Patient-derived Xenograft Models Research Articles

DIPG-27. MATCHED PRIMARY-RELAPSE PEDHGG PATIENT-DERIVED XENOGRAFT MODELS TO IDENTIFY THERAPY RESISTANCE PROFILES

Abstract BACKGROUND We established patient-derived xenografts (PDXs) from matched primary-relapse pairs of pediatric high-grade gliomas (pedHGGs) in collaboration with the Swedish Childhood Tumor Biobank. Further, we used human iPSC derived cortical organoids to evaluate interactions between glial and tumor cells under treatment pressure potentially lost in xenograft models. Using this setup, we hope to follow tumor relapse evolution and to identify novel mechanisms behind treatment evasion and relapse formation. METHODS Tumoral singel cell suspensions from matched primary-relapse pairs are injected orthotopically in immunodeficient NOG mice. We establish in vitro 2D models in stem cell conditions from the first generation of animals transplanted. Lentivirus-based labelling with GFP/Luciferase was done on PDX-derived cell lines to allow cell tracking and to quantify efficacy of drug screening efforts. Co-culture of these cells with normal cells in 3D assembloids were performed using 150-250 days old cortical organoids. Single-cell RNA sequencing and bulk RNA sequencing (RNA-seq) on patient biopsies, their corresponding PDXs from first generation of mice, and PDX-derived cell lines, was performed to track potential changes in vivo and in vitro and to further validate primary-relapse specific evolution. RESULTS 2D and 3D cell cultures offered efficient labeling and we quantified differences in sensitivity to standard therapy between primary and relapse samples. PDX models and assembloids highlighted pedHGG growth patterns, modes of invasion and cell interactions, especially astrocyte-tumor cell interactions. These 3D models also allow for further validation of targeted drug testing based on multiomics data (WGS, methylation and RNA-seq) from the patient biopsies. CONCLUSION PedHGG primary-relapse in vitro 2D cell line/ in vivo 3D assembloid models together with PDXs is a robust approach to study different aspects of relapse-specific tumor biology. Our results offer insights aiding the development of novel therapeutics inhibiting relapse mechanisms already in the first line of treatment.

MDB-57. PROTEOMIC ANALYSIS REVEALS AMBRA1 AS A NEW CRL3REN/KCTD11 SUBSTRATE PROMOTING SONIC HEDGEHOG-DEPENDENT MEDULLOBLASTOMA

Abstract BACKGROUND Sonic Hedgehog (SHH) signaling pathway is fundamental for a proper embryonic development and its uncontrolled activation is responsible for various cancers. One the most relevant SHH-dependent tumors is medulloblastoma (MB), a highly heterogeneous brain malignancy that predominantly occurs in childhood. Understanding the complex networks that govern SHH aberrant activation represents a dramatic challenge for the development of efficient personalized therapies. Here, we identify a new role of the pro-autophagic protein AMBRA1 as interactor of REN/KCTD11 (here REN), the substrate-receptor subunit of the Cullin3-RING ubiquitin ligase complex (CRL3REN) and tumor suppressor lost in ~30% of human SHH-MBs. We show a novel mechanism of action of AMBRA1 in SHH-MB pathogenesis whereby its highly expression, due to loss of REN, drives the activation of GLI1, the final effector of SHH signaling. METHODS Proteomic analysis, co-immunoprecipitation, ubiquitylation and cycloheximide assays have been performed to demonstrate that AMBRA1 is a substrate of ubiquitylation of CRL3REN and to define AMBRA1 mechanism of action in SHH-MB tumorigenesis. Transcriptomic analysis, in vitro and in vivo proliferation assays have been carried out on murine and patient-derived xenograft (PDX) models to examine the effect of AMBRA1 modulation on SHH-MB growth. RESULTS We demonstrate that CRL3REN binds and induces polyubiquitylation and proteasome-mediated degradation of AMBRA1. Interestingly, we found that genetic depletion of AMBRA1 strongly reduces SHH-dependent MB growth both in vitro and in vivo, an effect associated with the decrease in the expression of GLI1 and other SHH target genes. We hypothesize that AMBRA1 could impact on the stability of GLI1 by counteracting the activity of E3 ubiquitin ligases regulating GLI proteins. Remarkably, transcriptomic analysis of SHH PDX upon AMBRA1 knockdown revealed an impairment of SHH activity and an upregulation of cerebellum differentiation. CONCLUSION Our findings identify AMBRA1 as a novel CRL3REN substrate and promising therapeutic target for SHH-dependent cancer.

