Abstract Introduction: ACR-2316 is a potent and selective dual WEE1/PKMYT1 inhibitor rationally designed using Acrivon's generative phosphoprotemics AP3 platform. Currently advancing in a Phase 1 clinical study in AP3-identified solid tumor types, ACR-2316 was engineered for superior single-agent activity and high selectivity resulting in potent DNA damage and complete tumor regression across preclinical in vivo models. This study demonstrates that ACR-2316 not only damages the nuclear and mitochondrial genomes, but also stimulates the innate immune system, leading to complete tumor regression and lasting immune memory in mice when combined with PD-L1 blockade. Results: To investigate the mechanisms of immune activation by ACR-2316, we performed AP3-based proteomic profiling of xenograft tumors from ACR-2316 treated mice and observed strong upregulation of innate immune signaling pathways, including type I interferon. These findings were further validated at the cellular level, where ACR-2316 treatment led to the activation of double stranded RNA and DNA sensing machinery RIG-I, MDA5 and cGAS. Furthermore, we found evidence of mitochondrial DNA fragmentation with ACR-2316 treatment, suggesting that this may serve as an additional immune sensor.In a syngeneic colorectal cancer model, ACR-2316 monotherapy resulted in dose-dependent tumor growth inhibition. In combination with anti-PD-L1, ACR-2316 exhibited striking synergy, leading to complete tumor regression in mice. To assess the durability of this response, tumor cells were re-injected into tumor-free mice that were previously treated with the combination therapy. All animals remained tumor-free for over 200 days through four sequential tumor re-challenges, demonstrating strikingly robust and durable immune memory. To dissect the mechanism of this durable immunity, we systematically depleted key immune cell subsets in tumor re-challenged mice. While depletion of either CD4+ or CD8+ T cells alone did not enable tumor growth, co-depletion of both subsets resulted in tumor formation. This suggests that the immune memory generated by the combination treatment of ACR-2316 and anti-PD-L1 is co-dependent on both CD4+ and CD8+ T cell subsets. Conclusions: Our findings reveal the dual role of ACR-2316 in inducing tumor intrinsic DNA damage and promoting immune activation through multiple immune sensing mechanisms, resulting in permanent immune memory co-dependent on CD4+ and CD8+ T cell subsets. This provides a strong rationale for combining ACR-2316 with immune checkpoint inhibitors in the clinical setting. ACR-2316 is in a phase 1 monotherapy trial and has already shown initial clinical activity with tumor shrinkage and a confirmed partial response during dose escalation across solid tumors predicted by our AP3 platform to be sensitive to ACR-2316. Citation Format: Taronish Dubash, Joelle Baddour-Sousounis, Amira Elbakry, Jessica Hopkins, Subodh Kumar, Yingchun Spring Liu, Ahmed Youssef, Kate Rappard, Ignacio Arribas Diez, Georgia Mista, Marc Isaksson, Francisco Santana, Luka Romero, Zachary Best, Nina Lipjankic, Anna-Maria Alves, Daphne García-López, Portia Lombardo, Calvin Yang, Emma Ahrman, Valentina Siino, Magnus E. Jakobsson, Helen Nilsson, Ayesha Murshid, Lei Shi, Caroline Wigerup, Michail Shipitsin, Joon Jung, David Proia, Kristina Masson, Peter Blume-Jensen. Treatment with ACR-2316, a potential first- and best-in-class WEE1/PKMYT1 inhibitor, combined with anti-PD-L1 induces complete tumor regression with durable immune memory [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 3789.

