinases, and transcriptional regulation of acknowledged target genes. Furthermore, we investigated the effect of Smad2 and Smad3 knockdown on the TGFβ-regulated transcriptome by microarray analysis. Our results identify Smad3 as a key factor to trigger TGFβ-regulated events and ascribe tumor suppressor as well as oncogenic activities to this protein. (Mol Cancer Res 2009;7(8):1342–53)

Background Phosphoinositide 3-kinase alpha (PI3Kα) H1047R mutations are activating oncogenic events that occur in ~15% of breast cancers (BC). Early generation PI3Kα inhibitors target both wild-type (WT) and mutant PI3Kα and, as a result, their efficacy may be limited by on-target WT PI3Kα-mediated toxicities, including hyperglycemia, skin rash, and diarrhea. LOXO-783 is an oral, potent and highly mutant-selective, brain-penetrant allosteric PI3Kα H1047R inhibitor that is currently in phase 1 testing. Preclinically, LOXO-783 as a single agent is highly selective for PI3Kα H1047R over WT PI3Kα and other PI3K isoforms, and induces single-agent tumor regressions in ER+, HER2- PI3Kα H1047R-mutant breast cancer models without causing hyperglycemia or increases in plasma insulin/C-peptide. LOXO-783 also demonstrates brain penetration in vivo with dose-dependent tumor growth inhibition in brain metastasis models. Here we report the efficacy of LOXO-783 with SOC treatments in preclinical breast cancer models. Methods Cell proliferation assays and in vivo studies to evaluate combination effects were performed in various PI3Ka H1047R mutant HR+, HER2- and triple negative breast cancer models. For each combination in vitro, a combination index (CI) based on the Loewe Additivity Method was calculated (CI>2 antagonism, 0.5>CI< 2 additivity, CI< 0.5 synergy). For the in vivo studies, the Bliss Independence Method was used to evaluate the statistical significance of the combination effects. Results Combining LOXO-783 with either fulvestrant (FUL; CI at 50% inhibition = 0.28) or imlunestrant (CI at 50% inhibition = 0.43) showed increased efficacy in cell proliferation assays using the HR+, HER2-, PI3Kα H1047R-mutant T47D model. LOXO-783 also demonstrated an additive effect in combination with these endocrine therapies in vivo. Similar results were observed in a T47D model engineered to express ESR1 D538G, as well as in an HR+, HER2- PI3Kα double in-cis mutant model (H1047R/D350G) also harboring ESR1 D538G and derived from a patient who had progressed on prior letrozole plus taselisib. Moreover, LOXO-783 plus abemaciclib demonstrated an additive effect in vitro (CI at 50% inhibition = 0.61), and in T47D xenograft and PDX models in vivo. Combinations of LOXO-783 with abemaciclib plus imlunestrant resulted in a mean tumor regression of –48.1%; LOXO-783 with abemaciclib plus FUL showed mean tumor regression of –43.9% in T47D xenografts. Similar efficacy was not observed in the absence of LOXO-783 (mean tumor regression was –7.3% with abemaciclib plus imlunestrant, and 3.2% with abemaciclib plus FUL). We observed comparable results in PDX models. These data collectively demonstrate the additive effect of LOXO-783 with SOC treatments. Extending these studies to additional treatment settings, LOXO-783 was similarly efficacious as a single agent in abemaciclib-resistant and abemaciclib/FUL double-resistant models, and was additive in combination with paclitaxel in a triple negative breast cancer model in vitro and in vivo. Conclusions LOXO-783 shows additive effects when combined with SOC in breast cancers harboring the PI3Kα H1047R-mutation (as single or double in-cis mutations) in both HR+ and triple negative settings. LOXO-783 is also efficacious in ESR1 mutant as well as in abemaciclib and abemaciclib/FUL double-resistant models. A phase 1 trial of LOXO-783 alone or in combination with anticancer therapies is ongoing (PIKASSO-01; NCT05307705). Citation Format: Loredana Puca, Michele S. Dowless, Carmen M. Perez-Ferreiro, Maria Jesus Ortiz-Ruiz, Gregory P. Donoho, Andrew Capen, Lysiane Huber, Sarah M. Bogner, Dongling Fei, Jason R. Manro, Chun Ping Yu, Wei Guo Xu, Rui Wang, Shuang Chen, Mark A. Hicks, Parisa Zolfaghari, Andrew Faber, Raymond Gilmour, Monica D. Ramstetter, Matthew T. Chang, Maria Jose Lallena, Xuequian Gong, David M. Hyman, Lillian M. Smyth, Barbara J. Brandhuber, Barry S. Taylor, Anke Klippel. LOXO-783: A potent, highly mutant selective and brain-penetrant allosteric PI3Kα H1047R inhibitor in combination with standard of care (SOC) treatments in preclinical PI3Kα H1047R-mutant breast cancer 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 P4-08-02.

