Apoptosis, or programmed cell death, plays a critical role in maintaining tissue homeostasis by eliminating damaged or abnormal cells. Dysregulation of apoptosis pathways is a hallmark of cancer, allowing malignant cells to evade cell death and proliferate uncontrollably. Targeting apoptosis pathways has emerged as a promising therapeutic strategy in cancer treatment, aiming to restore the balance between cell survival and death. The MDM2 inhibitor alrizomadlin, the Bcl-2/Bcl-xL inhibitor pelcitoclax, and the IAP family inhibitor dasminapant were evaluated both individually and in combinations with standard of care and investigational anticancer small molecules in a spheroid model of solid tumors. The multi-cell type tumor spheroids were grown from human endothelial cells and mesenchymal stem cells combined with human malignant cells that were either established or patient-derived cell lines from the NCI Patient-Derived Models Repository. The malignant cell lines were derived from a range of solid tumors including uterine carcinosarcoma, synovial sarcoma, rhabdomyosarcoma, soft tissue sarcoma, malignant fibrous histiocytoma, malignant peripheral nerve sheath tumor (MPNST), pancreas, ovary, colon, breast, and small cell lung cancer. Interactions were observed from combinations of the apoptosis pathway targeted agents. Additionally, interactions were observed from combinations of the apoptosis pathway targeted agents with other agents, including PARP inhibitors, the XPO1 inhibitor eltanexor, and the PI3K inhibitor copanlisib. Enhanced activity was also observed from combinations of the apoptosis pathway targeted agents with MAPK pathway targeted agents, including the MEK inhibitor cobimetinib as well as adagrasib and MRTX1133, which specifically target the KRAS G12C and G12D variants, respectively.

The KRAS gene is among the most frequently altered genes in cancer and the KRAS protein was long deemed undruggable. Recent strategies to target oncogenic KRAS have included both direct inhibition of the KRAS protein and indirect inhibition of its activity by targeting upstream and downstream signaling pathway mediators. A high-throughput screen of multi-cell type tumor spheroids was designed to identify active combinations of targeted small molecules and KRAS pathway inhibitors. Inhibitors of the non-receptor protein tyrosine phosphatase SHP2 and the guanine nucleotide exchange factor SOS1 were tested to evaluate indirect upstream pathway inhibition, while sotorasib directly inhibited the KRAS G12C variant. As single agents, sotorasib and the SHP2 inhibitor batoprotafib (TNO155) exhibited selectivity towards spheroids with KRAS G12C, whereas the SOS1 inhibitor BI-3406 showed varying activity across KRAS variants. Vertical inhibition of the RAS/MEK/ERK pathway by targeting SHP2 or SOS1 and the downstream kinases MEK (trametinib) or ERK (temuterkib) was highly effective. Inhibition of upstream tyrosine receptor kinases with nintedanib in combination with batoprotafib or BI-3406 was also effective, and in combination with sotorasib, demonstrated synergy in spheroids harboring KRAS G12C. Dual inhibition of the RAS/MEK/ERK and PI3K/AKT/mTOR pathways with batoprotafib or sotorasib with either the mTORC1/2 inhibitor sapanisertib or the AKT inhibitor ipatasertib demonstrated combination activity, primarily in spheroids harboring KRAS G12C. Combination of the BCL-2 inhibitor venetoclax with sotorasib, batoprotafib or BI-3406 resulted in additive and synergistic cytotoxicity. Lastly, concurrent inhibition of the KRAS pathway with sotorasib and batoprotafib demonstrated combination activity in spheroids containing KRAS G12C. SIGNIFICANCE KRAS variants are oncogenic drivers for a range of human cancers. Multiple combinations of small molecule agents that target RAS signaling were screened and reduced the viability of multi-cell type spheroid models for a variety of human solid tumors. Combinations warranting further testing were identified. ### Competing Interest Statement The authors have declared no competing interest.

