Multiple Human Cancer Cell Lines Research Articles

Abstract B007: Identification of Asporin as a HER3 ligand exposes a therapeutic vulnerability in metastatic prostate cancer

Abstract Background: Rates of prostate cancer incidence and advanced-stage diagnoses are increasing. Despite curative-intent therapies, nearly all patients with advanced disease progress to metastatic castration-resistant prostate cancer (mCRPC), a lethal form of the disease. There is an urgent unmet need to understand the mechanisms driving prostate cancer progression and identify alternative therapies for patients with mCRPC. It is well-established that cancer-associated fibroblasts (CAF) in the tumor microenvironment secrete factors that enable cancer cells to establish metastases. Asporin (ASPN) is an extracellular protein secreted by a subset of CAF and is associated with worse oncologic outcomes, yet the mechanisms remain poorly understood. In this study, we sought to identify the molecular interactions between the CAF-secreted ASPN and metastatic prostate cancer cells. Methods: ASPN-induced HER2/HER3 activation and downstream signaling was identified by RNA-sequencing and then confirmed by immunoblotting in multiple human metastatic prostate cancer cell lines. Binding interactions were assessed computationally by AlphaFOld2 with Rosetta refinement and substantiated by co-immunoprecipitation assays. CRISPR-cas9 targeting of HER2 and HER3 and therapeutic inhibition of HER2 with Tucatinib were used to determine the role of HER2/HER3 in ASPN-induced signaling and migration. The efficacy of Tucatinib and antibody-drug conjugates (ADC) targeting HER2 (Trastuzumab-deruxtecan) and HER3 (Patritumab-deruxtecan) were assessed in multiple metastatic prostate cancer cell lines in vitro. Trastuzumab-deruxtecan was assessed in PC3 xenografts in vivo. Prostate cancer metastases from 33 patients were assessed by IHC for HER2 and HER3 and by RNAscope for ASPN. Results: We report that ASPN is a novel ligand of HER3 and induces HER3 heterodimerization with its preferred dimerization partner, HER2. ASPN activates established ErbB-associated pathways including Phosphoinositide 3-kinase (PI3K), Mitogen-activated protein kinase (MAPK), and calcium signaling, in multiple metastatic prostate cancer cell lines to promote cell migration. Genetic and molecular inhibition of HER2/HER3 mitigates ASPN-induced signaling and cell migration, suggesting these receptors are required for paracrine activation by ASPN. Importantly, small molecule and ADC therapies targeting HER2/HER3 significantly diminished prostate cancer cell growth in vitro, with enzalutamide-resistant cells showing increased sensitivity. Trastuzumab-deruxtecan nearly resolved tumors in an in vivo xenograft model. Lastly, ASPN+ CAF in the TME of HER2/HER3-expressing metastatic prostate cancer is frequently observed in patient samples, supporting the clinical relevance of these findings. Conclusions: Collectively, these findings indicate ASPN functions as a HER3 ligand to induce cellular migration, and inhibition with anti-HER2/HER3 therapies highlights potential clinical utility for patients with HER2-expressing metastatic prostate cancer. Citation Format: Amanda B. Hesterberg, Hong Yuen Wong, Jorgen Jackson, Monika Antunovic, Brenda L. Rios, Evan Watkins, Riley E. Bergman, Brad A. Davidson, Sarah E. Ginther, Diana Graves, Elliott F. Nahmias, Jared A. Googel, Lillian B. Martin, Violeta Sanchez, Paula Gonzalez Ericsson, Quanhu Sheng, Benjamin P. Brown, Jens Meiler, Ben H. Park, Kerry R. Schaffer, Jennifer B. Gordetsky, Paula J. Hurley. Identification of Asporin as a HER3 ligand exposes a therapeutic vulnerability in metastatic prostate cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor-body Interactions: The Roles of Micro- and Macroenvironment in Cancer; 2024 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(22_Suppl):Abstract nr B007.

Distinct cellular uptake patterns of two anticancer unsymmetrical bisacridines and their metabolic transformation in tumor cells

Unsymmetrical bisacridines (UAs) represent a novel class of anticancer agents. Their high cytotoxicity towards multiple human cancer cell lines and inhibition of human tumor xenograft growth in nude mice signal their potential for cancer treatment. Therefore, the mechanism of their strong biological activity is broadly investigated. Here, we explore the efflux and metabolism of UAs, as both strongly contribute to the development of drug resistance in cancer cells. We tested two highly cytotoxic UAs, C-2028 and C-2045, as well as their glucuronic acid and glutathione conjugates in human cancer cell lines (HepG2 and LS174T). As a point of reference for cell-based systems, we examined the rate of UA metabolic conversion in cell-free systems. A multiple reaction monitoring (MRM)-mass spectrometry (MS) method was developed in the present study for analysis of UAs and their metabolic conversion in complex biological matrices. Individual analytes were identified by several features: their retention time, mass-to-charge ratio and unique fragmentation pattern. The rate of UA uptake and metabolic transformation was monitored for 24 h in cell extracts and cell culture medium. Both UAs were rapidly internalized by cells. However, C-2028 was gradually accumulated, while C-2045 was eventually released from cells during treatment. UAs demonstrated limited metabolic conversion in cells. The glucuronic acid conjugate was excreted, whereas the glutathione conjugate was deposited in cancer cells. Our results obtained from cell-free and cell-based systems, using a uniform MRM-MS method, will provide valuable insight into the mechanism of UA biological activity in diverse biological models.