ACR-2316 is an internally discovered, clinical stage, potentially first- and best-in-class WEE1/PKMYT1 (MYT1) inhibitor specifically designed using Acrivon’s Predictive Precision Proteomics (AP3) platform. Here, we delineate the differentiated profile by which ACR-2316 achieves superior preclinical efficacy compared to benchmark WEE1 or MYT1 inhibitors. Intracellular global pathway activity modulation, kinase activity, and target engagement (TE) were assessed by mass-spectrometry based AP3, AlphaLISA, immunofluorescence, and Western blot (WB). Cell viability was assessed by CellTiter Glo (CTG). MYT1 stably overexpressing cells were generated by selection following transfection of MYT1 cDNA. Replication stress, DNA damage, and cell death were assessed by flow cytometry, CTG, and live cell imaging. In vivo efficacy and TE were evaluated in a human cancer cell line-derived xenograft (CDX) model. Through AP3 profiling, we previously uncovered WEE1 inhibitor-induced resistance mechanisms that were quenched by MYT1 inhibition. ACR-2316 was rationally designed using AP3 to suppress this resistance mechanism alongside potent WEE1 inhibition and robust activation of CDK1, CDK2, and PLK1, to induce potent tumor cell death. In cellular TE assays, ACR-2316 displayed more potent WEE1 TE than all benchmark WEE1 inhibitors (azenosertib, adavosertib, Debio0123), while simultaneously targeting MYT1. The impact of MYT1 TE was evidenced by a >3X loss of sensitivity induced by MYT1 overexpression, which was restored by co-treatment with the MYT1 inhibitor lunresertib. AP3 profiling in tumor cells and WB in human keratinocytes confirmed that ACR-2316, in contrast to lunresertib, does not modulate the canonical BRAF-MAPK pathway components, which may be involved in the dose-limiting skin rash observed clinically for lunresertib. Further differentiating from benchmark WEE1 or MYT1 inhibitors, ACR-2316 more potently induced DNA damage, premature mitotic entry, and cell death. Accordingly, ACR-2316 exhibited superior potency in cell viability compared to all benchmark inhibitors, including the WEE1/MYT1 inhibitor SGR-3515 (based on previously reported data), across a broad range of human cancer cell lines. In human CDX models, azenosertib and lunresertib resulted in only modest growth inhibition at their maximum tolerated/formulable doses, while ACR-2316 demonstrated complete tumor regression at doses associated with potent WEE1 and balanced MYT1 inhibition, highlighting the importance of dual targeting. WEE1 inhibitor-induced MYT1 activation constitutes a resistance mechanism that may limit the clinical efficacy of WEE1 inhibition. ACR-2316 is a potent, selective WEE1/MYT1 inhibitor that displays superior preclinical efficacy via its differentiated profile optimized by AP3 pathway-based structure-activity relationships in the intact cell. Acrivon’s ongoing Phase 1 ACR-2316 monotherapy trial in solid tumors has already demonstrated clinical activity during dose escalation prior to reaching Recommended Phase 2 Dose. Portia Lombardo, Anna-Maria Alves, Martina Pasetto, Mohamed Eldeeb, Zachary Best, Reina Improgo, Nina Lipjankić, Subodh Kumar, Ruban Cornelius, Kate Rappard, Uthira Muralitharan, Valentina Siino, Ignacio Arribas Diez, William Dahlberg, Shahrzad Rafiei, Kailash Singh, Ayesha Murshid, Joelle Baddour-Sousounis, Magnus E. Jakobsson, Michail Shipitsin, Helén Nilsson, Caroline Wigerup, Lei Shi, David Proia, Kristina Masson, Peter Blume-Jensen. ACR-2316 is a novel, differentiated, clinical-stage WEE1/PKMYT1 inhibitor designed by Acrivon’s Generative Phosphoproteomics AP3 Platform for optimal pro-apoptotic pathway effects in tumor cells resulting in superior preclinical activity [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C112.

