Oncogenic Driver Research Articles

Abstract 3223: Embryonic ectoderm development (EED) inhibitor APG-5918 (EEDi-5273) and MDM2 inhibitor alrizomadlin (APG-115) synergistically inhibit tumor growth in preclinical models of prostate cancer (PCa)

Abstract Advanced PCa, especially castration-resistant PCa, represents an urgent unmet treatment need. Dysregulation of the polycomb repressive complex 2 (PRC2), which mediates trimethylation of histone H3 at lysine 27 (H3K27me3) and leads to gene silencing, is common in PCa and correlates with poor prognosis. EED, a core PRC2 component, is crucial for histone methyltransferase activity. MDM2, a negative regulator of p53, is often amplified or overexpressed in PCa and associated with metastasis and poor clinical outcomes. We evaluated antitumor activity and molecular mechanisms of EED inhibitor APG-5918/EEDi-5273 and MDM2 selective inhibitor alrizomadlin in preclinical PCa models. Human castration-resistant (22Rv1) and androgen-dependent (LNCaP) PCa cell lines were used in antiproliferation assays. Cell viability was determined by CellTiter-Glo luminescent assays. Western blotting was used to explore mechanisms of action. Subcutaneous 22Rv1 and LNCaP cell line-derived xenograft (CDX) models were established to evaluate antitumor effects of APG-5918 and alrizomadlin in vivo. Single-agent APG-5918 or alrizomadlin had limited inhibition on PCa cell growth; combined, they demonstrated synergistic antitumor activity. In the 22Rv1 CDX model, single-agent alrizomadlin (100 mg/kg) or APG-5918 (100 mg/kg) administered for 26 days exerted no/limited antitumor activity, with treatment-to-control (T/C) values of 93.39% and 49.89%, respectively. In contrast, the doublet enhanced antitumor activity (T/C value, 33.22%), with a synergistic index of 1.40. In the LNCaP CDX model, APG-5918 (100 mg/kg) administered for 28 days exhibited minimal antitumor effects (T/C value on last day of dosing, 80.32%). Alrizomadlin (100 or 50 mg/kg) showed limited antitumor activity (T/C value, 47.61%). Combined treatment significantly enhanced antitumor activity (T/C value, 15.69%), with a synergistic index of 2.44. Regarding putative mechanisms, western blot analyses showed that APG-5918 downregulated oncogenic drivers and DNA methylation factors (UHRF1, DNMT1) and on-target-related pharmacodynamic markers (H3K27me3, EED, EZH2). Alrizomadlin markedly downregulated UHRF1 and DNMT1, and upregulated p53 and p21 expression. Combined treatment further enhanced downregulation of DNMT1, UHRF1, cell cycle pathway proteins (pRb, CDK6), antiapoptotic protein MCL-1, and synergistically increased cleavage of PARP-1, a marker of apoptosis. In summary, we show that targeting EED and MDM2 synergistically enhances antitumor activity in PCa cell lines and CDX models. Mechanistically, this combination likely modulated pathways related to DNA methylation, cell cycle, and apoptosis. The findings provide a scientific rationale for future clinical development of APG-5918 and alrizomadlin for PCa patients. Citation Format: Yan Yin, Yun Yang, Eric Liang, Baisong Li, Hengbang Wang, Dajun Yang, Yifan Zhai. Embryonic ectoderm development (EED) inhibitor APG-5918 (EEDi-5273) and MDM2 inhibitor alrizomadlin (APG-115) synergistically inhibit tumor growth in preclinical models of prostate cancer (PCa) [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 3223.

Abstract 5064: Wild type KRAS amplification as de novo oncogenic driver in non-small cell lung cancer

Abstract Introduction: Oncogenic KRAS mutations define subsets of non-small cell lung cancer (NSCLC) with unique clinicopathologic and genomic features, and the recent development of direct KRAS inhibitors has changed the treatment landscape of these patients (pts). However, whether KRAS amplification is oncogenic and defines unique subset of potentially targetable NSCLCs is currently unknown. Methods: Clinicopathologic and genomic data were abstracted from multiple independent cohorts of NSCLCs which underwent tumor genomic profiling at the Dana-Farber Cancer Institute (Cohort 1), AACR-GENIE v.13, TCGA, and other 212 studies (Cohort 2). Whole transcriptome sequencing and quantitative proteomics data from the The Cancer Genome Atlas (TCGA) and the Cancer Cell Lines Encyclopedia (CCLE) were used to correlate KRAS amplification with KRAS RNA levels and protein expression. KRAS doxycycline-inducible models were generated to determine in vitro and in vivo oncogenicity of wild-type KRAS overexpression. Results: Among 15,341 pts with NSCLC identified, KRAS amplification was identified in 355 (2.4%) cases. Compared to KRAS not amplified cases, pts with KRAS amplified NSCLC were more likely to be males (52.4% vs 45.6%, p=0.01), have a history of smoking (82.1% vs 64.2%, p<0.01), and higher median pack-years (35 vs 23.5, p<0.01). KRAS amplification was also associated with higher median aneuploidy levels (as fraction of genome altered) (30.4% vs 14.3%, p<0.01), tumor mutational burden (p<0.01), and increased median PD-L1 expression (20% vs 5%, p=0.01). KRAS amplified cases were enriched with KRAS, POLE2, SLC34A2 and PALB2 mutations, while EGFR mutations were mutually exclusive (Q<0.1). Among NSCLC samples with transcriptomic and proteomic profiling from the TCGA and CCLE cohorts, KRAS amplification was associated with significantly increased KRAS mRNA (p<0.01) and protein expression (p<0.01), compared to KRAS diploid samples. Among pts with clinical outcomes data available (N=9,335), median overall survival (OS) from the date of diagnosis was significantly shorter in pts with KRAS amplified vs KRAS diploid genotype in both KRAS mutant (HR: 0.71, P=0.02) and KRAS WT (HR: 0.74, P=0.02) NSCLCs. In KRAS doxycycline-inducible models (BEAS-2B cells) we demonstrated increased cell proliferation and MAPK activation upon doxycycline-induced KRAS expression. In vivo, sustained expression of KRAS in animals constantly fed with doxycycline-diet triggered tumor formation and development of metastasis, indicating that WT KRAS overexpression is oncogenic. Conclusion: KRAS amplification defines a novel subset of NSCLCs characterized by distinct clinicopathologic and genomic features, and worse survival. Developing novel therapeutic strategies to target KRAS amplification may improve outcomes in pts with NSCLC. Citation Format: Biagio Ricciuti, Sandra V. Michelina, Joao Alessi, Xinan Wang, Federica Pecci, Alessandro Di Federico, Malini Gandhi, Giuseppe Lamberti, Enrico Patrucco, Lynette Sholl, Andrew Aguirre, Chiara Ambrogio, Mark Magdi Awad. Wild type KRAS amplification as de novo oncogenic driver in non-small cell lung cancer [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 5064.