Dual inhibition of SUMOylation and MEK conquers MYC-expressing KRAS-mutant cancers by accumulating DNA damage

BackgroundKRAS mutations frequently occur in cancers, particularly pancreatic ductal adenocarcinoma, colorectal cancer, and non-small cell lung cancer. Although KRASG12C inhibitors have recently been approved, effective precision therapies have not yet been established for all KRAS-mutant cancers. Many treatments for KRAS-mutant cancers, including epigenome-targeted drugs, are currently under investigation. Small ubiquitin-like modifier (SUMO) proteins are a family of small proteins covalently attached to and detached from other proteins in cells via the processes called SUMOylation and de-SUMOylation. We assessed whether SUMOylation inhibition was effective in KRAS-mutant cancer cells.MethodsThe efficacy of the first-in-class SUMO-activating enzyme E inhibitor TAK-981 (subasumstat) was assessed in multiple human and mouse KRAS-mutated cancer cell lines. A gene expression assay using a TaqMan array was used to identify biomarkers of TAK-981 efficacy. The biological roles of SUMOylation inhibition and subsequent regulatory mechanisms were investigated using immunoblot analysis, immunofluorescence assays, and mouse models.ResultsWe discovered that TAK-981 downregulated the expression of the currently undruggable MYC and effectively suppressed the growth of MYC-expressing KRAS-mutant cancers across different tissue types. Moreover, TAK-981-resistant cells were sensitized to SUMOylation inhibition via MYC-overexpression. TAK-981 induced proteasomal degradation of MYC by altering the balance between SUMOylation and ubiquitination and promoting the binding of MYC and Fbxw7, a key factor in the ubiquitin–proteasome system. The efficacy of TAK-981 monotherapy in immunocompetent and immunodeficient mouse models using a mouse-derived CMT167 cell line was significant but modest. Since MAPK inhibition of the KRAS downstream pathway is crucial in KRAS-mutant cancer, we expected that co-inhibition of SUMOylation and MEK might be a good option. Surprisingly, combination treatment with TAK-981 and trametinib dramatically induced apoptosis in multiple cell lines and gene-engineered mouse-derived organoids. Moreover, combination therapy resulted in long-term tumor regression in mouse models using cell lines of different tissue types. Finally, we revealed that combination therapy complementally inhibited Rad51 and BRCA1 and accumulated DNA damage.ConclusionsWe found that MYC downregulation occurred via SUMOylation inhibition in KRAS-mutant cancer cells. Our findings indicate that dual inhibition of SUMOylation and MEK may be a promising treatment for MYC-expressing KRAS-mutant cancers by enhancing DNA damage accumulation.

Synthesis, biological evaluation, and molecular docking of novel 1,3,4-substituted-thiadiazole derivatives as potential anticancer agent

In an attempt to develop potent anti-cancer agents, a new 1,3,4-substituted-thiadiazole derivatives (8b-g), starting from 4-substituted-thiazol-2-chloroacetamides (4b-g), were synthesized and evaluated for their cytotoxic effects on multiple human cancer cell lines, including the hepatocellular carcinoma (HEPG-2), human lung carcinoma (A549), human breast carcinoma (MCF-7) and pseudo-normal human embryonic liver (L02) cancer cell lines by an MTT assay. Among all synthesized compounds, compound 8d showed the potent anti-cancer activities with GI50 values of 2.98, 2.85 and 2.53 μM against MCF-7, A549 and HepG-2 cell lines respectively as compared to standard drug Doxorubicin. Furthermore, molecular modelling studies have spotlighted the anchoring role of 1,3,4-substituted-thiadiazole moiety in bonding and hydrophobic interaction with the key amino acid residues. Therefore, these results can provide promising starting points for further development of best anti-cancer agents.Graphical

MicronuclAI: Automated quantification of micronuclei for assessment of chromosomal instability.

Chromosomal instability (CIN) is a hallmark of cancer that drives metastasis, immune evasion and treatment resistance. CIN results from chromosome mis-segregation events during anaphase, as excessive chromatin is packaged in micronuclei (MN), that can be enumerated to quantify CIN. Despite recent advancements in automation through computer vision and machine learning, the assessment of CIN remains a predominantly manual and time-consuming task, thus hampering important work in the field. Here, we present micronuclAI , a novel pipeline for automated and reliable quantification of MN of varying size, morphology and location from DNA-only stained images. In micronucleAI , single-cell crops are extracted from high-resolution microscopy images with the help of segmentation masks, which are then used to train a convolutional neural network (CNN) to output the number of MN associated with each cell. The pipeline was evaluated against manual single-cell level counts by experts and against routinely used MN ratio within the complete image. The classifier was able to achieve a weighted F1 score of 0.937 on the test dataset and the complete pipeline can achieve close to human-level performance on various datasets derived from multiple human and murine cancer cell lines. The pipeline achieved a root-mean-square deviation (RMSE) value of 0.0041, an R 2 of 0.87 and a Pearson's correlation of 0.938 on images obtained at 10X magnification. We tested the approach in otherwise isogenic cell lines in which we genetically dialed up or down CIN rates, and also on a publicly available image data set (obtained at 100X) and achieved an RMSE value of 0.0159, an R 2 of 0.90, and a Pearson's correlation of 0.951. Given the increasing interest in developing therapies for CIN-driven cancers, this method provides an important, scalable, and rapid approach to quantifying CIN on routinely obtained images. We release a GUI-implementation for easy access and utilization of the pipeline.

Abstract 5582: Discovery and preclinical characterization of ISM8001, a covalent and selective FGFR2/FGFR3 dual inhibitor with strong monotherapy anti-tumor activity against advanced solid tumors