Protein phosphorylation, a post-translational modification regulated by protein kinases, plays a crucial role in controlling most aspects of biological processes. However, understanding how the more than 500 human protein kinases selectively phosphorylate their specific substrates and contribute to disease-driving signaling pathways in the intact cell remain a critical challenge. The challenge becomes particularly acute in the analysis of large-scale mass spectrometry-based phosphoproteomic datasets, where most phosphorylation sites remain unassigned due to the lack of annotated kinase-substrate relationships (KSRs). To address this, Acrivon Therapeutics has developed a proprietary generative KSR prediction model (“KaiSR”) that is an ensemble of multiple transformer models fine-tuned on protein language model ESM-2. Each fine-tuned model reaches area under the precision-recall curve (AUPRC) values ranging from 83% to 92% on their respective holdout datasets. KaiSR can perform zero-shot predictions with high accuracy on unseen kinases as demonstrated through its performance on data from public and in-house kinase perturbation experiments. The predicted substrates from our model were further cross validated through in-house in vitro recombinant kinase assays on selected kinases. We have successfully applied KaiSR, a fully integrated and core component of Acrivon’s Predictive Precision Proteomics (AP3) Generative Phosphoproteomics platform, for streamlined, differentiated drug discovery, and the rational design and prioritization of novel compounds through pathway-based structure-activity relationship (SAR) in the intact cell. AP3 was recently used for designing the in-house discovered, potentially first- and best-in-class compound ACR-2316, a WEE1/PKMYT1 dual inhibitor currently in a phase 1b trial. Using KaiSR, we further confirmed the rationally AP3-designed activation of CDK1, CDK2, and PLK1 through potent and balanced inhibition of WEE1 and PKMYT1 in pre-clinical models with ACR-2316, which resulted in superior single agent efficacy of ACR-2316 via robust pro-apoptotic mitotic catastrophe. As an example of another application, we demonstrated KaiSR’s utility in identifying novel drug targets through a combined analysis of kinase activity inference based on our expanded KSRs on tumor-normal paired phosphoproteomic data from Clinical Proteomic Tumor Analysis Consortium (CPTAC) and patient matched survival data. In conclusion, Acrivon’s AP3 platform and generative AI model KaiSR uncover kinome-wide signaling networks generating unprecedented insights enabling a unique approach to target identification, streamlined drug discovery, and clinical development. Acrivon continues to leverage KaiSR and the full suite of its AP3 Generative Phosphoproteomics platform to expand and progress its proprietary pipeline of novel therapeutic candidates. Corey K. Xu, Bryan Thornlow, Ignacio A. Diez, Ahmed Youssef, Valentina Siino, Portia Lombardo, Zachary Best, Reina Improgo, Lei Shi, Magnus E. Jakobsson, Spring Y. Liu, Helen Nilsson, Caroline Wigerup, Joon Jung, Kristina Masson, Peter Blume-Jensen. Acrivon Therapeutics’ generative ensemble model (KaiSR) accurately predicts and expands proprietary, actionable kinase-substrate relationships globally for the human kinome [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr C053.

Proper cell cycle progression requires coordinated activation of the cell cycle kinases CDK1, CDK2, and PLK1. WEE1 and PKMYT1 kinases act as negative regulators of CDK1/2 through inhibitory phosphorylation of CDK1/2-Y15 and CDK1-T14. Using Acrivon’sPredictive Precision Proteomics (AP3) Generative Phosphoproteomics platform, we previously identified resistance mechanisms for WEE1 inhibition and demonstrated that resistance could be overcome by balanced co-inhibition of PKMYT1. Here, we present unbiased, global pharmacodynamic (PD) and in vivo analyses evaluating ACR-2316, our clinical stage, potentially first and best-in-class WEE1/PKMYT1 inhibitor rationally designed by AP3 and optimized for superior single agent activity. ACR-2316 was evaluated in PD and efficacy studies using ovarian and lung xenograft models. The clinical WEE1 inhibitors azenosertib and Debio0123, and the PKMYT1 inhibitor lunresertib were administered in parallel at maximum tolerated/formulable doses. Ovarian cancer xenograft tumors were characterized by AP3 phospho-and pan-proteomic profiling for unbiased analyses of global effects on cell cycle and signaling. Drug-regulated biomarkers were evaluated by xenograft tumors immunostaining. Effects of ACR-2316 and other compounds on cells were evaluated by FACS and Western blot. In ovarian and lung cancer xenograft models, treatment with ACR-2316 uniquely resulted in complete regressions, while treatment with WEE1 inhibitors (azenosertib, Debio0123) or the PKMYT1 inhibitor lunresertib, resulted in only stable disease at maximum tolerated/formulable doses. Using AP3, ACR-2316 was designed to induce potent activation of CDK1, CDK2, and PLK1 to ensure tumor cell death. PD analyses through AP3 profiling of human xenograft tumors treated with ACR-2316, compared to benchmark WEE1 or PKMYT1 inhibitors, revealed stronger activation of CDK1/2 and PLK-1, resulting in dysregulated mitotic signaling. Immunostaining of ACR-2316-treated xenograft tumors demonstrated stronger target engagement, increased phosphorylation of CDK1, CDK2, and PLK1 substrates, decreased WEE1 protein expression, and superior activation of DNA damage response, mitotic, and apoptotic markers compared to single-target WEE1 or PKMYT1 inhibitors. In cells, inhibition of CDK1, CDK2, or PLK1 largely reversed incomplete replication exit, premature mitosis, and apoptosis induced by ACR-2316. Co-inhibition of PLK1 with ACR-2316 restored protein expression of RRM2, a critical regulator of the DNA replication nucleotide pool. ACR-2316 is a potential first- and best-in-class dual inhibitor of WEE1 and PKMYT1, rationally designed using Acrivon’s proprietary AP3 platform to deliver superior single-agent efficacy. Potent activation of PLK1, together with CDK1/2, is essential for the superior activity of ACR-2316. ACR-2316 has shown clinical activity in its ongoing Phase 1b monotherapy trial in solid tumors during dose escalation. Subodh Kumar, Joelle Baddour-Sousounis, YingChun Liu Liu, Mohamed Eldeeb, Jessica Hopkins, Kate Rappard, Calvin Yang, Sam Saliba, Eunice Kwon, Georgia Mitsa, Martina Pasetto, Taronish Dubash, Portia Lombardo, William Dahlberg, Amira Elbakry, Zachary Best, Luka Romero, Maria R. Zabala, Nina Lipjankic, Valentina Siino, Ahmed Youssef, Yan He, Reina Improgo, Anil Prasad, Sibgat Choudhury, Magnus E. Jakobsson, Helen Nilsson, Caroline Wigerup, Lei Shi, Ayesha Murshid, Joon Jung, David Proia, Michail Shipitsin, Kristina Masson, Peter Blume-Jensen. Global pharmacodynamic effects uncovered with AP3 phosphoproteomic profiling of novel WEE1/PKMYT1 inhibitor ACR-2316 reveals the critical importance of PLK1 for ACR-2316’s superior preclinical activity and differentiated mechanism of action [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2025 Oct 22-26; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2025;24(10 Suppl):Abstract nr A097.