Abstract The potential of novobiocin to augment cancer chemotherapy was explored in the late 1980s and early 1990s in tumor cells and tumor-bearing mice and in Phase 1 clinical trials with cyclophosphamide or cisplatin. The molecular target of novobiocin was identified to be DNA POLθ which has a role in repair of DNA double strand breaks (DSB) during mitosis. Genetic alterations which may increase or decrease POLθ inhibitor effects have been elucidated. Thirty patient-derived tumor cell lines with known BRCA, ATM, ATR, POLQ, XRCC1, PALB2, PARP1, and LIG3 alterations were screened in a complex spheroid assay (tumor cells, endothelial cells, mesenchymal stem cells) with a POLθ inhibitor (novobiocin, ART-558, or RP6685), alone or in simultaneous combination with a FDA approved or investigational anticancer small molecule, using a 7-day exposure and a CellTiter-Glo 3D luminescence endpoint. As single agents, the POLθ inhibitors had little or no cytotoxicity. Potentiation of the topoisomerase II inhibitors doxorubicin (DOX) and etoposide by the POLθ inhibitors ART-558 or RP6685 was observed in spheroids grown from the serous endometrial adenocarcinoma 922993-354-T-J3, which has ATM and BRCA2 variants. The 379773-124-R-J2 endometrioid endometrial adenocarcinoma which harbors mutant POLθ demonstrated antagonism with ART-558 in combination with PARP inhibitors and less than additive to additive cytotoxicity in combination with DOX. Activity of the Chk1/2 inhibitor prexasertib was potentiated by either ART-558 or RP6685 in the 922993-354-T-J3 complex spheroids. The combination of POLθ inhibitor ART-558 and the Chk1/2 inhibitor prexasertib produced up to 1 log increase in cytotoxicity in the 922993-354-T-J3 complex spheroids. KRAS G12D inhibitor MRTX-133 cytotoxicity against 186277-243-T-J2 colon adenocarcinoma with the KRAS G12D variant was potentiated by either ART-558 or RP6685. Potential combinations to advance into in vivo studies will be presented. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Beverly A. Teicher, Thomas S. Dexheimer, Thomas Silvers, Nathan P. Coussens, J Paul Eder, James H. Doroshow. Polymerase θ (POLθ) inhibitors (novobiocin, ART-558, RP6685) were tested with 22 approved and investigational agents in multi-cell type spheroids of genetically selected patient-derived human tumor cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB215.

Abstract The NCI60 screen was established in the 1980s and introduced in the 1990s as a screening service for the worldwide cancer research community. The screen features 60 human tumor cell lines that represent 9 cancer types including non-small cell lung, colon, central nervous system, ovarian, renal, prostate and breast, as well as leukemia and melanoma. On a weekly basis, synthetic molecules, natural products, and biologics are screened against the 60-cell line panel to evaluate their anticancer potential. On average, more than 13,000 compounds are screened annually for investigators throughout the world. Cell growth and cytotoxicity were assessed by the sulforhodamine B (SRB) assay, performed in a 96-well format, that measures cellular protein content with an absorbance readout and enabled unprecedented throughput in the 1980s. Although the SRB assay supported a robust screening platform for three decades, its sensitivity and throughput are limited. Recently the NCI’s DCTD introduced the HTS384 NCI60 as a modernized screening platform. While the same 60 cell lines are evaluated, the HTS384 NCI60 is fully automated and performed in a 384-well format to enable a higher throughput. Additionally, the 48-h test agent exposure period of the SRB assay was increased to 72 h, which may result in lower 50% growth inhibition values (GI50) for some agents. A highly sensitive CellTiter-Glo luminescence readout for cell viability, based on cellular ATP content, was selected to enable comparisons of the NCI60 monolayer data to 3D cell culture models where the same assay chemistry can be applied. The HTS384 NCI60 was evaluated by screening a library of 1,003 U.S. Food and Drug Administration-approved and investigational oncology agents at five concentrations. Assay performance of the 60 cell lines was assessed from the controls of 30 - 60 microplates per cell line. The average doubling time of the 60 cell lines during the 72-h exposure period ranged between 19 h for NCI-H460 (17 - 23 h, n = 57) to 117 h for MDA-MB-468 (43 - 272 h, n = 60). The average signal-to-background ratio of all 60 cell lines was 89 (3,413 microplates) and ranged from 6 for OVCAR-5 (3 - 13, n = 54) to 229 for SF-539 (141 - 566, n = 60). The average coefficient of variation for microplate vehicle controls (n = 14) of all 60 cell lines (n = 3,413) was 5.2% and ranged from 3.2% for SK-MEL-28 (1.4 - 7.7%, n = 60) to 11.1% for HL-60(TB) (5.2 - 20.2%, n = 59). The average Z’-factor value of all 60 cell lines (n = 3,413) was 0.82 and ranged between 0.66 for HL-60(TB) (0.39 - 0.84, n = 59) to 0.89 for MALME-3M (0.79 - 0.95, n = 59), SF-295 (0.49 - 0.97, n = 59), SK-MEL-28 (0.74 - 0.95, n = 60), and UO-31 (0.8 - 0.94, n = 54). The development and performance of the HTS384 NCI60 screen will be described. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Nathan P. Coussens, Thomas S. Dexheimer, Eric M. Jones, Thomas Silvers, John R. Britt, Ronald C. Taylor, Mark W. Kunkel, James H. Doroshow, Beverly A. Teicher. Development and performance of the National Cancer Institute’s HTS384 NCI60 screen: A modernized platform to support drug discovery and development by the worldwide cancer research community [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 3090.