Abstract Oncogenic alterations in fibroblast growth factor receptors 2 and 3 (FGFR2 and FGFR3) are key drivers in intrahepatic cholangiocarcinoma, urothelial carcinoma and multiple other solid tumors. Currently approved pan-FGFR inhibitor therapy is limited by off-isoform toxicity (FGFR1-mediated hyperphosphatemia and FGFR4-mediated diarrhea) and acquired FGFR2 or FGFR3 kinase domain resistance mutations. The second generation of FGFR inhibitors currently undergoing clinical evaluation such as RLY-4008 and LOXO-435 are primarily focused on achieving selectivity against either the FGFR2 or FGFR3 isoform, which may narrow their clinical potential. To overcome this limitation and to allow a favorable toxicity profile, we designed and evaluated the anti-cancer effects of a novel and selective FGFR2/FGFR3 dual inhibitor, ISM8001. Biochemically, ISM8001 showed superior inhibition against FGFR2 and FGFR3 with >90-fold and >800-fold selectivity of FGFR2 over FGFR1 and FGFR4, respectively. As an irreversible inhibitor, ISM8001 displayed strong affinity and high efficiency of covalent bond formation with FGFR2/3. Additionally, ISM8001 showed broad inhibition against multiple FGFR2/3 mutant isoforms, especially gatekeeper mutation (FGFR2-V564F and FGFR3-V555M). ISM8001 inhibited cell proliferation of multiple human cancer cell lines with FGFR2/3 aberration with IC50 <10 nM while IC50 >1 µM was observed in cell lines without FGFR2/3 alterations. Furthermore, ISM8001 demonstrated robust in vivo anti-tumor efficacy in multiple cell line-derived xenograft mouse models including the SNU-16 gastric cancer model (FGFR2-amplification), the AN3CA endometrial cancer model (FGFR2 N549K mutation) and the RT4 bladder cancer model (FGFR3-fusion), with tumor growth inhibition varying from 80%~120% while no significant serum phosphate increase or body weight loss. ISM8001 also displayed strong anti-tumor effects in xenograft models of Ba/F3-FGFR2/3 engineered cell lines with different gatekeeper mutations. ISM8001 is classified as a BCS II compound with favorable DMPK properties and druggability, high permeability, low efflux ratio and low DDI risk. In vivo PK profiles showed moderate to low clearance and high oral bioavailability across preclinical species. In addition, ISM8001 showed a promising in vitro safety profile with favorable selectivity in 468-kinase and Cerep-44 panel studies, and no hERG concerns. Results of 28-day non-clinical toxicology studies in rat and dog showed a safety window of approximately 2-5 fold based on efficacious exposure in different models. Taken together, these data support the clinical development of ISM8001 as a novel, selective FGFR2/FGFR3 dual inhibitor for the potential tissue-agnostic therapy of advanced solid tumors with FGFR2/3 aberrations. Citation Format: Yihong Zhang, Yazhou Wang, Xin Cai, Jinxin Liu, Hui Cui, Junwen Qiao, Xiaoxia Lin, Xiao Ding, Supriya Bavadekar, Sujata Rao, Man Zhang, Feng Ren, Alex Zhavoronkov. Discovery and preclinical characterization of ISM8001, a covalent and selective FGFR2/FGFR3 dual inhibitor with strong monotherapy anti-tumor activity against advanced solid 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 5582.

Abstract 1927: Resistance to RAS-GTP inhibition in models of pancreatic ductal adenocarcinoma arises downstream of RAS effectors

Abstract More than 90% of pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS that drive it into an active, GTP-bound state (KRAS(ON)), producing excessive signaling via MAPK and other effector pathways. RMC-7977 is a potent inhibitor of GTP-bound RAS proteins (RAS(ON)), including both wild type and mutant variants of KRAS, NRAS, and HRAS. The related investigational agent, RMC-6236, is a first-in-class, potent, orally bioavailable, RASMULTI(ON) inhibitor currently in Phase 1/1b clinical trials (NCT05379985). Exposure to RMC-7977 suppressed the MAPK biomarker p-ERK, reduced cell growth, and induced apoptosis in multiple human RAS-addicted cancer cell lines. Assessment of RMC-7977 in a series of PDAC preclinical model systems revealed broad anti-tumor activity and treatment was well tolerated at translatable doses. The K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) autochthonous mouse model was utilized to study the effects of Ras-GTP inhibition in both tumor and normal tissues. At doses that drove anti-tumor activity, RMC-7977 dosing produced a metronomic effect on RAS signaling in tumor and normal tissue, with full pathway inhibition at 4 h post treatment that was restored by 24 h. This pattern yielded tumor-selective effects on apoptosis and proliferation, consistent with RAS oncogene addition in PDAC. RMC-7977 treatment drove regressions and an unprecedented ~3-fold extension of overall survival in KPC mice. After an initial response, all tumors eventually developed resistance on treatment, affording the opportunity to study acquired resistance mechanisms to RAS-GTP inhibition. Strikingly, in all resistant tumors, acute inhibition of MAPK and PI3K signaling was still apparent following treatment. These results indicate that RAS signaling re-activation mechanisms that have been reported as clinical mechanisms of resistance to mutant-selective KRASG12C inhibitors (e.g. second-site mutations or upregulation of upstream RTK) may be insufficient to confer resistance to a compound that inhibits all RAS isoforms. Rather, in this model we observed activation of alternative oncogenic mechanisms downstream of RAS effector signaling, including frequent focal amplification of Myc, focal amplification of Jun, and/or activation of Yap/Taz/Tead signaling in most resistant KPC tumors. Together these results demonstrate preclinical activity of RMC-7977 at tolerable doses, producing unprecedented responses even in highly chemoresistant PDAC models such as the KPC mouse. The observed range of potential resistance mechanisms suggests that RAS-GTP inhibition imposes a narrow evolutionary path to the development of acquired resistance in this context. These preclinical findings may help inform the ongoing development of investigational RAS inhibitors and the design of potential combinatorial treatment strategies to forestall resistance. Citation Format: Urszula N. Wasko, Jingjing Jiang, Yingyun Wang, Lorenzo Tomassoni, Lingyan Jiang, Marie Menard, Alvaro Curiel-Garcia, Tanner C. Dalton, Margo Orlen, Cole Edwards, Clint Stalnecker, Julien Dilly, Stephanie Chang, Stephen A. Sastra, Carmine F. Palermo, Timour Baslan, Sha Tian, Matthew Holderfield, Elsa Quintana, Zhengping Wang, Jacqueline A. Smith, Andrea Califano, David Wildes, Andrew J. Aguirre, Robert H. Vonderheide, Ben Z. Stanger, Scott W. Lowe, Channing J. Der, Mallika Singh, Kenneth P. Olive. Resistance to RAS-GTP inhibition in models of pancreatic ductal adenocarcinoma arises downstream of RAS effectors [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 1927.

Human Breast Cancer Cell Lines Differentially Modulate Signaling from Distant Microenvironments, Which Reflects Their Metastatic Potential.