Abstract 4496: A computational initiative to determine the role of dietary polyphenols against oncogenic KRAS mutants

Abstract Introduction: Two oncogenic driver mutants (G12C and G14D) of KRAS have been reported to drive tumorigenesis in several cancers and targeting the switch I/II domain of mutant KRAS oncoprotein can be an efficient strategy for inhibiting the mutated KRAS-driven tumors. Modern clinical therapeutics are often associated with several adverse side effects and the eventual development of multi-drug resistance. Herein, exhaustive computational strategies are employed in designing dietary polyphenol-based small molecule inhibitors against such oncogenic KRAS tumors. Methodology: Integrated bioinformatics strategies such as pharmacokinetic property prediction, analysis of medicinal chemistry profile, density functional theory (DFT) study, molecular docking, molecular dynamics simulation (MDS) and principal component analysis were performed to screen 930 dietary polyphenols to identify potent inhibitors against the G12C and G14D mutants. Sotorasib, an FDA-approved KRAS chemotherapeutic inhibitor was used as a reference drug in this study. Results: Out of the 930 dietary polyphenols screened, based on the properties of pharmacokinetic descriptors and medicinal chemistry profile, 512 molecules were shortlisted for further studies. Five dietary polyphenols viz. Quercetin, Luteolin, Peonidin 3-O-glucoside, Myricetin, and Kaempferol identified via DFT and MDS exhibited the highest binding affinity and optimum energy profiles with our selected mutants G12C and G14D. An all-atom MDS study executed over 200 ns each revealed that Quercetin, Luteolin, and Peonidin 3-O-glucoside show favorable thermodynamic stability with significantly less conformational perturbations with G12C mutant. By contrast, mutant G12D complexed with Kaempferol and Myricetin exhibited the least thermodynamic flexibility and stable backbone trajectory, when compared to clinically standardized KRAS inhibitor Sotorasib. The efficient and higher binding stability of the polyphenols targeting the KRAS mutants, G12C and G14D switches I/II is mainly attributed to the formation of hydrogen bonds and other hydrophobic interactions at the molecular level. All five evaluated polyphenols qualified the parameters of cell-line cytotoxicity hinting at remarkable chemotherapeutic efficacy. Conclusion: Computer-aided drug designing performed here aided the identification of natural dietary polyphenolic inhibitors as potential drug candidates exhibiting stable binding dynamics like Sotorasib, which thus awaits validation through further in-vitro analyses in inhibiting KRAS-driven tumorigenesis. Citation Format: Prarthana Chatterjee, Rohit Karn, Sohini Chakraborty, Saptarshi Sinha, Pradipta Ghosh, Satarupa Banerjee. A computational initiative to determine the role of dietary polyphenols against oncogenic KRAS mutants [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 4496.

Abstract 6898: Cholangiocarcinoma patient-derived models in the NCI Patient-Derived Models Repository

Abstract Cholangiocarcinoma (CHOL) is an aggressive rare malignancy arising in the biliary tract with a 5-year relative survival rate of 10% and a 60-70% recurrence rate following surgical intervention. Advances in treatment strategies are hampered by the lack of well annotated and characterized preclinical models. The National Cancer Institute’s Patient-Derived Models Repository (NCI PDMR; https://pdmr.cancer.gov) has developed a national repository of Patient-Derived Models (PDMs) comprised of patient-derived xenografts (PDXs), in vitro patient-derived tumor cell cultures (PDCs) and cancer associated fibroblasts (CAFs) as well as patient-derived organoids (PDOrg). These PDMs are clinically annotated with molecular information available in an easily accessible database for the extramural community. To date, 94 patient CHOL tumor specimens (resections, biopsies, rapid autopsy) have been received from 55 unique patients with an overall PDX take rate of 36.3% (80 assessable specimens). There are currently 15 CHOL PDX models available for the research community to request with 14 more in final QC. Nine of the 15 public PDX models were generated from unique lesions in two rapid autopsy patients and while histo-morphologically consistent, some genomic heterogeneity is observed between the unique anatomical locations likely due to tumor evolution during metastasis. In addition, 27 in vitro PDOrgs, PDCs, and CAFs have been generated from patient or PDX material matched to most of the existing PDX models to allow for comparative translation research. The genetic landscape of the CHOL PDXs includes oncogenic drivers in NRAS, IDH1, ERBB2, TP53, KRAS and FGFR2 fusions; OncoKB likely oncogenic variants including ARID1A, AXIN1 and BAP1; and CDKN2A deep deletions representing most of the common alterations in CHOL. Early passage preclinical PDX and in vitro models for CHOL, and many other cancer types, with translational relevant features along with associated NextGen sequencing and patient treatment history are available from the NCI PDMR. Availability of preclinical models that can be used to study these cancers and improve preclinical drug screening is of high importance to better understand the biology of these cancers and prioritize novel therapeutics from bench to clinic. Funded by NCI Contract No. HHSN261200800001E Citation Format: Yvonne A. Evrard, Cindy R. Timme, Biswajit Das, Gareth Bliss, Carrie Bonomi, Carley Border, Ting-Chia Chang, Alice Chen, Li Chen, Michelle A. Crespo-Eugeni, Kevin Cooper, Natalie Czarra, Isabella Czernia, Kelly Dougherty, Marion Gibson, Tara Grinnage-Pulley, Shahanawaz Jiwani, Keegan Kalmbach, Chris A. Karlovich, Chelsea McGlynn, Michael Mullendore, Matthew Murphy, Rini Pauly, Kevin Plater, Jessica Steed, Luke Stockwin, Shannon Uzelac, Peter I-Fan Wu, Dianne L. Newton, P. Mickey Williams, Melinda G. Hollingshead, James H. Doroshow. Cholangiocarcinoma patient-derived models in the NCI Patient-Derived Models Repository [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 6898.

Abstract 7266: VGLL4 is the target of the 3p25 homozygous deletion and presents a novel therapeutic vulnerability for TEAD1/4 but not TEAD1 inhibitors

Abstract The Hippo pathway, a critical cell proliferation regulator, remains an underexplored oncogenic pathway in precision oncology. It is executed through YAP1/TAZ co-activators and the TEAD transcription factor family (TEAD1-4), driving expression of tumor-promoting genes. Palmitic acid site TEAD inhibitors have shown strong pre-clinical and clinical activity, but identification of genetic markers of vulnerability and patient selection remain an urgent and open question. Sporos undertook a broad bioinformatic analysis of Hippo pathway components and regulatory genes to identify genetic alterations that drive TEAD activity and potentially act as targetable vulnerabilities. Here, we present a key finding from this investigation. The 3p25 locus undergoes frequent homozygous deletion in solid tumors especially lung squamous cell carcinoma. While VHL was long assumed to be the target tumor suppressor gene of this deletion - we show that VGLL4 is in the only gene in the peak of GISTIC statistically significance in lung squamous cell carcinoma and VGLL4 and ATG7 are both in the peak in Renal Clear Cell Carcinoma (RCC). These data unambiguously establish that VGLL4 deletion is a major oncogenic driver in lung squamous cell carcinoma (SCC) and a contributor in RCC. Because VGLL4 is the major negative relator of the YAP/TEAD transcriptional complex and directly competes with YAP for TEAD binding - we hypothesize that Lung SCC with VGLL4-homozygous deletion would be uniquely dependent on YAP/TEAD activity and show selective sensitivity to TEAD inhibitors. While we were unable to locate a Lung SCC VGLL4-deleted PDX, we identified a VGLL4/ATG7 homozygous deleted RCC PDX, KI2552, in the CrownBio collection. We show that treatment with SPR1, Sporos’s novel TEAD1/4 inhibitor, decreases tumor growth and dramatically extends survival of mice bearing VGLL4/ATG7-deleted KI2552 RCC PDX. On the other hand, VT103, a strong TEAD1 inhibitor which shows comparable pre-clinical activity to SPR1 in the NCI-H226 mesothelioma CDX - did not show any activity in KI2552 PDX. These data emphasize the importance of targeting paralogs besides TEAD1 to broaden anti-neoplastic activity beyond mesothelioma and suggest that VGLL4-homozygous deletion confers TEAD inhibitor sensitivity even in carcinomas where it isn’t the principal driver and portent exceptionally anti-neoplastic activity in neoplasms where it is. Given the wide distribution of the 3p25 homozygous deletion - our findings open a large new potential responder population to TEAD inhibitors and warrant inclusion of VGLL4 in NSG diagnostic sequencing panels. Citation Format: Florian Muller, Selvi Kunnimalaiyaan, Parth Mangrolia, Jill Olson. VGLL4 is the target of the 3p25 homozygous deletion and presents a novel therapeutic vulnerability for TEAD1/4 but not TEAD1 inhibitors [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 7266.