Abstract The alternative splicing of mRNA precursors allows one gene the capacity to yield multiple protein isoforms, each with distinct functions. Although this intricate process is subject to stringent regulation under normal physiological conditions, aberrant alternative splicing can generate atypical proteins and contribute to various diseases, including cancer. By activating splicing factors, Cdc-like kinases (CLKs) serve as pivotal regulators of alternative splicing and are considered promising therapeutic targets for various tumors. In this study, we investigated the activity of two CLK inhibitors, cirtuvivint (SM08502) and CC-671, in combination with precision oncology agents or conventional chemotherapeutics. Thirty highly characterized patient-derived cancer cell lines were selected from the National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov/models/database.htm). The cell lines were derived from a range of cancer types including head and neck, bladder, pancreas, endometrial, colon, and melanoma, and contained clinically relevant variants of KRAS (G12C, G12D). Multi-cell type tumor spheroids were grown from a ratio of 6:2.5:1.5 malignant cells, endothelial cells, and mesenchymal stem cells, respectively. Following three days of growth, the spheroids were treated with single agents or combinations at concentrations up to their clinical Cmax value, if reported. After seven days of continuous drug exposure, cell viability was assessed using the CellTiter-Glo 3D assay. As a single agent, cirtuvivint showed concentration-dependent activity in all spheroid types with 1 – 3 logs of cytotoxicity, whereas CC-671 did not show activity in all spheroid types and achieved no more than 1 log of cytotoxicity. Among the standard chemotherapeutic drugs, doxorubicin and SN-38 demonstrated additive and greater-than-additive cytotoxicity in various spheroid types when combined with either CLK inhibitor. Several of the targeted oncology drugs exhibited noteworthy additive and greater-than-additive cytotoxicity when combined with a CLK inhibitor. These agents included the XPO1 inhibitor, eltanexor, the MCL-1 inhibitor, tapotoclax, the α-isoform-specific PI3K inhibitor, inavolisib, and the pan-PI3K inhibitor, copanlisib. Notably, combinations of the CLK inhibitors with the KRAS G12D variant-specific inhibitor, MRTX-1133, showed selective activity against all tumor cell lines harboring this genetic variant. Interestingly, a strong antagonistic interaction between paclitaxel and CC-671 was observed in all thirty spheroid types, while no such interaction was observed with cirtuvivint. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas Steven Dexheimer, Thomas Silvers, Nathan P. Coussens, Steven D. Gore, James H. Doroshow, Beverly A. Teicher. Combinations of Cdc-like kinase (CLK) inhibitors with targeted oncology agents or standard chemotherapy in patient-derived multi-cell type tumor spheroids [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 4608.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that regulates genes of drug transporters and metabolic enzymes to detoxify small molecule xenobiotics. It has a complex role in cancer biology, influencing both the progression and suppression of tumors by modulating malignant properties of tumor cells and anti-tumor immunity, depending on the specific tumor type and developmental stage. This has led to the discovery and development of selective AhR modulators, including BAY 2416964 which is currently in clinical trials. To identify small molecule anticancer agents that might be combined with AhR antagonists for cancer therapy, a high-throughput combination screen was performed using multi-cell type tumor spheroids grown from malignant cells, endothelial cells, and mesenchymal stem cells. The AhR selective antagonists BAY 2416964, GNF351, and CH-223191 were tested individually and in combination with twenty-five small molecule anticancer agents. As single agents, BAY 2416964 and CH-223191 showed minimal activity, whereas GNF351 reduced the viability of some spheroid models at concentrations greater than 1 µM. The activity of most combinations aligned well with the single agent activity of the combined agent, without apparent contributions from the AhR antagonist. All three AhR antagonists sensitized tumor spheroids to TAK-243, an E1 ubiquitin-activating enzyme inhibitor. These combinations were active in spheroids containing bladder, breast, ovary, kidney, pancreas, colon, and lung tumor cell lines. The AhR antagonists also potentiated pevonedistat, a selective inhibitor of the NEDD8-activating enzyme E1 regulatory subunit, in several tumor spheroid models. In contrast, the AhR antagonists did not enhance the cytotoxicity of the proteasome inhibitor bortezomib.