WEE1 and PKMYT1 are essential kinases that safeguard genomic integrity by regulating CDK1 and CDK2. ACR-2316 is a potent, selective WEE1/PKMYT1 inhibitor rationally designed using Acrivon’s Predictive Precision Proteomics (AP3) platform. ACR-2316 was uniquely designed, optimized, and advanced from initial lead to first-in-human dosing in < 15 months by AP3 to deliver complete tumor regression and pro-apoptotic tumor cell death through potent activation of CDK1, CDK2, and, importantly, PLK1. Here, we leveraged AP3-based global differential phosphoproteomic profiling and computational analyses to map and quantify the ACR-2316-regulated CDK1/2 and PLK1 substrates for unbiased understanding of drug action. AP3-based data-independent acquisition mass spectrometry was conducted using human cancer cells treated with vehicle or ACR-2316, -/+ CDK1 or CDK2 inhibition. Kinase activity inference analyzed by AP3 machine learning leveraging curated kinase-substrate relationships revealed that ACR-2316 strongly activated CDK1 and CDK2 followed by mitotic and DNA damage response kinases. We quantified hundreds of CDK1 and CDK2 substrates, regulated by ACR-2316, associated with S-phase DNA replication and replicative stress (CDK2) and with G2/M phase mitotic chromosome re-organization and segregation (CDK1). Multiparametric flow cytometry confirmed that ACR-2316 triggers DNA damage, premature mitosis, and S/G2-M cell cycle arrest. CellTiter-Glo and live cell imaging assays demonstrated robust inhibition of cell proliferation and potent cell death across human cancer cell lines. Inhibition of CDK1 or CDK2 partially rescued these phenotypes, confirming on-target mechanism of action identified through AP3. Chromatin immunoprecipitation sequencing showed unique intronic regions were susceptible to ACR-2316-induced double strand breaks. Finally, multiplexed immunofluorescence for alpha-tubulin and DNA revealed a large ACR-2316-induced increase in the number of mitotic aberrations in human cancer cells compared to vehicle control, and inhibition of only WEE1 or PKMYT1. The unique capabilities of our generative AI-driven AP3 platform used to rationally design ACR-2316 enabled a comprehensive analysis of ACR-2316-regulated CDK1/2-and PLK1-induced pathways underlying its differentiated, potent anticancer activity, as demonstrated in head:head preclinical studies against clinical benchmark inhibitors. ACR-2316 has advanced into the clinic ahead of schedule uniquely enabled by AP3 and is currently in a Phase 1 clinical trial in subjects with AP3-selected advanced solid tumors. Acrivon anticipates reporting initial ACR-2316 clinical Phase 1 data second half of 2025. Lei Shi, Martina Pasetto, Mohamed Eldeeb, Reina Improgo, Shahrzad Rafiei, Maria Rodriguez Zabala, Calvin Yang, Ahmed Youssef, Georgia Mitsa, Nina Lipjankić, Anna-Maria Alves, Portia Lombardo, Jessica Hopkins, Bryan Thornlow, William Dahlberg, Everett Hay, Ignacio Arribas Díez, Marc Isaksson, Zachary Best, Chris S. Balagtas, Corey Xu, Magnus E. Jakobsson, Helén Nilsson, Joon Jung, Caroline Wigerup, David A. Proia, Kristina Masson, Peter Blume-Jensen. Detailed mechanistic understanding of ACR-2316, a novel, clinical-stage WEE1/PKMYT1 inhibitor, rationally designed for superior single-agent activity through potent activation of CDK1, CDK2, and PLK1 using Acrivon’s machine learning-driven AP3 platform [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 357.