DIPG-82. RADIOSENSITIZATION WITH RAD52 INHIBITION IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Diffuse midline glioma (DMG) is one of the most devastating childhood cancers that limited response to radiation therapy (RT). There is a critical need for new therapeutics that enhance the radiation effect. We, therefore, tested the hypothesis that targeting RAD52 activity sensitizes the radiation response of DMG. METHODS Genome-wide CRISPR/Cas9 screening was performed to identify the potential therapeutic target. RAD52 inhibition was used by shRNA-mediated RAD52 depletion and treatment with RAD52 inhibitor, D-I03. The protein expression, cell proliferation, DNA damage marker expression, DNA repair pathway, and transcriptional alteration were analyzed by western blotting, MTS assay, colony formation assay, immunocytochemistry, DNA repair pathway assay, and RNA sequencing. Mice with DMG patient-derived xenograft (PDX) models were treated with RAD52 inhibitor alone or combination with RT. RESULTS Genome-wide CRISPR/Cas9 screening identified RAD52 as a potential therapeutic target in DMG cells. RAD52 inhibition suppressed DMG cell proliferation. Importantly, RAD52 inhibition in combination with RT further increased the radiosensitivity of DMG cells. Immunocytochemistry of DNA double-strand breaks (DSB) marker γH2AX and repair marker 53BP1 showed that RAD52 inhibition sustained DNA damage with high levels of γH2AX at 24 hours following radiation while the level of 53BPI was decreased, thereby inhibiting DNA DSB repair. Western blotting also revealed that RAD52 inhibition causes a sustained level of phosphorylated Rad50 and γH2AX in irradiated DIPG cells over 24 hours. DNA repair assay showed that RAD52 inhibition suppressed homologous recombination DNA repair pathway. RNA sequencing showed that RAD52 inhibition downregulated genes associated with the DNA repair pathway. Finally, the combination therapy of RAD52 inhibitor and RT further suppressed tumor growth and increased survival of mice bearing DMG PDXs, outperforming either monotherapy. CONCLUSIONS these results highlight RAD52 inhibition as a potential radiosensitization and provide a rationale for developing combination therapy with radiation in the treatment of DMG.

IMMU-18. ENHANCING GD2.CAR T CELL THERAPY FOR DIFFUSE MIDLINE GLIOMA WITH LSD1 AND HDAC INHIBITORS

Abstract BACKGROUND Chimeric antigen receptor T cells against the glycolipid GD2 (GD2.CARTs) have shown promise against Diffuse Midline Glioma (DMG) in clinical trials but are not yet curative. Previous studies in neuroblastoma demonstrate that histone deacetylase inhibitor (HDACi) treatment can increase GD2 on tumor cells and improve targeting by GD2.CARTs. Our lab previously found that simultaneous HDACi and lysine-demethylase 1 inhibitor (LSD1i) treatment in DMG cells causes synergistic alterations in gene expression and reduces DMG growth. Here, we find that treating GD2-low DMG cell lines with combinations of LSD1i and HDACi improves DMG response to GD2.CARTs. METHODS Surface expression of GD2 before and after LSD1i/HDACi treatment were analyzed using immunofluorescence microscopy and flow cytometry. The cytotoxicity of GD2.CARTs against DMG cells was determined from co-culture experiments and in patient-derived orthotopic xenograft models using flow cytometry and bioluminescent imaging. RESULTS Since low target antigen expression is one mechanism by which tumor cells can escape CAR T cell therapy, we profiled GD2 expression on 9 different H3K27M-mutant DMG cell lines. This analysis shows that only 5 of the 9 cell lines exhibit high GD2 expression while the remaining 4 lines have low GD2 expression. When treating these cell lines with GD2.CARTs, we find that low GD2 expression on DMG cells correlates with tumor cell escape and resistance to GD2.CARTs. Treating GD2-low DMG cells with LSD1i+HDACi increases GD2 expression and enhances tumor cell targeting by GD2.CARTs in subsequent co-culture assays. Further studies in mice bearing GD2-low DMG tumors were initially GD2.CART-insensitive but showed reduced tumor growth when pre-treated with LSD1i+HDACi before intravenous GD2.CART infusion. CONCLUSIONS These studies suggest that variable GD2 expression is a major driver of DMG resistance to GD2.CARTs and that LSD1i+HDACi pre-treatment may improve DMG patient responses to GD2.CART therapy by increasing target antigen expression.