Figure S1. Representative brightfield images for assay optimized cell densities from a DMSO-treated well on Day 10.

Combination of DNA-PK inhibitors with trabectedin. A, Top, Mean Bliss matrix scores (n = 546) were calculated from the concentration matrix [(5 concentrations of drug A) × (6 concentrations of drug B) = (30 combination concentrations)] of each combination tested (n = 21) in all complex tumor spheroid models (n = 26). Mean Bliss matrix scores from the combination of the DNA-damaging drug trabectedin with the DNA-PK inhibitor nedisertib are highlighted in blue, whereas all other combinations are shown in light gray. A, Bottom, A mean Bliss score plot (n = 104) calculated from the combination concentration matrix of trabectedin with nedisertib across all complex tumor spheroid models (n = 26). The data are colored as a heat map, where blue indicates synergy, yellow indicates additivity, and red indicates antagonism. B, Concentration–response curves (top, mean ± SD, n = 4 technical replicates) from combinations of trabectedin with nedisertib and corresponding mean Bliss score plots (bottom, n = 4 technical replicates) showing the scores from each combination's concentration matrix and colored as a heat map. Data are shown for complex tumor spheroids grown with the malignant cell lines (from left): ASPS-1 (alveolar soft part sarcoma), HS-SY-2 (synovial sarcoma), and NCI-H211 (SCLC). C, Top, Mean Bliss matrix scores are highlighted from the combination of trabectedin with the DNA-PK inhibitor VX-984 across all complex tumor spheroid models (blue, n = 26), whereas all other combinations are shown in light gray. C, Bottom, A mean Bliss score plot (n = 104) calculated from the combination concentration matrix of trabectedin with VX-984 across all complex tumor spheroid models (n = 26). D, Concentration–response curves (top, mean ± SD, n = 4 technical replicates) from combinations of trabectedin with VX-984 and corresponding mean Bliss score plots (bottom, n = 4 technical replicates) showing the scores from each combination's concentration matrix and colored as a heat map. Data are shown for complex tumor spheroids grown with the malignant cell lines (from left): ASPS-1 (alveolar soft part sarcoma), HS-SY-2 (synovial sarcoma), and NCI-H211 (SCLC).

Table S1. Top-ranking drug combinations by mean Bliss matrix score.