Abstract WEE1 and PKMYT1 kinases play essential roles in cell cycle checkpoints and DNA damage response. Genetic interaction has been documented between WEE1 and PKMYT1, and clinical trials have reported clinical activity with inhibitors of WEE1 (adavosertib, azenosertib) and PKMYT1 (lunresertib). Here, we report the discovery and characterization of ACR-2316, a dual inhibitor of WEE1 and PKMYT1 specifically designed for optimal selectivity through co-crystallography and superior single agent activity uniquely enabled by Acrivon Predictive Precision Proteomics (AP3). Mass spectrometry-based AP3 profiling was conducted across several novel WEE1 and PKMYT1 inhibitor leads generated through co-crystallography-based rational drug design. Selective leads originating from one series were further optimized using AP3 for biological structure-activity relationship analysis. AP3 profiling revealed WEE1 inhibitor-upregulated phosphorylation sites across a subset of phosphoproteins that were oppositely regulated (quenched) by PKMYT1 inhibitors. Consensus sites for these included CDK1 T14, a direct PKMYT1 phosphorylation site, as well as CHK1 S296. A particular lead compound, ACR-2316, demonstrated a desirable potent, balanced ratio of cellular WEE1 (IC50 = 2 nM, IC90 = 10 nM) and PKMYT1 (IC20 = 44 nM) inhibition resulting in superior activation of the mitotic kinases CDK1, CDK2, and PLK1 compared to adavosertib and lunresertib, based on annotated kinase substrate relationships. ACR-2316 is more selective than adavosertib, azenosertib, and lunresertib based on >200 kinases profiled by AP3 and 468 kinases assessed by KINOMEscan. Cell cycle analyses demonstrated a drastic ACR-2316-induced S-G2/M accumulation qualitatively distinct from adavosertib or lunresertib. In a 19-cancer cell line proliferation assay (CellTiter-Glo), ACR-2316 demonstrated greater potency in all cell lines tested compared to adavosertib and lunresertib (mean IC50 = 70, 252 and 364 nM, respectively). Superior anti-cancer activity of ACR-2316 was observed in 12 ovarian cancer patient-derived xenograft models tested ex vivo (CellTiter-Glo 3D) compared to azenosertib and lunresertib (mean IC50 = 9, 248 and 1620 nM, respectively). Across human tumor xenograft mouse models, oral administration of ACR-2316 demonstrated superior, durable, dose-dependent efficacy compared to azenosertib and lunresertib and was well tolerated at all doses. Complete responses observed with ACR-2316 were associated with strong WEE1 and intermediate PKMYT1 inhibition in tumors. In conclusion, ACR-2316 is a potent, selective dual WEE1/PKMYT1 inhibitor with superior single-agent activity compared to clinical WEE1 or PKMYT1 inhibitors. ACR-2316 is progressing through IND-enabling studies in preparation for clinical monotherapy development. Citation Format: Caroline Wigerup, Helén Nilsson, Lei Shi, Joon Jung, Joelle Baddour-Sousounis, Ruban Cornelius, Nina Lipjankic, Uthira Muralitharan, Valentina Siino, Ignacio Arribas Diez, Zachary Best, Martina Pasetto, William Dahlberg, Shahrzad Rafiei, Portia Lombardo, Magnus E. Jakobsson, Reina Improgo, Christina Scherer, John van Duzer, David A. Proia, Kristina Masson, Peter Blume-Jensen. ACR-2316: A potentially first-in-class, potent, selective WEE1/PKMYT1 inhibitor rationally designed for superior single agent activity through synergistic disruption of cell cycle checkpoints [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 1977.