Abstract 1974: Discovery of TRX-211-399, a potent, CNS-penetrant, highly selective inhibitor of EGFR exon 20 insertion mutations

Abstract Lung cancer is the leading cause of cancer-related deaths worldwide, accounting for approximately 2 million new cases and 1.8 million deaths annually. Various types of EGFR-activating mutations are one of the most important oncogenic drivers in non-small cell lung cancer (NSCLC). Among NSCLC patients with EGFR mutations, 5-10% of patients have EGFR exon 20 insertion (ex20ins) mutations that are insensitive to clinically available EGFR tyrosine kinase inhibitors (TKIs). Recently, as mobocertinib (EXKIVITY®) failed to improve progression-free survival in Phase 3 clinical trial, there remains a high unmet medical need to develop new drugs with wider therapeutic window to treat NSCLC harboring EGFR ex20ins mutations. Herein, we report a novel EGFR-TKI, TRX-211-399, specifically designed to target ex20ins mutations. TRX-211-399 potently inhibited the proliferation of not only Ba/F3 cells overexpressing near-loop (A767dupASV, S768dupSVD) and far-loop (H773insNPH, H773dupH) mutations but also patient-derived NSCLC cell line. In particular, TRX-211-399 demonstrated high selectivity against wild-type EGFR. With favorable DMPK profiles, once-daily oral administration of TRX-211-399 showed significant tumor regression in multiple Ba/F3 xenograft models carrying different EGFR exon20ins mutations. Furthermore, in a PC-9-luc cell intracranial xenograft model, TRX-211-399 induced substantial anti-tumor effects and prolonged survival without body weight loss. TRX-211-399 is currently undergoing further preclinical evaluation as a potential candidate for clinical development for patients with EGFR exon20ins mutations. Citation Format: Kwangwoo Chun, Sumin Lim, Joohyun Lee, Seulgi Choi, Seongrak Kim, Youngil Choi, Areum Park, Bohee Kim, Yongseo Park, Eunhyun Choi, Sungwon Lee, Koo Lee. Discovery of TRX-211-399, a potent, CNS-penetrant, highly selective inhibitor of EGFR exon 20 insertion mutations [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 1974.

Abstract 2667: Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer

Abstract Lung cancer, including lung adenocarcinoma (LUAD), is the leading cause of cancer-related deaths in the United States. KRAS mutations are the most prevalent oncogenic driver alteration in human LUAD. We have previously shown that high expression of Mucin 5 AC (MUC5AC), a main airway secretory mucin, is significantly associated with poor prognosis. Mucins are essential for mucociliary clearance and the maintenance of airway homeostasis, however, their overproduction could negatively affect the lung immune microenvironment and has been shown to promote airway diseases such as chronic obstructive pulmonary disease (COPD) and lung cancer. Previously, we have shown that genetic deletion of MUC5AC in a murine model of K-ras mutant lung adenocarcinoma (KM-LUAD) impedes tumor development and reduces pro-tumor inflammatory and immune phenotypes. These findings indicate that MUC5AC is a potential druggable target against KM-LUAD. In this study, we sought a translational approach and inhibited MUC5AC in our K-rasG12D mutant mouse model of LUAD via RNA interference (RNAi). Six-week-old mice were given RNAi (5mg/kg) or vehicle (saline) via biweekly intratracheal administration for 8 weeks. This approach led to a significant decrease in tumor burden (40%) and a trending increase in total white blood cell counts, predominantly macrophages in the lung tumor microenvironment which was associated with phenotypic changes in macrophages characterized by reduced expression of Fizz1, and M2 pro-tumor macrophage marker. Additionally, RNAi-treated mice had reduced expression of Siglec-E, a mucin receptor and known inhibitor of the TLR4 endocytosis and promoter of NF-ĸB signaling, as well as a significant decrease in IL-6, a product of the NF-ĸB pathway and promoter of STAT3 signaling which we have previously shown to have an essential role in promotion of KM-LUAD. In summary, these findings elucidate a potential mechanism by which MUC5AC protects tumor cells from apoptosis and immune-mediated cytotoxic activity but also highlights how MUC5AC RNAi is an effective indirect preventive and therapeutic strategy in high-risk individuals (smokers with COPD) and patients with early stage KM-LUAD, respectively. Citation Format: Melvin Zarghooni, Maria T. Grimaldo, Avantika Krishna, Michael J. Clowers, Linda Phan, Yasmina H. Rezai, Arnav Gaitonde, Carlos Rodriguez Reyna, Walter Velasco Torrez, Erik Bush, Seyed Javad Moghaddam. Mucin 5AC inhibition via intratracheal RNAi delivery as an effective alternative strategy for prevention and treatment of Kras mutant lung cancer [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 2667.

Abstract 3164: The in vivo Hollow Fiber model is a valuable tool in drug development of selective KRas inhibitors

Abstract The proto-oncogene KRas is a well-described small GTPase that functions as a molecular switch for major physiological signaling pathways involved in cell proliferation, differentiation and survival. It has been shown that activating mutations in KRas are among the most common oncogenic drivers of tumorigenesis. Missense mutations of KRas result in constitutive activation due to impaired hydrolysis of GTP which enhances tumor-promoting downstream signaling pathways. Most KRas mutations are located in exon 2 or 3 including the most frequently altered glycine 12, which is present in most pancreatic cancers as well as in colorectal cancers and lung adenocarcinomas. A panel of cancer cell lines harboring different KRas mutations such as G12C, G12V or G12D was selected to test KRas inhibitors in several cellular assays: phosphorylation assays, 2D and 3D proliferation assays. Applying the cellular pERK AlphaLISA assay showed high selectivity of several inhibitors for specific KRas mutants, while RAF and MEK inhibitors did not show significant selectivity in the tested cancer cell lines. The KRas inhibitors were then examined in a cellular 2D and a 3D spheroid proliferation assay to determine the inhibitory effect on cell growth. These cellular assays revealed a high selectivity for a specific KRas mutant, which was even more pronounced in the 3D format than in the 2D setup. The transferability of the cellular data obtained to in vivo was tested in the Hollow Fiber mouse model. The Hollow Fiber model allows the simultaneous study of multiple cell lines implanted in separated drug-(but not cell-) permeable fibers in a single mouse with a study duration of only 16 days. AMG510 (Sotorasib), a drug approved for tumors with a G12C Kras mutation, and MRTX1133, which is currently in clinical phase and shows activity in tumors with a G12D Kras mutation, were screened for their inhibitory effect on the pancreatic tumor cells MiaPaCa-2 (G12C Kras mutation), AsPC-1 (G12D Kras mutation) and BxPC-3 (wt Kras) in the in vivo Hollow Fiber model in female NMRI nude mice. While the growth of BxPC-3 tumor cells was not affected by either inhibitor, the growth of AsPC-1 tumor cells in particular was selectively and significantly inhibited by MRTX1133. In summary, the in vivo Hollow Fiber model is a fast and cost-effective model that can play an prominent role in drug development as a link between in vitro and in vivo xenograft studies by providing rapid and transferable evidence for in vivo efficacy. Citation Format: Philipp Metzger, Claus-Werner Franzke, Ezgi Dikici, Gregor Sommerkamp, Franziska Fimm-Todt, Jan E. Ehlert, Cynthia Obodozie, Holger Weber. The in vivo Hollow Fiber model is a valuable tool in drug development of selective KRas inhibitors [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 3164.