The malignant cell lines grown as complex tumor spheroids for this study. The names of both patient-derived and established cell lines are listed along with the tumor type they were derived from and key genetic alterations

Sarcoma cell lines that were in the screen

Multicellular spheroids comprised of malignant cells, endothelial cells, and mesenchymal stem cells served as an in vitro model of human solid tumors to investigate the potentiation of DNA-damaging drugs by pharmacologic modulation of DNA repair pathways. The DNA-damaging drugs, topotecan, trabectedin, and temozolomide were combined with varied inhibitors of DNA damage response enzymes including PARP (olaparib or talazoparib), ATM (ataxia telangiectasia mutated; AZD-1390), ATR (ataxia telangiectasia and Rad3-related protein; berzosertib or elimusertib), and DNA-PK (DNA-dependent protein kinase; nedisertib or VX-984). A range of clinically achievable concentrations were tested up to the clinical Cmax, if known. Mechanistically, the types of DNA damage induced by temozolomide, topotecan, and trabectedin are distinct, which was apparent from the response of spheroids to combinations with various DNA repair inhibitors. Although most combinations resulted in additive cytotoxicity, synergistic activity was observed for temozolomide combined with PARP inhibitors as well as combinations of the ATM inhibitor AZD-1390 with either topotecan or trabectedin. These findings might provide guidance for the selection of anticancer agent combinations worthy of further investigation. Significance: Clinical efficacy of DNA-damaging anticancer drugs can be influenced by the DNA damage response in tumor cells. The potentiation of DNA-damaging drugs by pharmacologic modulation of DNA repair pathways was assessed in multicellular tumor spheroids. Although most combinations demonstrated additive cytotoxicity, synergistic cytotoxicity was observed for several drug combinations.

Abstract Dysregulation of the phosphatidylinositol 3-kinase (PI3K) pathway plays a significant role in cancer, and inhibitors offer a promising strategy for targeted therapies. The selectivity of current inhibitors varies against different PI3K isoforms or subtypes. For example, alpelisib and inavolisib predominantly target the α-isoform, whereas duvelisib targets the δ- and γ-isoforms. By contrast, copanlisib is a pan-PI3K inhibitor, exhibiting activity against all isoforms. In combinations with other targeted agents, we investigated the activity of these four PI3K inhibitors against multicellular spheroids. The multicellular spheroids, grown from 60% malignant cells, 25% endothelial cells, and 15% mesenchymal stem cells, served as a model for human solid tumors that incorporates both malignant and stromal components. Thirty multicellular spheroid models were grown from various malignant cell types, including twenty-seven patient-derived cancer cell lines obtained from the NCI Patient-Derived Models Repository (https://pdmr.cancer.gov/) and three established cell lines from the NCI-60 human tumor cell line panel. After three days of spheroid growth, anticancer agents were introduced at concentrations up to their reported clinical Cmax value, if known. After seven days of drug exposure, cell viability was measured using the CellTiter-Glo 3D assay. Among the various drug combinations evaluated, simultaneous targeting of the PI3K and RAS pathways was highly effective, particularly against malignant cell lines with an activated RAS pathway. Notably, additive and/or synergistic activities were observed when either the α-isoform-specific PI3K inhibitors (alpelisib and inavolisib) or the pan-PI3K inhibitor (copanlisib) was combined with either the MEK inhibitor selumetinib, the ERK inhibitor ravoxertinib, or the type II pan-RAF kinase inhibitor DAY101. Additionally, combinations of the same three PI3K inhibitors with variant-specific inhibitors, such as vemurafenib (BRAF V600E), sotorasib (KRAS G12C), or MTRX-1133 (KRAS G12D) demonstrated selective activity against cell lines carrying the variant. Another effective combination with PI3K inhibitors was achieved with vertical inhibition of the PI3K pathway through the addition of the mTORC1/2 kinase inhibitor sapanisertib. Finally, combinations of a CDK4/6 inhibitor (abemaciclib or palbociclib) with a PI3K inhibitor demonstrated synergistic activities, although they were not as potent as other combinations. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas S Dexheimer, Thomas Silvers, Nathan P Coussens, Rabih Said, Beverly A Teicher, James H Doroshow. Combinations of PI3K inhibitors with targeted oncology agents in multicellular spheroid models [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 C045.

Figure S5. Heat maps of Bliss synergy scores across the combination dose-response matrices for all twenty-six cell lines grown as multicellular complex spheroids exposed to each DNA damaging agent (A, TMZ; B, topotecan; C, trabectedin) in combination with DNA-PK inhibitors, nedisertib or VX-984.