Metastasis is the stage at which the prognosis substantially decreases for many types of cancer. The ability of tumor cells to metastasize is dependent upon the characteristics of the tumor cells, and the conditioning of distant tissues that support colonization by metastatic cells. In this report, we investigated the systemic alterations in distant tissues caused by multiple human breast cancer cell lines and the impact of these alterations on the tumor cell phenotype. We observed that the niche within the lung, a common metastatic site, was significantly altered by MDA-MB-231, MCF7, and T47 tumors, and that the lung microenvironment stimulated, to differing extents, an epithelial-to-mesenchymal transition (EMT), reducing proliferation, increasing transendothelial migration and senescence, with no significant impact on cell death. We also investigated the ability of an implantable scaffold, which supports the formation of a distant tissue, to serve as a surrogate for the lung to identify systemic alterations. The scaffolds are conditioned by the primary tumor similarly to the lung for each tumor type, evidenced by promoting a pro-EMT profile. Collectively, we demonstrate that metastatic and non-metastatic breast cancers condition distant tissues, with distinct effects on tumor cell responses, and that a surrogate tissue can distinguish the metastatic potential of human breast cancer cell lines in an accessible site that avoids biopsy of a vital organ.

Abstract A039: Silencing MICAL2 expression in pancreatic cancer cells reduces IL-1A expression and inhibits tumor growth through a CD8+ T cell dependent mechanism

Abstract Introduction: Pancreatic cancer is characterized by desmoplastic, fibroinflammatory stroma. The crosstalk between cancer cells and their surrounding tumor microenvironment (TME) promotes disease progression, metastasis, and chemoresistance. We identified MICAL2 as a super-enhancer-associated gene in pancreatic cancer. MICAL2 (molecule interacting with Cas-L) proteins are flavin monooxygenases that promote actin depolymerization and indirectly regulate SRF transcription. In other work, we have found that MICAL2 promotes pancreatic cancer growth and progression. This study evaluated how MICAL2 pancreatic cancer cell expression modulates the PDAC tumor microenvironment. Methods: RNA-Seq analysis performed on human pancreas cancer cells (AsPC-1) before and after MICAL2 KD revealed differential regulation of IL-1α. We validated the expression of IL-1α in multiple human and mouse pancreas cancer cell lines by q-PCR. We next exposed human (h) and mouse (m) PSCs to conditioned media (CM) from cancer cells before and after MICAL2 KD and checked the expression of a-SMA, fibronectin, and Col1A and IL-6 genes by qPCR. KrasG12D/+; Trp53R172H/+; Pdx1-cre (KPC) cells with and without MICAL2 were orthotopically injected to assess the in vivo tumor growth and metastasis. Sc-RNA seq and Immunophenotyping were performed on Shcontrol (ShCNT) and MICAL2 (M2KD) KD tumors. CD8+ T cell depletion was done using an antibody-mediated approach. The adoptive transfer of CD8+ T cells extracted from MICAL2 KD tumors was done by tail vein injection. Results: MICAL2 KD resulted in the downregulation of the IL-1α gene in both human and mouse pancreas cancer cell lines (50% reduction, p<0.05). Human and mouse pancreatic stellate cells (hPSCs and mPSCs) co-cultured with cancer cells CM showed significant downregulation of a-SMA, fibronectin, Col1A, and IL-6 upon MICAL2 KD as compared to hPSCs and mPSCs co-cultured with ShCNT cells. Orthotopic injections of KPC MICAL2 KD cells led to decreased tumor growth as compared to ShCNT (0.26 gm vs. 1 gm, p = 0.008). Immunofluorescence (IF) revealed less α-SMA, PDGF-α, and IL-6 expression and reduced deposition of collagen and fibronectin in MICAL2 KD tumors. Single-cell RNA seq and flow cytometry studies of tumors harvested from both groups showed significantly increased infiltration of activated cytotoxic CD8+ T cells in MICAL2 KD tumors which was further confirmed by IF. Antibody-mediated depletion of CD8+ T cells in MICAL2 KD tumors abrogated their reduced tumor growth in both immune-hot and immune-cold KPC cell lines. Adoptive transfer of CD8+ T cells extracted from M2KD tumors significantly reduced ShCNT flank tumor growth. Conclusion: These data reveal that MICAL2 expression mediates tumor-stromal crosstalk through IL1A and creates an immunosuppressive environment in PDAC and suggests MICAL2 may be an attractive immunomodulatory target for pancreatic cancer therapy. Ongoing work is focused on dissecting the role of MICAL2 in priming the metastatic niche through the remodeling of the TME. Citation Format: Bharti Garg, EvanGeline Mose, Edgar Esparza, Jay Patel, Sarah Sass, Alexei Martsinkovskiy, Dawn Jacquish, Herve Tiriac, Andrew M. Lowy. Silencing MICAL2 expression in pancreatic cancer cells reduces IL-1A expression and inhibits tumor growth through a CD8+ T cell dependent mechanism [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A039.