Abstract 5902: Targeting MAPK-driven tumors via inhibition of MAP2K4

Abstract MAPK pathway activation is a feature of multiple tumor types. Drugging KRAS and BRAF, the two main oncogenic drivers in the MAPK pathway, has proven successful in the clinic. Inhibition of the downstream effectors, MEK and ERK, can also induce tumor regression. Despite this, many tumors are intrinsically resistant to MAPK pathway inhibitors, or acquire resistance under selective pressure to drug treatment. This creates a need for combination treatments to improve clinical responses. A synthetic lethal (SL) interaction between inhibition of the MAPK pathway and blockade of JNK-JUN signaling has recently been described1. Specifically, data from yeast genetics and CRISPR knockout experiments in human cells have identified MAP2K4 as a potential therapeutic target that could be combined with MAPK inhibitors. To date, however, no potent MAP2K4 inhibitors with in vitro and cellular selectivity against key anti-targets have been reported. Here, we describe the development of potent covalent inhibitors of MAP2K4 kinase activity. Biochemical and cell-based assays show that the compound(s) are selective for MAP2K4 versus anti-targets including MAP2K7 and ERK kinases. The combination of MAP2K4 and MEK/ERK inhibitors was effective in cell lines driven by MAPK signaling. These data provide the rationale for further development of MAP2K4 inhibitors to advance our understanding of this novel drug combination.

Abstract 5900: INCB161734: A novel, potent, and orally bioavailable KRAS G12D selective inhibitor demonstrates antitumor activity in KRAS G12D mutant tumors

Abstract KRAS G12D (G12D) is one of the most frequent oncogenic driver mutations, and is especially common in pancreatic (PDAC) and colorectal (CRC) cancers. Patients with G12D-mutated disease experience poor treatment outcomes, representing a significant unmet medical need. G12D mutation results in constitutively active signaling, including hyperactivation of the ERK and PI3K pathways, which drive cell proliferation and survival. Here we describe INCB161734, a novel, potent, selective, and orally bioavailable small molecule G12D inhibitor that demonstrates in vivo efficacy in G12D-bearing tumor models. INCB161734 binds to both the GDP and GTP forms of the G12D mutant at the switch II pocket with picomolar affinity (KD), and exhibits >80-fold selectivity over wildtype (WT) KRAS. INCB161734 potently inhibits SOS1-dependent GDP/GTP exchange activity in cell-free assays (IC50 <3 nM), and exhibits >40-fold selectivity for G12D versus WT KRAS. Additionally, INCB161734 demonstrates high selectivity for G12D over WT in various cellular assays using G12D mutant versus WT cancer derived cell lines. INCB161734 potently inhibits ERK phosphorylation (a correlate for KRAS activity), with a mean IC50 of 14.3 nM (range: 1.9-45.2 nM) across 7 human and 3 mouse G12D cell lines; mean 21.5% inhibition was observed at 1 μM (maximum tested concentration) across 14 WT cell lines. Likewise, INCB161734 inhibits proliferation of G12D mutant cell lines, with a mean IC50 of 154 nM (range: 8.3-318 nM) across the same 7 human G12D cell lines; <30% inhibition (mean 13%) was observed at 1 μM across the same 14 WT cell lines. Treatment with INCB161734 induces caspase 3/7 cleavage in a PDAC cell line (G12D HPAC), with EC50 <100 nM; caspase 3/7 cleavage in a WT cell line (NCI-H838) was not induced at doses as high as 5 μM. In addition, INCB161734 induced cell cycle arrest (S-phase inhibition) in the G12D HPAC cell line, with IC50 12.8 nM; S-phase inhibition in the WT NCI-H838 cell line only occurred at much higher doses (IC50 >3.3 μM). INCB161734 demonstrates excellent oral bioavailability with good absorption, low clearance, and low metabolic turnover. Orally dosed INCB161734 clearly shows target engagement, generating continuous near-maximal KRAS inhibition in G12D HPAC mouse xenograft tumors for almost the entire dosing interval. INCB161734 is efficacious against multiple types of G12D-mutated tumors and xenografts, resulting in significant tumor growth inhibition, growth arrest and/or regression in multiple PDAC (HPAC, Panc0403, and 2838c3) and CRC (CT26, GP2D, and LS513) mouse tumor models. These preclinical results demonstrate that INCB161734 is a potent, selective, and orally bioavailable KRAS G12D inhibitor, strongly efficacious against KRAS G12D mutant tumors. The potential benefit of INCB161734 for patients with KRAS G12D mutant disease is under investigation in ongoing clinical trials. Citation Format: Matthew R. Farren, Valerie Roman, Alexandra Gallion, Abdellah Allali-Hassani, Alexander Sokolsky, Weixi Kong, Amanda Smith, Hui Wang, Gina Correa, Marc Deller, Leslie B. Epling, Jessica Procak, Guofeng Zhang, Katherine Pecko, Keith Kennedy, Jason Boer, Kerri Kurzeja-Lipinski, Maryanne Covington, Kwang-Jong Chen, Renee Wallower, Jennifer Rocha, Rina Pan, Anthony Perry, Brad Yuska, Xiaozhao Wang, Ricardo Macarron, Sunkyu Kim. INCB161734: A novel, potent, and orally bioavailable KRAS G12D selective inhibitor demonstrates antitumor activity in KRAS G12D mutant tumors [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 5900.