Abstract ACR-368 (prexasertib) is a clinically advanced CHK1/2 inhibitor which has demonstrated durable activity across a proportion of patients with advanced solid tumors. Genomic biomarkers have proven unsuccessful in predicting response to ACR-368, limiting its clinical success. Using AP3, we previously developed a response-predictive proteomics-based test for ACR-368 (ACR-368 OncoSignature) for the identification of patients sensitive to ACR-368 monotherapy treatment as demonstrated in blinded preclinical studies. A Phase 2 clinical trial is ongoing where patients are treated with ACR-368 monotherapy based on OncoSignature-predicted sensitivity (NCT05548296). Here, we demonstrate the utility of AP3 for the identification of a key druggable resistance mechanism to ACR-368 and how to overcome that with low dose gemcitabine (gem), providing OncoSignature negative patients with a new potential therapeutic option. Five ovarian cancer cell lines were rendered durably resistant to ACR-368 by culturing in the presence of clinically relevant concentrations of ACR-368. Matched parental and ACR-368 resistant cell line pairs were profiled using AP3 mass spectrometry. Comprehensive pathway reconstitution and kinase activity analyses were performed to identify drug resistance mechanisms in an unbiased manner. Downregulation of DNA damage repair pathway activity was causally linked to the ACR-368-resistant phenotype, suggesting agents that restore replication stress around the CHK1/2 signaling axis may re-sensitize to ACR-368. To test this, a panel of ovarian cancer cell lines with intrinsic or drug-induced resistance to ACR-368 were screened for cell growth inhibition by ACR-368 combined with gem. Gem synergized with ACR-368 in 12/13 cell lines at low doses (1-40 nM). Moreover, Western blot analysis demonstrated protein markers of replication stress were induced by low dose gem (3-30 nM), suggesting a correlation between gem-induced replication stress and synergy with ACR-368. Comet assays showed that DMSO, gem (3 nM), or ACR-368 (100 nM) had minimal impact (4.4%, 4.5%, and 11.4%, respectively) on % comet tail DNA in ACR-368 resistant cells, while the gem combination led to 35% comet tail DNA (p<0.001). Finally, in a human tumor xenograft mouse model, low dose gem demonstrated a dose-dependent increase in replication stress markers (Cyclin E, pCHK1 S345) from 0.3-3 mg/kg, which allometrically scales to 1-10 mg/m2 in humans. These data supported a dose escalation Phase 1b/2 clinical study of low dose gem with ACR-368 to evaluate the efficacy and safety of the combination in ACR-368 OncoSignature negative patients (NCT05548296). This shows the potential of AP3 for unbiased elucidation of actionable drug resistance mechanisms and rapid clinical implementation in our trials, which have recently confirmed clinical activity. Citation Format: Helén Nilsson, Lei Shi, Magnus E. Jakobsson, Joelle Baddour-Sousounis, Shahrzad Rafiei, Uthira Muralitharan, Zachary Best, Valentina Siino, Francisco J. Santana, Ignacio Arribas Diez, Kailash Singh, Portia Lombardo, William Dahlberg, Subodh Kumar, Ahmed Youssef, Reina Improgo, Corey Xu, Joon Jung, Jung-Min Lee, Ayesha Murshid, Michail Shipitsin, Jesper V. Olsen, Kristina Masson, David A. Proia, Caroline Wigerup, Peter Blume-Jensen. Acrivon predictive precision proteomics (AP3) uncovers mechanism of resistance to ACR-368, a clinical-stage CHK1/2 inhibitor, and identifies rational combination treatment [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 4749.

Supplementary Table 3. siRNA oligos used for transfections.

Figure legends corresponding to Supplementary Figures 1-4.

Supplementary Table 2. Nucleotide sequences of primers used for qPCR reactions.

Supplementary Table 1. Antibodies used for western blotting.

Supplementary Figure 1. Knockdown of IGF2R does not affect HIF2A transcription.

Supplementary Figure 3. Knockdown or chemical inhibition of Akt does not affect HIF2A expression.

Supplementary Figure 4. Inhibition of mTORC1 has no effect on HIF-2α downstream target genes.

Supplementary Figure 2. Hypoxia increases Akt activity in neuroblastoma cells.