Abstract B144: Discovery and preclinical evaluation of novel oral WEE1 degraders

Abstract Background: WEE1 is a key component of cell cycle checkpoints as it inhibits cell cycle progression through phosphorylation of CDK1. Inhibition of WEE1 could induce premature mitotic entry of cells with damaged DNA, leading to mitotic catastrophe and cell death. WEE1 small molecule inhibitors (SMIs) have demonstrated clinical benefits particularly in gynecological malignancies. However, limited antitumor activity and multifaceted toxicity profile from SMIs warranted the discovery of novel WEE1-targeting modalities to afford better therapeutic index towards a greater patient population. Here, we report the discovery of a novel oral, heterobifunctional WEE1 degrader NXD01 and its preclinical evaluation. Methods: We discovered NXD01 through our AI-empowered, prediction-guided compound optimization platform neoDegrader. We quantified NXD01-mediated WEE1 protein degradation by western blot and evaluated the anti-proliferative activity of NXD01 by CellTiter-Glo assay in multiple human cancer cell lines. We further assessed downstream effects of WEE1 degradation on phosphorylation of its target CDK1, and markers of cell cycle and apoptosis by western blot and flow cytometry. We studied the in vivo antitumor activity of NXD01 by oral gavage in the SK-UT-1 (human uterine leiomyosarcoma cell) xenograft model, followed by PK/PD analysis of the tumor tissues. Results: In SK-UT-1 cells, NXD01 potently degraded WEE1 protein at a DC50 of 1.4 nM with no obvious hook effect and achieved complete degradation within 4 h of treatment (DC90 = 4.5 nM). Marked reduction in CDK1-Y15 phosphorylation and increased γ-H2AX levels (a marker for DNA damage) and PARP cleavage (apoptosis) were all detected for NXD01 at 100 nM, one-tenth the effective concentration for the WEE1 SMI azenosertib (ZN-c3). Treatment of NXD01 (but not ZN-c3) at 100 nM greatly increased the proportion of cells in the G2/M phase indicating mitotic arrest. These results translated into a more potent anti-proliferative effect by NXD01 compared to ZN-c3 in vitro: the IC50 was 1.3 nM vs 37 nM for MOLT4, a T cell leukemia line, as well as across 10 gynecological cancer cell lines (NXD01 IC50: 10-210 nM; ZN-c3 IC50: 100-975 nM). NXD01 exhibited good exposure after oral administration in mice (dose normalized AUC0-t: 765 mg∙h/mL, t1/2: 3 h, F%: 15.7%). NXD01 showed strong in vivo antitumor activity in the SK-UT-1 xenograft model, achieving 96% tumor growth inhibition (TGI) at 30 mg/kg BID, as compared to 81% TGI for ZN-c3 at 60 mg/kg QD. Analysis of tumor samples suggested that 85% of in-tumor WEE1 protein had been degraded by NXD01 at 12 h post dose. Conclusion: We have discovered a structurally-novel, orally bioavailable, cereblon-mediated, heterobifunctional degrader of WEE1 protein NXD01. NXD01 displayed stronger in vitro and in vivo antitumor activity than currently the most advanced WEE1 small molecule inhibitor azenosertib, making NXD01 a differentiated candidate with strong potential for clinical development. Citation Format: Weiqiang Xing, Meixin Tao, Fan Huang, Liuge Gu, Min Cao, Chunhao Wang, Jianxiong Diao, Chunming Du, Xinyu Bai, Mingchen Chen, Xin Zheng, Ying Lei, Yang Xie, Taylor B. Guo. Discovery and preclinical evaluation of novel oral WEE1 degraders [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 B144.

Abstract C074: VPS34 inhibition delays activation-induced STING degradation to prolong STING signaling and improve anti-tumor efficacy in preclinical models

Abstract Background: The STING/interferon (IFN) response pathway has been a target of cancer immunotherapy for many years, as a method for reactivating the anti-tumor immune response. Stimulation of the STING pathway leads to activation and infiltration of immune cells into the tumor, and cancer cell death. However, multiple STING agonists have failed in the clinic due to poor efficacy or toxicity1. STING activation leads to its degradation within a few hours through trafficking to the lysosome, as a mechanism to control immune cell hyperactivation2. Although this safety mechanism is important for preventing autoimmunity and chronic inflammation, it diminishes the continuous activation of immune-mediated cancer cell killing, especially in an immunosuppressive tumor microenvironment. VPS34 is a lipid kinase which plays an integral role in endosomal trafficking and autophagy3. Previous publications have demonstrated a role for VPS34 inhibition in increased effector immune cell infiltration, which synergizes with anti-PD1 therapy, demonstrating a link to immune activation4. Herein, we demonstrate that the in vivo combination efficacy with a VPS34 inhibitor and a STING agonist is due to reduced degradation of activated STING, through the inhibition of VPS34-mediated endosomal trafficking. Materials and Methods: Cancer cell lines were cultured using recommended medium. STING activity was measured by Western blot and via a CisBio pTBK1 kit. ADU-S100/MSA-2 were sourced from MedChemExpress. CCL5, CXCL10 and IFNb ELISA’s were purchased from R&D systems. Primers used to measure mRNA expression were obtained from IDT. Results: DP-9244 is a potent and selective VPS34 inhibitor. The combination of DP-9244 with the STING agonist, ADU-S100, enhances IFN-mediated gene expression and cytokine secretion in the murine melanoma cell line, B16F10, and in the human and murine myeloid cell lines THP-1 and DC2.4, demonstrating combination activity in both cancer and immune cells. This combination activity is STING-dependent as demonstrated by the complete loss of IRF and NFκB activity in the THP-1 STING-/- cell line when treated with ADU-S100, DP-9244 or the combination. Mechanistically, VPS34 inhibition delays degradation of activated STING and prolongs downstream signaling of pTBK1 in multiple human and murine cancer cell lines. Importantly, the in vitro effects extend to in vivo efficacy in the B16F10 model, in which the combination of DP-9244 and ADU-S100 results in increased tumor growth control and number of mice responding to treatment. Conclusions: These data provide a strong preclinical rationale to combine a VPS34 inhibitor with a STING agonist as a method of improving STING pathway mediated anti-tumor efficacy.

Depletion of the Mitochondrial E3 Ligase MARCH5 Induces Synthetic Lethality to BCL2 Inhibitor (Venetoclax) Therapy in Cell Lines Representative of Diverse Blood Cancers