Three aryl-hydrocarbon receptor (AhR) selective inhibitors, BAY-2416964, GNF351 and CH-223191, were assayed alone and in combination with 25 approved or investigational anticancer agents in complex spheroids including tumor cells, endothelial cells, and mesenchymal stem cells. The tumor cell lines were from the National Cancer Institute’s Patient-Derived Models Repository (PDMR) collection (https://pdmr.cancer.gov/models/database.htm). The AhR is a ligand-activated helix-loop-helix transcription factor of the bHLH-PAS family. It is involved in the regulation of responses to planar aromatic hydrocarbons by activating the transcription of xenobiotic-metabolizing enzymes such as cytochrome P450 or by acting as an E3 ligase. Thus, the AhR activates detoxification pathways that dispose of small molecule toxins and damaged proteins. In the absence of xenobiotics, the AhR is located within the cytoplasm in complex with two Hsp90 molecules and co-chaperones. Upon xenobiotic binding, the AhR translocates to the nucleus and binds to the AhR nuclear translocator protein to form an active transcription factor complex and promote gene transcription. As single agents, the three AhR inhibitors showed little cytotoxicity at concentrations up to 10 µM. Combinations of AhR inhibitors with anticancer agents including doxorubicin, cisplatin, SN38, venetoclax, selinexor, and etoposide (https://dtp.cancer.gov/organization/dscb/obtaining/default.htm) produced primarily additive cytotoxicity in complex spheroids after a 7-day exposure. However, unexpected greater-than-additive effects were observed with AhR inhibitors in combination with proteasome pathway inhibitors. Bortezomib, a direct inhibitor of the chymotrypsin-like protease of the 26S proteasome, and pevonedistat, a NEDD8-activating enzyme inhibitor, produced additive or greater-than-additive cytotoxicity in some complex spheroids. However, the combination of BAY-2416964 with TAK-243, a ubiquitin activating enzyme inhibitor, produced profound greater-than-additive cytotoxicity in more than half of the 28 PDMR cell lines tested as complex spheroids. The combination resulted in an increase of 1- to 3-logs of excess cytotoxicity in malignancies including bladder cancer, pancreatic cancer, colon cancer, NSCLC, SCLC and glioblastoma. In vivo studies are under discussion. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: BEVERLY A. TEICHER, Jeffrey A. Moscow, Joel Morris, James H. Doroshow, Thomas S. Dexheimer, Nathan P. Coussens, Thomas Silvers, Rene Delosh, Zahra Davoudi, Russel Reinhart, Chad Ogle, Eric Jones. Aryl-hydrocarbon receptor inhibitors in combination with anticancer agents, especially proteasome pathway inhibitors, in a complex spheroid screen using patient-derived cell lines can result in greater-than additive cytotoxicity. [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 4555.