Abstract 3156: Unveiling the functional role of HDGFRP3 in pediatric medulloblastoma: Implications for growth, progression, and radioresistance

Abstract Introduction: Medulloblastoma (MB) is the most common malignant brain cancer in children. Although long-term survival has improved in recent years, only a modest percentage of patients survive high-risk disease. The quality of life for those who survive is substantially reduced due to the high toxicity associated with the radiation and chemotherapy. Therefore, identification of safe and potent therapeutic approaches that can sensitize the therapy response in MB without inducing toxicity will lead to more efficient treatment of MB patients. We recently discovered that targeting HDGFRP3, a protein highly expressed in MB, and can block MB growth and improve the efficacy of radiation therapy. HDGFRP3 is a multifaceted protein that plays important roles in diverse cellular processes. Our investigation sheds light on the functional role of HDGFRP3 in MB, focusing on its involvement in growth, progression, and radioresistance where we report HDGFRP3 as a novel oncogenic driver of these processes. Methods: Human MB cell lines (DAOY, D556, D425, and HDMB03) were purchased from the American Type Culture Collection and cultured in standard medium. MB cancer cells were transfected with siRNA specific to HDGFRP3. These knockdown cells were analyzed for cell viability, migration, invasion, colony formation, stemness, cell cycle, apoptosis assays for functional role assessment. Real-time qPCR and western blotting were performed to validate the downstream targets of HDGFRP3 in MB models. Results: Our findings demonstrate that HDGFRP3 functions as an oncogene in MB, as its overexpression promotes cancer growth, while its depletion results in decreased tumor growth in MB models. Furthermore, we provide evidence that HDGFRP3 knockdown results in decreased migratory potential of MB cells as well as a reduction in stemness. Interestingly, HDGFRP3 depletion improved the efficacy of standard of care radiation in multiple MB models. At present, in vivo studies are validate the oncogenic role of HDGFRP3 in preclinical animal models of MB. Conclusions: In conclusion, our data suggests HDGFRP3 facilitates MB growth, progression, stemness as well as resistance to radiation therapy. Our comprehensive investigation into HDGFRP3's functional role in MB presented here provides valuable insights into its potential as a therapeutic target and opens avenues for further research into novel treatment strategies for the pediatric brain tumors. Citation Format: Saif S. Nirzhor, Prabhakar Pitta Venkata, Shahad Abdulsahib, Panneerdoss Subbarayalu, Santosh Timilsina, Manjeet K. Rao. Unveiling the functional role of HDGFRP3 in pediatric medulloblastoma: Implications for growth, progression, and radioresistance [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 3156.

Abstract 6487: The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C models

Abstract KRASG12C is a potent oncogenic driver which results in downstream hyperactivation of MAPK signaling and unchecked oncogenic growth, while simultaneously increasing replication stress (RS) and accumulation of DNA damage. KRASG12C-specific inhibitors are approved and/or recommended in metastatic non-small cell lung cancer (NSCLC) and colorectal cancer (CRC), and while responses are robust, some patients are inherently resistant, and most responders acquire resistance. Resistance mechanisms include reactivation of MAPK signaling, and cancer cells which escape MAPKi are reported to have high levels of genomic instability. WEE1 is a kinase involved in cell cycle progression, allowing cells to repair damaged DNA before entering the next cell cycle phase. Consequently, inhibition of WEE1 leads to premature cell cycle entry and RS, thereby driving cancer cells through the cell cycle with unchecked accumulation of DNA damage, ultimately resulting in mitotic catastrophe and cell death. We hypothesized that high levels of genomic instability and RS in KRASG12C mutant and KRASG12C inhibitor-resistant tumors present a vulnerability to WEE1 inhibition and provide rationale for combining azenosertib, a novel, selective, and orally bioavailable WEE1 inhibitor, with KRASG12C inhibitors. Here, we report azenosertib in combination with KRASG12C inhibitors as an effective therapeutic strategy for KRASG12C tumors. In vitro combination of azenosertib with multiple KRASG12C inhibitors demonstrated synergistic cell growth inhibition across a panel of KRASG12C cell lines in both 2D and 3D assays. In vivo treatment with azenosertib in KRASG12C inhibitor-sensitive cell line-derived xenograft (CDX) models of NSCLC (NCI-H358, NCI-H2122, NCI-H1792), CRC (SW837, SW1463), and pancreatic cancer (MIA PaCa-2) demonstrated significant monotherapy activity as well as synergistic tumor growth inhibition (TGI) when combined with KRASG12C inhibitors, including tumor regression in some models. Importantly, the KRASG12C inhibitor-resistant SW1573 NSCLC CDX model demonstrated synergistic TGI in the combo arm (84%) when compared against monotherapy azenosertib (31%) or adagrasib (58%). Similarly, treatment of a KRASG12C inhibitor-resistant patient-derived xenograft (PDX) model of CRC resulted in synergistic TGI in the combination arm (61%) relative to monotherapy azenosertib (17%) or adagrasib (2%). Finally, analysis of biomarkers from both in vitro and in vivo tumor samples demonstrated synergistic increases in protein markers of RS, DNA damage, and apoptosis with combination therapy. Taken together, our results suggest that the combination of azenosertib with KRASG12C inhibitors enhances tumor growth inhibition over single agent therapy and may be an effective treatment option for patients with KRASG12C tumors. Citation Format: Nathan M. Jameson, Gabriel Kim, Catherine Lee, Blake Skrable, Alexandra Shea, Monah Abed, Olivier Harismendy, Jianhui Ma, Doris Kim, Mark R. Lackner. The selective WEE1 inhibitor azenosertib shows synergistic anti-tumor activity with KRASG12C inhibitors in multiple KRASG12C 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 6487.

Abstract 1089: Endogenous HiBiT-tagging of PAX3-FOXO1 reveals downregulation of the fusion oncogene by CDK inhibitors and has synergy with vincristine

Abstract Background: Oncogenic fusion genes are attractive therapeutic targets due to their tumor-specific expression and driver roles in cancers. PAX3-FOXO1 (P3F) is the dominant oncogenic driver of fusion-positive rhabdomyosarcoma (FP-RMS) with no current targeted therapy. We developed methods to directly measure endogenous P3F protein levels amenable to high-throughput drug screens. Method: HiBiT tag, an 11 amino acid peptide of NanoLuc luciferase, was inserted into the endogenous P3F using CRISPR in FP-RMS cell lines RH4 and SCMC. Western blot was used for HiBiT tag validation. RNA-seq and ChIP-seq were used to assess transcriptomics and DNA binding of HiBiT-tagged P3F (P3F-HiBiT). High-throughput drug screen using Nano-Glo luciferase assay was performed using the Mechanism Interrogation PlatE (MIPE 5.0) drug library, a 2,480 drug library with known mechanisms of action. CellTiter-Glo was used to monitor cell viability. Mouse xenograft models of FP-RMS were used to investigate in vivo efficacy. Results: We validated HiBiT tagging of P3F by Western. Both P3F-HiBiT and unmodified P3F activated the same gene sets in fibroblasts by RNA-seq Gene Set Enrichment Analysis (GSEA). ChIP-seq using HiBiT antibody for P3F-HiBiT matched the genomic locations from ChIP-seq with P3F antibody in RH4 and SCMC. Using a cutoff value of > 90 (Area Under the Curve (AUC) of CellTiter-Glo minus AUC of Nano-Glo), in both RH4 and SCMC, we identified 182 compounds which downregulate P3F before cell death. Filtering for drugs with ≥ 3 hits for the same target identified 14 drug classes that suppressed P3F including HDAC inhibitors (3), BRD4 inhibitors (3), and CDK inhibitors (8). FP-RMS was sensitive to CDK1/2, CDK4/6, CDK9, and multi-CDK inhibitors. A multi-CDK inhibitor TG02, currently in human trials, downregulated P3F and RNA-seq GSEA showed marked suppression of P3F targets. TG02 also significantly delayed tumor progression in mouse xenograft model of FP-RMS without weight loss. Interestingly, the commonly used chemotherapeutic Vincristine (VCR) also downregulated P3F in vitro. TG02 with VCR showed synergy by Loewe analysis. In vivo, testing showed significant delay in tumor progression by the combination compared to TG02 or VCR alone. Of note, tumor RNA-seq GSEA following treatment with TG02, or VCR alone, and in combination significantly downregulated P3F targets. Conclusion And Future Directions: By HiBiT tagging the fusion oncogene P3F, we identified 182 drugs that suppress P3F levels of which TG02 was a top hit. TG02 showed in vivo efficacy indicating that FP-RMS is susceptible to CDK inhibition. We also found synergy between TG02 and VCR, resulting in a significant delay in FP-RMS tumor progression compared to single agents. Interestingly, we found that VCR alone can downregulate P3F and its targets. Combination therapy of TG02 with VCR shows promise for clinical translation in FP-RMS. Citation Format: Yong Yean Kim, Katrina Jia, Mehal Churiwal, Teresa Hawley, Silvia Pomella, Christine Evans, Raj Chari, David Milewski, Ranuka Sinniah, Young Song, Hsien-Chao Chou, Xinyu Wen, Craig Thomas, Michele Ceribelli, Jun Wei, Robert Hawley, Javed Khan. Endogenous HiBiT-tagging of PAX3-FOXO1 reveals downregulation of the fusion oncogene by CDK inhibitors and has synergy with vincristine [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 1089.