Combination therapies incorporating the BCL2 inhibitor, venetoclax (VEN), are standards of care in chronic lymphocytic leukemia (CLL) and acute myeloid leukemia (AML), and display promising activity against mantle cell lymphoma (MCL), multiple myeloma (MM) and many other hematologic malignancies. However, primary resistance is observed in some diseases and even across responsive malignancies, enduring cells typically persist, ultimately driving relapse and precluding cure. Genome wide CRISPR/Cas9 screens have the potential to identify novel mediators of venetoclax resistance. However, conventional knock-out/drop out (KO/DO) screens have several limitations including the frequent identification of common essential genes as false positives and the large scale required to detect DO with high statistical confidence. We devised a novel augmentation on the conventional CRISPR/Cas9 KO/DO screen utilizing two MCL cell lines (Jeko1, Z138) which were both resistant to apoptosis induced by VEN +/- ibrutinib (IBR)(Fig 1). Rather than detecting sensitizing hits through DO, we positively enriched for sensitizing hits by flow-sorting the rare (~10%) population of cells initiating apoptosis as indicated by AnnexinV positivity after brief (6hr) BH3-mimetic exposure. At this early time point, cells whose KO had led to a lowered apoptotic threshold could be identified without sufficient time for DNA degradation. DNA yield from dying cells approached 100% efficiency. sgRNA frequencies among the control cells and those undergoing early apoptosis were analyzed using MAGecKE-MLE. This method was anticipated to reduce common essential gene contamination, increase statistical power to detect sensitizing hits due to enrichment rather than DO of relevant genes, and identify targets likely to exhibit synthetic lethality (i.e. neither VEN nor KO alone are lethal, but combination achieves potent killing). The screens in both cell lines demonstrated significant depletion of common essential genes defined by DepMap by D14 (prior to treatment). Consistent with the increased power expected from a positive selection screen, both yielded >50 statistically significant VEN-sensitizing hits, after correction for multiple hypothesis testing. These included the expected KOs of MCL1 and BCL2L1 (BCL-X L), confirming the validity of the method. sgRNAs resulting in KO of the E3 ligase MARCH5 were among the most enriched within dying (VEN-sensitized) cells in both Jeko1 and Z138 screens, with either VEN only or VEN+IBR treatment (Fig 1). The screen method was repeated in Jeko1 cells using the MCL1 inhibitor AMG176 and BCL-X L inhibitor A1331853, and again yielded BCL2 as well as novel hits, including MARCH5 KO as a sensitizing hit to BCL-X L inhibition. KO of MARCH5 markedly sensitized multiple human blood cancer cell lines to venetoclax (Table 1), including sensitive and resistant lines, with and without functional TP53. In Jeko1 (MCL) cells, which are highly resistant to BCL2 or BCL-X L inhibition when wildtype (10-20% apoptosis at 6µM), MARCH5 KO induced marked sensitization to single-agent VEN (LC50 8nM) or A1331853 (LC50 375nM).By introducing a degron dTAG MARCH5 construct into MARCH5 KO Jeko1 cells, we demonstrated dose-dependent sensitisation to VEN with MARCH5 depletion, marked synergy (mean Bliss score: 22.97) and synthetic lethality. Degron dTAG-mediated depletion of MARCH5 demonstrated synergy with venetoclax in MOLM13 (AML) and Z138 (MCL) cell lines, and enhanced in vitro killing of MOLM13 by VEN + azacytidine, and killing of Jeko1 and Z138 by VEN + IBR. CRISPR “death” represents a powerful modification to conventional CRISPR KO screens, with reduced common essential gene false positives, increased statistical power and enrichment for synthetic lethal hits. We demonstrate the pan-hematologic, dose-dependent VEN-sensitization achieved by MARCH5 depletion across multiple blood cancer lines with and without normal TP53 function. MARCH5 depletion also sensitizes blood cancer cell lines to BCL-X L inhibition. Addition of MARCH5 depletion to VEN-combination treatment improved MCL and AML killing in vitro and this target should be prioritized for accelerated drug development.

DDX5 (p68) orchestrates β-catenin, RelA and SP1 mediated MGMT gene expression in human colon cancer cells: Implication in TMZ chemoresistance

DDX5 (p68) upregulation has been linked with various cancers of different origins, especially Colon Adenocarcinomas. Similarly, across cancers, MGMT has been identified as the major contributor of chemoresistance against DNA alkylating agents like Temozolomide (TMZ). TMZ is an emerging potent chemotherapeutic agent across cancers under the arena of drug repurposing. Recent studies have established that patients with open MGMT promoters are prone to be innately resistant or acquire resistance against TMZ compared to its closed conformation. However, not much is known about the transcriptional regulation of MGMT gene in the context of colon cancer. This necessitates studying MGMT gene regulation which directly impacts the cellular potential to develop chemoresistance against alkylating agents. Our study aims to uncover an unidentified mechanism of DDX5-mediated MGMT gene regulation. Experimentally, we found that both mRNA and protein expression levels of MGMT were elevated in response to p68 overexpression in multiple human colon cancer cell lines and vice-versa. Since p68 cannot directly interact with the MGMT promoter, transcription factors viz., β-catenin, RelA (p65) and SP1 were also studied as reported contributors. Through co-immunoprecipitation and GST-pull-down studies, p68 was established as an interacting partner of SP1 in addition to β-catenin and NF-κB (p50-p65). Mechanistically, luciferase reporter and chromatin-immunoprecipitation assays demonstrated that p68 interacts with the MGMT promoter via TCF4-LEF, RelA and SP1 sites to enhance its transcription. To the best of our knowledge, this is the first report of p68 as a transcriptional co-activator of MGMT promoter and our study identifies p68 as a novel and master regulator of MGMT gene expression.

Protein-encapsulated doxorubicin reduces cardiotoxicity in hiPSC-cardiomyocytes and cardiac spheroids while maintaining anticancer efficacy.

The chemotherapeutic doxorubicin (DOX) detrimentally impacts the heart during cancer treatment. This necessitates development of non-cardiotoxic delivery systems that retain DOX anticancer efficacy. We used human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), endothelial cells (hiPSC-ECs), cardiac fibroblasts (hiPSC-CFs), multi-lineage cardiac spheroids (hiPSC-CSs), patient-specific hiPSCs, and multiple human cancer cell lines to compare the anticancer efficacy and reduced cardiotoxicity of single protein encapsulated DOX (SPEDOX-6), to standard unformulated (UF) DOX. Cell viability assays and immunostaining in human cancer cells, hiPSC-ECs, and hiPSC-CFs revealed robust uptake of SPEDOX-6 and efficacy in killing these proliferative cell types. In contrast, hiPSC-CMs and hiPSC-CSs exhibited substantially lower cytotoxicity during SPEDOX-6 treatment compared with UF DOX. SPEDOX-6-treated hiPSC-CMs and hiPSC-CSs maintained their functionality, as indicated by sarcomere contractility assessment, calcium imaging, multielectrode arrays, and RNA sequencing. This study demonstrates the potential of SPEDOX-6 to alleviate cardiotoxic side effects associated with UF DOX, while maintaining its anticancer potency.

Characterization of CCoV-HuPn-2018 spike protein-mediated viral entry.

Canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018) was recently isolated from a child with pneumonia. This novel human pathogen resulted from cross-species transmission of a canine coronavirus. It has been known that CCoV-HuPn-2018 uses aminopeptidase N (APN) from canines, felines, and porcines, but not humans, as functional receptors for cell entry. The molecular mechanism of cell entry in CCoV-HuPn-2018 remains poorly understood. In this study, we demonstrated that among the nine APN orthologs tested, the APN of the Mexican free-tailed bat could also efficiently support CCoV-HuPn-2018 spike (S) protein-mediated entry, raising the possibility that bats may also be an alternative host epidemiologically important for the transmission of this virus. The glycosylation at residue N747 of canine APN is critical for its receptor activity. The gain of glycosylation at the corresponding residues in human and rabbit APNs converted them to functional receptors for CCoV-HuPn-2018. Interestingly, the CCoV-HuPn-2018 spike protein pseudotyped virus infected multiple human cancer cell lines in a human APN-independent manner, whereas sialic acid appeared to facilitate the entry of the pseudotyped virus into human cancer cells. Moreover, while host cell surface proteases trypsin and TMPRSS2 did not promote the entry of CCoV-HuPn-2018, endosomal proteases cathepsin L and B are required for the entry of CCoV-HuPn-2018 in a pH-dependent manner. IFITMs and LY6E are host restriction factors for the CCoV-HuPn-2018 entry. Our results thus suggest that CCoV-HuPn-2018 has not yet evolved to be an efficient human pathogen. Collectively, this study helps us understand the cell tropism, receptor usage, cross-species transmission, natural reservoir, and pathogenesis of this potential human coronavirus. IMPORTANCE Viral entry is driven by the interaction between the viral spike protein and its specific cellular receptor, which determines cell tropism and host range and is the major constraint to interspecies transmission of coronaviruses. Aminopeptidase N (APN; also called CD13) is a cellular receptor for HCoV-229E, the newly discovered canine coronavirus-human pneumonia-2018 (CCoV-HuPn-2018), and many other animal alphacoronaviruses. We examined the receptor activity of nine APN orthologs and found that CCoV-HuPn-2018 utilizes APN from a broad range of animal species, including bats but not humans, to enter host cells. To our surprise, we found that CCoV-HuPn-2018 spike protein pseudotyped viral particles successfully infected multiple human hepatoma-derived cell lines and a lung cancer cell line, which is independent of the expression of human APN. Our findings thus provide mechanistic insight into the natural hosts and interspecies transmission of CCoV-HuPn-2018-like coronaviruses.

Increased levels of eIF2A inhibit translation by sequestering 40S ribosomal subunits.

eIF2A was the first eukaryotic initiator tRNA carrier discovered but its exact function has remained enigmatic. Uncharacteristic of translation initiation factors, eIF2A is reported to be non-cytosolic in multiple human cancer cell lines. Attempts to study eIF2A mechanistically have been limited by the inability to achieve high yield of soluble recombinant protein. Here, we developed a purification paradigm that yields ∼360-fold and ∼6000-fold more recombinant human eIF2A from Escherichia coli and insect cells, respectively, than previous reports. Using a mammalian in vitro translation system, we found that increased levels of recombinant human eIF2A inhibit translation of multiple reporter mRNAs, including those that are translated by cognate and near-cognate start codons, and does so prior to start codon recognition. eIF2A also inhibited translation directed by all four types of cap-independent viral IRESs, including the CrPV IGR IRES that does not require initiation factors or initiator tRNA, suggesting excess eIF2A sequesters 40S subunits. Supplementation with additional 40S subunits prevented eIF2A-mediated inhibition and pull-down assays demonstrated direct binding between recombinant eIF2A and purified 40S subunits. These data support a model that eIF2A must be kept away from the translation machinery to avoid sequestering 40S ribosomal subunits.

Otilonium Bromide acts as a selective USP28 inhibitor and exhibits cytotoxic activity against multiple human cancer cell lines

USP28 contributes to tumorigenesis through modulating the lifespan of oncogenic factors such as c-Myc and ΔNp63, and it has been identified as a potential target for anti-cancer drug development. Currently, although quite a number of USP28 inhibitors have been developed, they all are still in preclinical research stage. Besides, none of them exhibits satisfying inhibition selectivity against USP28 over its closest homologue USP25. Here in this manuscript, a high-throughput screening aiming to discover USP28 inhibitors with novel scaffold and enhanced inhibition selectivity were conducted. After the primary screening and the second round of validation, Otilonium Bromide, an approved drug for treating irritable bowel syndrome, was identified to inhibit USP28′s activity with the IC50 value at 6.90 ± 0.90 μM. Besides, the drug exhibits a 3–4 folds inhibition selectivity against USP28 over USP25. According to the enzymatic kinetics analysis data and the hydrogen–deuterium exchange mass spectrometry results, Otilonium Bromide could bind to the allosteric pocket of USP28 and inhibit its activity in a reversible and non-competitive mode. The following performed cell-based assays revealed that the drug could cause cytotoxicity against human colorectal cancer cells and lung squamous carcinoma cells potentially through down-regulating USP28′s oncogenic substrates c-Myc and/or ΔNp63. Meanwhile, since Otilonium Bromide has been found to preferentially distribute to gastrointestinal tissues, we then evaluated its potential in the combination treatment of colorectal cancer cells with Regorafenib, which is an approved drug for colorectal cancer therapy. As expected, Otilonium Bromide could significantly enhance the sensitivity of colorectal cancer cells to Regorafenib.