There is a major need in oncology drug development to establish predictive preclinical assays with high translational relevance to patient responses. The National Cancer Institute’s Patient-Derived Models Repository (https://pdmr.cancer.gov) offers a collection of highly characterized models from a variety of cancer types including rare and recalcitrant malignancies and tumors from patients of diverse ancestry. This collection includes matched sets of patient-derived cell lines, organoids, and xenografts (PDXs), which allows comparisons of drug responses from in vitro and in vivo assays performed with the same patient-derived tumor model. A high-throughput screen was conducted using matched sets of patient-derived organoids and cell lines. Patient-derived cell lines were grown as 3D multicellular spheroids mixed with endothelial cells and mesenchymal stem cells. The patient-derived organoids were 100% tumor cells and were plated in 5% basement membrane extract supplemented with growth factors and cytokines. All drugs were tested at concentrations up to their reported clinical Cmax values and cell viability for individual drug treatments and drug combinations were assayed using CellTiter-Glo 3D after seven days drug exposure. Prior to the endpoint viability measurements, growth curves for spheroid median volume and organoid median surface area were calculated from a series of non-invasive brightfield images collected every 12 hours. For some drug combinations, differential responses were observed between the matched organoids and multicellular spheroids, potentially reflecting the contribution of the stromal component in the spheroids. Overall, the drug-dependent growth responses observed from the two 3D in vitro models (i.e., multicellular spheroids and organoids) were frequently comparable to those observed in vivo from PDXs. For example, the in vitro activities of several drug combinations including: BAY1895344 + temozolomide, erlotinib + cediranib, entinostat + talazoparib, and selumetinib + abemaciclib, demonstrated good agreement with the responses observed in vivo. However, among the drug combinations tested ixazomib + panobinostat showed the greatest cytotoxicity in vitro but had no activity in the matched PDX models. The availability of matched patent-derived cell lines, organoids and PDXs provides an opportunity to learn about the features of assay methodologies and data analyses that influence the successful translation of preclinical results between in vitro and in vivo systems. The results of this study are encouraging, but also highlight discrepancies that will be important to investigate, understand and address in order to improve translational capacity of future assays. This project was funded in part with federal funds from the NCI, NIH, under contract no. HHSN261201500003I. Citation Format: Thomas S. Dexheimer, Thomas Silvers, Rene Delosh, Russell Reinhart, Chad Ogle, Zahra Davoudi, Eric Jones, Debbie Trail, John Carter, Justine Mills, Kyle Georgius, Howard Stotler, Michelle Norris, Shannon Uzelac, Suzanne Borgel, Tiffanie Minor, Luke Stockwin, Michael Mullendore, Kevin Plater, Keegan Kalmbach, Jessica Steed, Matthew Murphy, Gareth Bliss, Carrie Bonomi, Kelly Dougherty, Marion Gibson, Kevin Cooper, Dianne Newton, Cindy R. Timme, Yvonne A. Evrard, Melinda G. Hollingshead, Nathan P. Coussens, Ralph E. Parchment, James H. Doroshow, Beverly A. Teicher. Combination therapies in matched 3D in vitro and in vivo preclinical models of rare and recalcitrant cancers from the National Cancer Institute’s Patient-Derived Models Repository. [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 5720.

Combination of ATR inhibitors with topotecan or trabectedin. Concentration–response curves (top, mean ± SD, n = 4 technical replicates) with corresponding mean Bliss score plots (bottom, n = 4 technical replicates) showing the scores from each combination's concentration matrix and colored as a heat map (blue indicates synergy; yellow indicates additivity; red indicates antagonism). Combinations of the DNA-damaging drug topotecan with the ATR inhibitors berzosertib (A) and elimusertib (B). Combinations of the DNA-damaging drug trabectedin with either berzosertib (C) or elimusertib (D). Data are shown for complex tumor spheroids grown with the malignant cell lines (from left): 287954-098-R-J1 (Ewing sarcoma), NCI-H841 (SCLC), DMS 114 (SCLC), COR L88 (SCLC), and 425362-245-T-J1 (melanoma).

Combination of PARP inhibitors and temozolomide. A, Mean Bliss matrix scores were calculated from each combination's concentration matrix [(5 concentrations of drug A) × (6 concentrations of drug B) = (30 combination concentrations)]. The scores (n = 546) are graphed for all combinations of DNA-damaging drugs with DNA repair inhibitors tested (n = 21) in all complex tumor spheroid models (n = 26). B, The same data shown in A) but combinations of the DNA-damaging drug temozolomide with either PARP inhibitor olaparib or talazoparib are highlighted in blue (n = 52), whereas all other combinations are shown in light gray. C, A scatter plot of the mean Bliss matrix scores from the temozolomide combinations with olaparib and talazoparib (Pearson r = 0.73, two-tailed P < 0.0001). Concentration–response curves (top, mean ± SD, n = 4 technical replicates) from combinations of temozolomide with either olaparib (D) or talazoparib (E) and corresponding mean Bliss score plots (bottom, n = 4 technical replicates) showing the scores from each combination's concentration matrix and colored as a heat map (blue indicates synergy; yellow indicates additivity; red indicates antagonism). Data are shown for complex tumor spheroids grown with the malignant cell lines (from left): VA-ES-VJ (epithelioid sarcoma), SYO-1 (synovial sarcoma), 287954-098-R-J1 (Ewing sarcoma), 349418-098-R (NSCLC), and DMS 114 (SCLC).