Abstract 4967: Predicting progression-free survival (PFS) in first-line (1L) immune checkpoint inhibitor (ICI)-treated patients (pts) with advanced non-small cell lung cancer (aNSCLC): Machine learning (ML) application in real-world data

Abstract Introduction: Anti-programmed cell death (ligand)-1 ICIs are the standard of care 1L treatment for pts with aNSCLC without actionable oncogenic driver mutations. Heterogeneity in ICI utilization and outcomes is observed in real-world pts. By leveraging ML in electronic health records (EHRs), we identified predictors influencing key clinical outcomes, and developed a nomogram to predict likelihood of median PFS in 1L ICI-treated aNSCLC pts for potential risk stratification for interventions. Methods: 1L ICI-treated aNSCLC pts without ALK/ROS1/KRAS/BRAF/EGFR alterations were identified in a US oncology EHR database (ConcertAI; Jan 2015-Feb 2023). Survival ML models were trained on 112 clinical and demographic features with 5-fold nested cross-validation. The top predictors, determined by the best-performing ML model through SHapley Additive exPlanations and clinical judgement, were used to create a Cox proportional hazard (CPH) nomogram of median PFS. All models were evaluated using the concordance index (c-index). Patients were categorized as having high and low risk of progression/death at median PFS according to median risk predicted by the nomogram. Results: The study cohort had 4668 pts (median PFS: 6.1 months; 3811 events). The CPH nomogram predicting 6-month PFS had a c-index of 0.60 with the top 10 predictors identified using the XGBoost model (c-index: 0.62). Nomogram predictors included increased number of metastases, ECOG PS, cough suppressant use, WBC counts, and NLR (Table). Median probability of 6-month PFS for low- and high-risk groups was 35.9% and 19.4%, respectively. Conclusions: Future research should validate these findings and evaluate the opportunity to guide clinical practice to optimize outcomes in aNSCLC pts treated with 1L ICIs. Summary of top 10 clinical predictors at baseline Mean HR (95% CI) Max nomogram points Total metastases, n 1.15 (1.11-1.19) 100 ECOG PS, ordinal 1.14 (1.10-1.19) 55 Basophils, % 0.81 (0.73-0.90) 52 Albumin, g/dL 0.81 (0.75-0.87) 46 Chloride, mmol/L 0.98 (0.97-0.99) 46 Cough suppressants/expectorants, n 1.08 (1.03-1.14) 42 NLR 1.03 (1.02-1.05) 33 WBC counts, 103 cells/mL 1.02 (1.01-1.03) 29 PD-L1 expression, % 1.00 (1.00-1.00) 28 Presence of liver metastases 1.26 (1.15-1.39) 24 ECOG PS, Eastern Cooperative Oncology Group performance status; NLR, neutrophils-to-lymphocytes ratio; PD-L1, programmed cell death-ligand 1; WBC, white blood cell. Citation Format: Deep K. Hathi, James Harnett, Ning Wu, Zhenxing Xu, Weishen Pan, T G. Hager, Ruben G. Quek, Shivani Aggarwal, Young Kim, Petra Rietschel, Frank Seebach, Fei Wang, Ying Li. Predicting progression-free survival (PFS) in first-line (1L) immune checkpoint inhibitor (ICI)-treated patients (pts) with advanced non-small cell lung cancer (aNSCLC): Machine learning (ML) application in real-world data [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 4967.

Abstract 1968: Discovery of a highly potent and selective FGFR2 inhibitor for FGFR2-driven cancers

Abstract Fibroblast growth factor receptor 2 (FGFR2) is a clinically validated target and frequently altered in many solid tumors. The resulting oncogenic driver alterations often drive multiple solid tumors. Therefore, targeting FGFR2 has broad therapeutic potential. Currently, only pan-FGFR inhibitors are approved for FGFR2-driven intrahepatic cholangiocarcinoma, but not for other FGFR2-driven cancers. Due to the severe on-target toxicity associated with inhibition of FGFR1 and FGFR4, the use of the pan-FGFR inhibitors has been very limited. Thus, developing a selective FGFR2 inhibitor addresses the unmet medical need as the treatment options for FGFR2-altered cancers remain limited. Here we report the discovery and characterization of a highly selective FGFR2 inhibitor, ACE-16229210. This molecule potently inhibits FGFR2 (IC50 = 6.2 nM) and exhibits excellent selectivity (>133-fold) over other FGFR1 and FGFR4 in biochemical assays. It also potently (IC50 <1 nM) and selectively (≥500-fold) inhibits FGFR2-induced ERK phosphorylation in multiple cancer cell lines harboring FGFR2 fusions, amplification, and mutations, but not those harboring FGFR1, FGFR3, or FGFR4 genetic alterations (IC50 > 460 nM). Furthermore, this molecule, when evaluated in a panel of 24 cell lines, demonstrates potent anti-proliferative activity against the cell lines harboring FGFR2, but not FGFR3, and especially FGFR1, or FGFR4 genetic alterations. Thus, ACE-16229210 is a potent and selective FGFR2 inhibitor. Interestingly, it exhibits the most potent activity against gatekeeper and molecular brake mutations followed by fusions, amplification, and activation loop mutations. This molecule shows a robust broad spectrum of antitumor activity with significant tumor regression at low doses (1-10 mg/kg) in several tumor xenograft and PDX models representing the major FGFR2 relevant tumor histologies including gastric, breast, ovarian, and endometrial cancers harboring clinically important FGFR2 driver alterations with a well-defined pharmacokinetic/pharmacodynamic relationship. This molecule also demonstrates strong synergistic effects when combined with irinotecan and fulvestrant in a gastric tumor model and a breast cancer PDX model harboring FGFR2 amplification, respectively. Finally, ACE-16229210 did not significantly affect serum phosphorus levels in animals at the exposure levels that are >100-fold higher than the efficacious AUC, further confirming its excellent selectivity over FGFR1. Taken together, these in vitro and in vivo studies show that ACE-16229210 is a potent and selective FGFR2 inhibitor with the potential to be developed into a more effective treatment option for multiple cancers harboring FGFR2 oncogenic driver alterations. Citation Format: Wenqian Li, Bin Liu, Junmei Wang, Tinggui Yin, Kuo-Long Yu, Sanjeev Kumar, Zhiming Wen, Genshi Zhao, Weitao Pan. Discovery of a highly potent and selective FGFR2 inhibitor for FGFR2-driven cancers [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 1968.