Deciphering the involvement of the Hippo pathway co-regulators, YAP/TAZ in invadopodia formation and matrix degradation

Invadopodia are adhesive, actin-rich protrusions formed by metastatic cancer cells that degrade the extracellular matrix and facilitate invasion. They support the metastatic cascade by a spatially and temporally coordinated process whereby invading cells bind to the matrix, degrade it by specific metalloproteinases, and mechanically penetrate diverse tissue barriers by forming actin-rich extensions. However, despite the apparent involvement of invadopodia in the metastatic process, the molecular mechanisms that regulate invadopodia formation and function are still largely unclear. In this study, we have explored the involvement of the key Hippo pathway co-regulators, namely YAP, and TAZ, in invadopodia formation and matrix degradation. Toward that goal, we tested the effect of depletion of YAP, TAZ, or both on invadopodia formation and activity in multiple human cancer cell lines. We report that the knockdown of YAP and TAZ or their inhibition by verteporfin induces a significant elevation in matrix degradation and invadopodia formation in several cancer cell lines. Conversely, overexpression of these proteins strongly suppresses invadopodia formation and matrix degradation. Proteomic and transcriptomic profiling of MDA-MB-231 cells, following co-knockdown of YAP and TAZ, revealed a significant change in the levels of key invadopodia-associated proteins, including the crucial proteins Tks5 and MT1-MMP (MMP14). Collectively, our findings show that YAP and TAZ act as negative regulators of invadopodia formation in diverse cancer lines, most likely by reducing the levels of essential invadopodia components. Dissecting the molecular mechanisms of invadopodia formation in cancer invasion may eventually reveal novel targets for therapeutic applications against invasive cancer.

Abstract 42: In vivo subcutaneous and orthotopic cancer xenograft modeling in the SRG immunodeficient rat

Abstract Human cancer xenografts are a vital tool for understanding tumor biology, growth kinetics, and therapeutic efficacy using animal models. Historically, immunodeficient mice have been the standard rodent species for cancer xenograft modeling. However, an immunodeficient rat that supports a wide variety of human cancer cell types would provide a larger rodent strain for easier surgical manipulation, serial blood sampling, and provide a single model in which efficacy, pharmacokinetics, and toxicology can be performed. We have created a Sprague Dawley Rag2 -/-, Il2rg -/- rat (SRGTM OncoRat®) that provides a highly supportive environment for growing tumors of human origin. The SRG rat lacks B, T, and NK cells and readily supports the growth of multiple human cancer cell lines. The SRG rat is more immunodeficient than the Nude rat, suggesting it may be permissive to a wider variety of human cancer types. Here we demonstrate the utility of the SRG rat for both subcutaneous and orthotopic xenograft modeling. The SRG rat supports the growth of both lung and liver orthotopic cancers. In addition, the SRG rat supports the growth of orthoptic human glioblastoma multiforme in the brain. We use in vivo imaging to visualize tumor establishment and growth in subcutaneous, orthotopic, and metastatic models. Furthermore, our data show the ability of the SRG rat to support the growth of multiple different human cancer cell types subcutaneously in two different matrices, Matrigel® and VitroGel®. These data confirm that the SRG rat is an excellent host for studying human cancer. Our data demonstrate that the SRG rat has a high utility for studies using in vivo imaging, orthotopic tumor implantation, and standard subcutaneous tumor modeling. As the most immunodeficient rat commercially available, the SRG rat supports the growth of multiple human cancer types in a larger rodent strain relative to commercially available mouse models. Citation Format: Diane Begemann, Aida Javidan, Cynthia Dunn, Nicolas Johnston, R. Grace Walton, Valeriya Steffey, Ian Corbin, Niveen Fulcher, Cleusa De Oliveira, Hu Xu, Mila Uzelac, Andrew Deweyert, John A. Ronald, Susanne Schmid, Matthew O. Hebb, Fallon K. Noto. In vivo subcutaneous and orthotopic cancer xenograft modeling in the SRG immunodeficient rat [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 42.

Abstract 377: RON receptor tyrosine kinase in head and neck cancer: a potential target for overcoming resistance to anti-EGFR therapy

Abstract Background: Although the epidermal growth factor receptor (EGFR) is overexpressed in 80-90% of Head and Neck Squamous Cell Carcinomas (HNSCCs), response to anti-EGFR targeted therapy remains low. Trans-activation of other receptor tyrosine kinases is a common mechanism of resistance to EGFR directed therapy. The RON receptor tyrosine kinase (Macrophage stimulating 1-receptor/MST1R) is a member of the c-MET oncogene family. While limited, prior studies by our group and others have reported elevated RON expression in over 60% of primary HNSCC samples and that receptor cross talk exists with RON and EGFR. Based on this information, we hypothesize that the pro-tumorigeneic role of RON and its crosstalk with EGFR contributes to HNSCC growth and to anti-EGFR therapeutic failure. To test this hypothesis, we examined the expression level of RON and EGFR in human and mouse HNSCC cell lines and in HNSCC patient samples from publicly available databases. Further, we investigated the role of RON in radiation resistance as radiation is a primary treatment modality for HNSCC. Results: Immunoblotting demonstrated strong RON expression in eight of nine human HNSCC cell lines tested and strong EGFR expression in six HNSCC cell lines. In exploring mRNA expression of RON using the Kaplan-Meier Plotter HNSCC RNA-seq dataset, high RON expression was associated with poorer overall survival (P = 0.054) in patients with Stage 3 HNSCC and low mutation burden. To assess the relationship between RON and EGFR, mRNA expression was mined in the GSE6631 dataset from the NCBI gene expression omnibus (GEO). Our analysis found RON to be significantly overexpressed in HNSCC samples compared to normal matched controls (P = 0.029). Interestingly, EGFR mRNA expression was negatively correlated (r = -0.2) with RON mRNA expression from the same patients. This inverse correlation between RON and EGFR expression was corroborated by immunoblotting data of HNSCC lines, UM-SCC-090 and 93-UV-147T which demonstrated low levels of EGFR and high levels of RON. As ionizing radiation is a pillar of HNSCC treatment and is often delivered concurrent with anti-EGFR therapy, we next examined the relative radiosensitivity in multiple murine and human cancer cell lines with and without RON expression using colony formation assays. Preliminary studies demonstrated significantly increased colony formation in high RON-expressing cells relative to low RON-expressing cells. Conclusion: RON is expressed at high levels in multiple HNSCC cell lines and in human samples. Protein expression data and in silica analyses support an inverse association between EGFR and RON. RON expression is associated with radiation resistance. Future studies will investigate interactions between RON and EGFR, and whether RON inhibition can increase anti-EGFR treatment and radiation sensitivity in HNSCC cells. Citation Format: Zhixin Lu, Vinita Takiar, Susan E. Waltz. RON receptor tyrosine kinase in head and neck cancer: a potential target for overcoming resistance to anti-EGFR therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 377.