Abstract Introduction ACR-368 (prexasertib) is a potent and selective CHK1/2 inhibitor with demonstrated durable, single-agent activity in patients with advanced solid tumors. Genomic biomarkers have been unsuccessful in predicting response to ACR-368 due in part to the complex genetic changes in cancer that translate into dysregulated protein signaling pathways. To address this challenge, we sought to identify protein-based, predictive biomarkers that measure the ACR-368-sensitive dysregulated signaling driving tumorigenesis using our proprietary approach, AP3 (Acrivon Predictive Precision Proteomics). Methods ACR-368 anticancer activity was measured in a panel of ovarian cancer cell lines using CellTiter-Glo. Quantitative phosphoproteomics was performed on ACR-368 sensitive and resistant ovarian cancer cell lines using data-independent acquisition mass spectrometry (DIA-MS). Kinase activity inference analysis and signaling pathway analyses were conducted to uncover ACR-368-regulated pathways associated with tumorigenesis. A quantitative, multiplexed immunofluorescent (IF) assay was developed based on three biomarkers, termed ACR-368 OncoSignature. Ovarian PDX studies were conducted in female athymic nude or CB-17 Scid mice. Results ACR-368 demonstrated diverse anti-proliferative activity in ovarian cancer cell lines. Phosphoproteome-profiling analysis from ACR-368 sensitive and resistant cells exposed to ACR-368 yielded >17,000 confidently localized phospho-sites (localization probability > 0.75), with 8272 being significantly regulated by ACR-368 (FC > 1.5, Q-value < 0.05, Limma t-test). Unsupervised hierarchical clustering analysis revealed overrepresentation of ATM/ATR and CDK1/2-associated substrate sites in the upregulated group, while the downregulated group showed overrepresentation of CHK1-associated sites. Pathway enrichment analysis highlighted the differential regulation of the homology-directed repair (HDR) pathway between sensitive and non-sensitive ovarian cancer cells. Through analysis of HDR-related signaling networks, three functionally orthogonal predictive biomarkers were assembled into a quantitative multiplex in situ assay for FFPE tissue that provides a direct readout of a tumor’s dependency on the signaling axis inhibited by ACR-368. Quantitation of these biomarkers in cancer cell lines using the IF-based OncoSignature demonstrated efficient classification based on ACR-368 sensitivity. The assay accurately predicted sensitivity to ACR-368 across ovarian cancer PDX models with an AUC of 0.9 (95% confidence interval: 0.71 to 1; p-value = 0.025). Conclusions Employing our AP3 platform, which combines MS-based phosphoproteomics and quantitative multiplexed-IF staining of drug-tailored biomarkers, we developed a response-predictive test for ACR-368 that enables identification of responders to ACR-368 treatment. A clinical trial (NCT05548296) evaluating the efficacy of ACR-368 based on the OncoSignature test status is currently recruiting in patients with platinum-resistant ovarian, endometrial, and urothelial cancer. Citation Format: Caroline Wigerup, Michail Shipitsin, Ayesha Murshid, Lei Shi, Magnus E. Jakobsson, Dorte Bekker-Jensen, Sibgat Choudry, James Dunyak, David Proia, Jesper V. Olsen, Kristina Masson, Peter Blume-Jensen. Identification of biomarkers predictive of sensitivity to the CHK1/2 inhibitor ACR-368 using high-resolution phosphoproteomics and development of an ACR-368-tailored patient responder identification 3-marker test, ACR-368 OncoSignature [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 C002.

Acute myeloid leukemia (AML) is a heterogeneous disease with variable patient responses to therapy. Selinexor, an inhibitor of nuclear export, has shown promising clinical activity for AML. To identify the molecular context for monotherapy sensitivity as well as rational drug combinations, we profile selinexor signaling responses using phosphoproteomics in primary AML patient samples and cell lines. Functional phosphosite scoring reveals that p53 function is required for selinexor sensitivity consistent with enhanced efficacy of selinexor in combination with the MDM2 inhibitor nutlin-3a. Moreover, combining selinexor with the AKT inhibitor MK-2206 overcomes dysregulated AKT-FOXO3 signaling in resistant cells, resulting in synergistic anti-proliferative effects. Using high-throughput spatial proteomics to profile subcellular compartments, we measure global proteome and phospho-proteome dynamics, providing direct evidence of nuclear translocation of FOXO3 upon combination treatment. Our data demonstrate the potential of phosphoproteomics and functional phosphorylation site scoring to successfully pinpoint key targetable signaling hubs for rational drug combinations.