Abstract 3233: EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma

Abstract METTL3 is the RNA methyltransferase predominantly responsible for the addition of N-6-methyladenosine (m6A), the most abundant epigenetic modification to mRNA. The prevalence of m6A and the activity and expression of METTL3 have been linked to the appearance and progression of numerous hematological and solid tumor cancers. EPICS has discovered and optimized small molecule inhibitors of METTL3 (“M3i”). The aim of the current study is to evaluate the efficacy of M3i in various CDX mouse tumor models. M3i compound potency was evaluated in vitro by measuring enzymatic inhibition of METTL3/14 by scintillation proximity assay (IC50 = 2 nM). The anti-proliferative effects of M3i were measured in cell viability assays following a 72h treatment of A549 (NSCLC, IC50 = 190 nM), SKOV3 (ovarian, IC50 = 9 nM) and FaDu (hypopharyngeal squamous cell carcinoma, IC50 = 24 nM) cells. Complementary data in all three cell lines demonstrates a clear concentration-response for a target engagement biomarker (m6A; IC50 = 2-8 nM, across cell lines) as well as M3i-induced changes in biomarkers of oncogenic drivers. M3i (30 mg/kg, IP, QD) was tested in vivo in mouse models using FaDu (subcutaneous implant) in addition to A549 and SKOV3 (both orthotopic implants); all animal models were performed by LabCorp (MI, USA). Daily M3i treatment significantly prolonged survival in all three disease models consistent with efficacy on tumor growth inhibition as measured by imaging (A549, SKOV3) or direct measurement of tumor volume in the case of subcutaneous implant (FaDu). M3i has previously been shown to be effective in mouse CDX and PDX models of AML. The current results extend the application of METTL3 inhibitors to include the treatment of various types of solid tumors. The efficacy of M3i as a single-agent on tumor growth inhibition and survival advances the validation of METTL3 as a cancer target and provides additional evidence for the applicability of mRNA epigenetic mechanisms to the treatment of solid tumors. Citation Format: Graeme Fraser, Catherine Sorlet, Nicolas Parmentier, Elodie Brunel, Christian Coremans, Sarah Rorive, Anne-France Hartiel, Killian Oukoloff, Guillaume Dutheuil. EP102: Pharmacological Inhibition of METTL3 causes tumor growth inhibition and prolongs survival in preclinical models of NSCLC, ovarian and squamous cell carcinoma [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 3233.

Abstract 5705: Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition

Abstract CDK2 is a cell cycle kinase that plays a critical role in controlling the G1 to S-phase transition and its abnormal activation through cyclin E1(CCNE1) amplification has been indicated as the primary oncogenic driver in advanced cancers including ovarian, endometrial, and gastric cancers. In addition, cyclin E1 overexpression has emerged as a key resistance mechanism to CDK4/6 inhibitors in hormone receptor-positive breast cancer patients. Therefore, CDK2 represents a promising novel oncology target. However, development of a potent and selective CDK2 inhibitor has been challenging due to the high homology of CDK2 with other CDKs, particularly CDK1, which is essential in normal cell cycle progression. NKT3447 is a novel, orally bioavailable small molecule CDK2 inhibitor with slow-off kinetics and is highly selective against CDK1 and other CDKs. In addition, NKT3447 exhibits a distinct mechanism of action, including suppression of the Thr160 activating phosphorylation on CDK2 and downregulation of cyclin E1. In biochemical assays, NKT3447 drives a concentration dependent disruption of the CDK2/cyclin E1 and CDK2/cyclin A2 complexes, but not the CDK1/cyclin B1 complex. Consequently, NKT3447 potently inhibits the phosphorylation of Rb (pRb) and induces G1 cell cycle arrest in CCNE1 amplified OVCAR3 cells, while showing minimal or no effect on G2/M phase distribution in all the cell lines tested, including OVCAR3 and KYSE520 (CDK1-dependent) cells. Importantly, NKT3447 has a slow-off kinetics, as demonstrated by NanoBRET target engagement assay, which leads to sustained pRb inhibition after treatment withdrawal. In addition, NKT3447 treatment reduces cyclin E1 protein levels and suppresses phospho-CDK2 (Thr160), which further represses CDK2 activity and prolongs the inhibition of CDK2 signaling. NKT3447 also demonstrates a selective anti-proliferation effect in a panel of human cancer cell lines with elevated cyclin E1 expression and/or dependency on CDK2 or cyclin E1 (DepMap). In line with cellular studies, pharmacodynamic study of NKT3447 in mouse tumor models recapitulates a significant repression of pRb, pCDK2 and cyclin E1. Furthermore, NKT3447 treatment results in a dose-dependent tumor growth inhibition or tumor regression in CCNE1 amplified OVCAR3 and MKN1 tumor models. In addition, the combination of NKT3447 with carboplatin exhibits enhanced anticancer effects in OVCAR3 and MFE-280 models. These results demonstrate that NKT3447 is a potent, selective CDK2 inhibitor with distinct properties and mechanism of action, which holds great potential to effectively treat patients with aberrant CDK2/Cyclin E activation in a variety of cancer types. NKT3447 is currently being investigated in a Phase 1 clinical trial in patients with advanced solid tumors. Citation Format: Jianlin Geng, Ke Liu, Zhiyong Yu, Wenfeng Sun, Hairong Wei, Wenjun Li, Jing Lu, Juan Deng, Liqing Geng, Zhihong Liu, Zhenhai Gao, Yan Lou. Discovery of a selective slow-off CDK2 inhibitor NKT3447 with distinct features of suppressing pCDK2, downregulating cyclin E, and achieving prolonged pathway inhibition [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 5705.