Supplementary Data from Smad3 Is a Key Nonredundant Mediator of Transforming Growth Factor β Signaling in Nme Mouse Mammary Epithelial Cells

Abstract Phosphoinositide 3-kinase alpha (PI3Kα) H1047R mutations are activating oncogenic events that occur in ~15% of advanced breast cancers. While there is one PI3Kα inhibitor FDA-approved for patients with PI3Kα-mutated breast cancer, and many others in clinical development, all of these agents inhibit wild-type PI3Kα and its mutated form with approximate equal potency. As a result, their efficacy is limited by toxicities associated with on target wild-type PI3Kα inhibition, notably hyperglycemia as well as cutaneous and GI toxicity. LOX-22783 is a highly potent, mutant-selective and brain-penetrant allosteric PI3Kα H1047R inhibitor. Here, we describe the preclinical profile of LOX-22783. H1047R selectivity was measured using biochemical kinase activity and cell-titer Glo and signal transduction assays. Tumor growth inhibition, pharmacokinetic and pharmacodynamic effects were assessed in in vivo studies using xenograft and patient-derived xenograft (PDX)-models. LOX-22783 inhibited growth and signaling responses in multiple H1047R-driven breast cancer cell lines and demonstrated high selectivity for H1047R mutated PI3Kα (EC50 values <5 nM) relative to wild-type PI3Kα (EC50 >250 nM) as well as the other wild-type PI3K isoforms (beta, gamma, and delta, all EC50 >250nM). In enzyme and cell-based assays, LOX-22783 dissociated from PI3Kα H1047R at a slower rate (3-6 hrs) compared to alpelisib (≤10 mins), potentially allowing for extended inhibition of PI3Kα H1047R by LOX-22783. LOX-22783 also normalized the EGF-stimulated membrane-localization of PI3Kα H1047R while alpelisib did not. LOX-22783 was highly kinome-selective when assayed at 3 µM, with no inhibitory activity on 17 lipid kinases or 374 protein kinases. In preclinical species, LOX-22783 demonstrated high oral bioavailability, including exposure in the CNS, a common site of metastases for patients with breast cancer. In vivo, LOX-22783 demonstrated dose-dependent tumor regression in H1047R breast cancer models without inducing hyperglycemia or other toxicities. Tumor pharmacodynamic analyses confirmed successful pathway inhibition. At doses resulting in 90% pathway inhibition, tumor regressions of ≥60% were observed. This wide therapeutic index is predicted to allow for maximizing dose intensity and efficacy in patients, without wild-type PI3Kα inhibition limiting target coverage for the H1047R mutant form. These data demonstrate that LOX-22783 potently and selectively inhibits mutant H1047R, but not wild-type PI3Kα, or other PI3K isoforms. LOX-22783 binds to an allosteric pocket distinct from the ATP binding site used by the approved and investigational PI3Kα inhibitors. We hypothesize that this profile will lead to differentiated efficacy and tolerability for patients with PI3Kα H1047R-mutated cancers, with the additional potential to address brain metastases. An IND submission is planned for 2022. Citation Format: Anke Klippel, Rui Wang, Loredana Puca, Andrew Lee Faber, Weihua Shen, Shripad V. Bhagwat, Kannan Karukurichi, Feiyu Fred Zhang, Carmen Perez, Ramon Rama, Ana Ramos, Yi Zheng, Zahid Bonday, James Thomas, Harold B. Brooks, Lisa J. Kindler, Sarah M. Bogner, Parisa Zolfaghari, Mark Hicks II, Sophie Callies, Brian Mattioni, Laurie LeBrun, Jim Durbin, Erin Anderson, Chris Mayne, Edward Kesicki, Gabrielle Kolakowski, Steven W. Andrews, Barbara J. Brandhuber. Preclinical characterization of LOX-22783, a highly potent, mutant-selective and brain-penetrant allosteric PI3Kα H1047R 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 P142.

Extracellular adenosine, produced through the activity of ecto-5'-nucleotidase CD73, elicits potent immunosuppressive effects, and its upregulation in tumor cells as well as in stromal and immune cell subsets within the tumor microenvironment is hypothesized to represent an important resistance mechanism to current cancer immunotherapies. Soluble CD73 (sCD73) enzymatic activity measured in patient serum or plasma at a baseline is reported to have prognostic as well as predictive relevance, with higher sCD73 activity associating with poor overall and progression-free survival in melanoma patients undergoing anti-PD1 monoclonal antibody treatment. Here, we report a novel NMR-based method that measures the ex-vivo kinetics of sCD73 activity with high specificity and reproducibility and is suitable for future high-throughput implementation. Unlike the existing assays, this method has the advantage of directly and simultaneously measuring the concentration of both the CD73 substrate and product with minimal sample manipulation or special reagents. We establish the utility of the assay for measuring the activity of sCD73 in human serum and show a strong linear correlation between sCD73 protein levels and enzyme activity. Together with our finding that sCD73 appears to be the predominant activity for the generation of adenosine in human blood, our results demonstrate a link between activity and protein levels that will inform future clinical application.