Abstract 562: Frequent copy number gain of MCL1 is a therapeutic target for osteosarcoma

Abstract Osteosarcoma (OS) is the most common primary malignant bone tumor that predominantly occurs in children, adolescents, and young adults. The treatment for OS that combines surgery with chemotherapy, which consists of a four-drug combination of adriamycin (DOX), cisplatin (CDDP), high-dose methotrexate (MTX), and ifosfamide, was established in 1970s, and it is still used as a standard therapy. Oncogenic driver mutations and fusion genes common to OS have not been identified, which is one of the reasons for the lack of success in drug development for OS. Herein, to explore the characteristic vulnerability in OS, molecular targeted drugs were screened against OS cell lines and patient-derived cells; obatoclax, a pan-BH3 mimetic, and a concomitant drug that enhances obatoclax-induced apoptosis, OSI906, a dual inhibitor of IGF1R and IR, were found. Fluorescence in situ hybridization (FISH) analysis revealed that copy number gains of MCL1, an antiapoptotic Bcl-2 family protein, were observed in 46.3% of formalin-fixed paraffin-embedded (FFPE) specimens from patients with OS (N = 41) and in 45.5% of OS cell lines (N = 11), thereby causing vulnerability to BH3 mimetics. IHC staining also revealed high expression of Mcl-1 protein in specimens with MCL1-amplification in FISH analysis. Pharmacological or genetic inhibition of Mcl-1 induced potent apoptosis in MCL1-amplified OS cells, which was enhanced by the co-inhibition with Bcl-xL. Moreover, chromosome 1q21.2-3, where MCL1 is located, contains several IGF1R/PI3K pathway-related genes, including PIP5K1A, TARS2, OUTD7B, and ENSA, whose copy numbers were also increased in MCL1-amplified OS cells, suggesting that 1q21.2-3-amplified OS cells readily activate downstream pathways via IGF1R. IGF signaling activation in 1q21.2-3-amplified OS cells negated the obatoclax-induced apoptosis, which was overcome by OSI906 treatment. Combined obatoclax and OSI906 administration remarkably suppressed MCL1-amplified OS tumor growth in the xenograft mouse model and resulted in tumor regression; however, none of the treatments reduced tumor growth of OS without MCL1 amplification. These results suggest that combining obatoclax with OSI906 could be a potential therapeutic intervention for 1q21.2-3-amplified OS, and that prior stratification of OS based on the 1q21.2-3 amplification status would be important for the success of this combination therapy. Thus, we elucidated the mechanism of action of drugs and their potential as a novel therapeutic strategy for approximately 50% of patients with OS. Moreover, a FISH analysis that can detect copy number gains of MCL1 in FFPE specimens from patients with OS was established to stratify patients eligible for this therapy. Citation Format: Satoshi Takagi, Ryohei Katayama. Frequent copy number gain of MCL1 is a therapeutic target for osteosarcoma [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 562.

Abstract 7203: Anti-HDGF antibody attenuates the rise of resistance to EGFR tyrosine kinase inhibitor in NSCLC patient-derived xenograft tumors

Abstract Mutant epidermal growth factor receptor (EGFR), a major oncogenic driver in non-small cell lung cancer (NSCLC), is found in the tumors of 15% (western countries) to 50% (Asian countries) of patients. EGFR-targeted therapy using tyrosine kinase inhibitors (TKIs) is a standard of care for those patients who developed metastatic or recurrent diseases. However, despite having an 80% initial response rate, most patients had partial tumor regression and their disease would progress when acquired resistance emerges. Thus, developing more effective treatment is an unmet need. The clinical response of these patients can be recapitulated in mouse models of patient-derived xenograft (PDX) tumor bearing sensitizing EGFR mutations. In this study, treating established PDX tumors with osimertinib, a third generation EGFR TKI, induced partial tumor regression (50% to 70% size reduction from baseline) followed by progression with a median progression-free survival (PFS) of 26 to 28 days. Interestingly, co-administering an antibody against hepatoma-derived growth factor (HDGF) with osimertinib resulted in enhanced tumor regression (98% to 100% size reduction from baseline) which translated into significantly extended animal survival with a median PFS of 103 or 135 days. Immunohistochemical and western blot examination of tumors collected at different treatment timepoints indicated that AKT/mTOR and MAPK pathways were reactivated after initial suppression in TKI monotherapy arm despite persistent EGFR inhibition. Since AKT/mTOR and MAPK pathways are key survival and proliferative signaling pathways, their reactivation can promote TKI tolerance of tumor cells and the emergence of TKI resistance. However, blocking HDGF attenuated the reactivation of these pathways which could partially explain the enhanced efficacy of osimertinib plus anti-HDGF antibody combination treatment seen in this study. Our results suggest tumor-derived HDGF could play a crucial role in the rise of acquired resistance to osimertinib. Identifying the exact cells and molecular pathways utilized by HDGF to promote tumor cell tolerance to TKI will help us understand how TKI sensitive tumor cells survive EGFR blockade and emerge as resistant cells. Citation Format: Cindy Q. Zhou, Ariel Li, Kaoru Ri, Ahmed Sultan, Hening Ren. Anti-HDGF antibody attenuates the rise of resistance to EGFR tyrosine kinase inhibitor in NSCLC patient-derived xenograft tumors [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 7203.

Abstract 6485: Profile guided low dose drug combination strategies and kinase activities with prognostic and therapeutic avenues in pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease, commonly characterized by multiple aberrant signaling. Phosphoproteomics provides a direct read-out of these complex signaling networks and the resultant clinically relevant phenotype, as well as a functional scaffold to identify new targets. In the absence of an oncogenic driver, low dose (LD) kinase inhibitor (KI) combinations against multiple (parallel) activated kinases might provide higher efficacy and reduce toxicity as compared to single drug treatment. Aims: 1) To identify targets and test combinations of multiple KIs at LDs for potential synergism in preclinical models. 2) To chart the phosphoproteome of 42 PDAC tumors to reveal signalling pathways that may be involved in PDAC progression. Methods: By using a two step phosphopeptide enrichment with phosphotyrosine immunoprecipitation and immobilized metal affinity chromatography, followed by label free MS analysis, we analyzed phosphoproteome data of 7 immortalized and 2 primary PDAC cell lines, and of 42 PDAC tumors. We used integrative inferred kinase activity (INKA) scoring of the maxquant output to identify hyperactive kinases. For the cell line panel, five KIs were selected based on targeting coverage of the INKA profiles. LD were set as IC20s for 2,3,4 drug combinations. Cell growth inhibition was assessed by SRB assay. Median-effect analysis was used to assess synergy. Functional testing was performed in immortalized 2D, xenoderived 2D and 3D cultures. Effective low dose 3 drug combinations were further validated using patient-derived xenografts. Results: High INKA scoring of multiple activities per cell line without clear outliers of single kinases underscores the need for combination therapies. Multiple LD combinations showed effective growth inhibition (70 to 92%) and synergism, mostly 3 drug combinations, which required targeting of several RTKs and downstream signaling. Different responses were further observed between epithelial and mesenchymal cell lines. These top performing combinations were further validated in PDAC organoids and in vivo. Clinical utility of these kinase targets was then confirmed in 42 tumor phosphoprofiles, which were characterized in different subtypes with distinct therapeutic options. Phosphoproteome signals and kinases activities with potential prognostic value and mutational associations were further described. Conclusion: Our INKA pipeline, which can rank kinase activities in individual tumors, is optimally suited to specifically prioritize actionable kinases with targeting purposes. Tailored LD combination strategies exhibited promising efficacy in preclinical models and may ultimately improve treatment outcomes. Next Steps: Multicellular patient-derived models will be used to further study and target the TME in PDAC tumors. Citation Format: Andrea Vallés Martí, Giulia Mantini, Cynthia Waasdorp, Richard R. de Goeij- de Haas, Alex A. Henneman, Sander R. Piersma, Thang V. Pham, Jaco C. Knol, Joanne Verheij, Frederike Dijk, Hans Halfwerk, Elisa Giovannetti, Connie R. Jiménez, Maarten F. Bijlsma. Profile guided low dose drug combination strategies and kinase activities with prognostic and therapeutic avenues in pancreatic ductal adenocarcinoma [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 6485.