EPEN-20. LEVERAGING ABERRANT EXTRACELLULAR VESICLE SIGNALING FOR TARGETED DRUG REPURPOSING IN PEDIATRIC EPENDYMOMA

Abstract Increasing evidence highlights the diverse functions of extracellular vesicles (EVs) as intercellular messengers in all facets of cancer progression, pointing towards a potential new avenue in cancer treatment. Limited understanding of tumor-specific deregulations and lack of suitable drug compounds have hampered clinical translation of targeted interference with EV signaling. The most aggressive types of intracranial ependymoma (EPN) in children and adults are resistant to chemotherapy. Despite enormous diagnostic advancements in classification and stratification, EPN lack recurrent effective molecular targets precluding access of affected patients to precision-based oncology therapeutics. Here, we characterized the proteome of ninety-five EPN and five matching healthy control brain tissue samples. Employing a mulit-omic data integration approach, we identified EVs as an excessively augmented pathway on the functional level. Isolation and molecular characterization of EPN EVs combined with single cell sequencing analysis, and ultra-high content imaging unraveled perivascular mesenchymal cell populations as predominant EV originators. Targeting EV pathways through chemical inhibition of sphingomyelinases (SMPD1, SMPD2, SMPD3) including re-purposed FDA approved compounds revealed growth inhibition, reduced invasion capacity in vitro and significant survival probability in vivo. Transcriptomic analysis and scanning electron microscopy delineated transition of mesenchymal-to neuroepithelial subpopulations in cells and patient-derived xenograft (PDX) models of EPN upon reduction of EV release. This study showcases the inhibition of EV signaling as a promising therapeutic strategy against mesenchymal tumor persister cells, which are the suspected origins of repeated EPN relapses. Our study provides a framework to leverage cell state-specific vulnerabilities and drug repurposing to attack the most aggressive cell populations in cancers with unfavorable prognosis.

ETMR-05. DEVELOPMENT OF NOVEL PRECLINICAL MODELS AND THERAPEUTIC STRATEGIES FOR ETMR

Abstract BACKGROUND Embryonal Tumor with Multilayered Rosettes (ETMR) is a very rare, aggressive pediatric brain tumor with a dismal 5-year overall survival rate. Few genetic aberrations in miRNA clusters and processing machinery have been detected in ETMRs with 90% of ETMR patients having the C19MC amplification. ETMRs show unique histopathological features which, together with the detection of C19MC or DICER alterations, are necessary for diagnosis. The cellular and molecular mechanisms underlying ETMR development are poorly understood, and targeted therapeutics have not been developed due to scarcity of preclinical models. To expand knowledge of ETMR biology and accelerate the design of novel and therapies, it is thus essential to develop preclinical models. METHODS We have generated two novel patient-derived ETMR cell lines from resected patient derived tumor samples (CTBP-03A and CBTP-139) and created three patient-derived xenograft (PDX) models (CTBP-03A, CBTP-139 and CBTP-38). We have used these unique patient-derived cell lines to identify mechanisms driving ETMR development and progression. To identify novel therapeutic targets, we conducted high-throughput drug screening (HTDS) utilizing 2480 approved and investigational drugs, against the CBTP-03A and CBTP-139 lines. RESULTS Among the candidate drugs showing an inhibitory effect on both lines, compounds were selected based on their activity, CNS penetration ability, mechanism of action, and bioavailability, if known. The top selected hits were screened in an all-versus-all format, totaling 1953 combinations. The rapidity of action and efficacy of the top choice compounds used as single agent or in double and triple combination was assessed by caspase glow and cell titer glow assays. This initial combination screen identified DNA replication stress, retinoid receptor agonist and FGF signaling as potential combinatorial targets. Data are currently being validated in vivo. CONCLUSIONS This is the first combinatorial HTDS on two novel patient-derived cell lines which highlighted potential drug combination strategies for the treatment of ETMR.

Targeting Patient-Derived Orthotopic Gastric Cancers with a Fluorescent Humanized Anti-CEA Antibody

BackgroundGastric cancer poses a major diagnostic and therapeutic challenge as surgical resection provides the only opportunity for a cure. Specific labeling of gastric cancer could distinguish resectable and nonresectable disease and facilitate an R0 resection, which could improve survival.MethodsTwo patient-derived gastric cancer lines, KG8 and KG10, were established from surgical specimens of two patients who underwent gastrectomy for gastric adenocarcinoma. Harvested tumor fragments were implanted into the greater curvature of the stomach to establish patient-derived orthotopic xenograft (PDOX) models. M5A (humanized anti-CEA antibody) or IgG control antibodies were conjugated with the near-infrared dye IRDye800CW. Mice received 50 µg of M5A-IR800 or 50 µg of IgG-IR800 intravenously and were imaged after 72 hr. Fluorescence imaging was performed by using the LI-COR Pearl Imaging System. A tumor-to-background ratio (TBR) was calculated by dividing the mean fluorescence intensity of the tumor versus adjacent stomach tissue.ResultsM5A-IR800 administration resulted in bright labeling of both KG8 and K10 tumors. In the KG8 PDOX models, the TBR for M5A-IR800 was 5.85 (SE ± 1.64) compared with IgG-IR800 at 0.70 (SE ± 0.17). The K10 PDOX models had a TBR of 3.71 (SE ± 0.73) for M5A-IR800 compared with 0.66 (SE ± 0.12) for IgG-IR800.ConclusionsHumanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright and specific labeling of gastric cancer PDOX models. This tumor-specific fluorescent antibody is a promising potential clinical tool to detect the extent of disease for the determination of resectability as well as to visualize tumor margins during gastric cancer resection.

Combination of menin and kinase inhibitors as an effective treatment for leukemia with NUP98 translocations.

Chromosomal translocations of the nucleoporin 98 (NUP98) gene are found in acute myeloid leukemia (AML) patients leading to very poor outcomes. The oncogenic activity of NUP98 fusion proteins is dependent on the interaction between Mixed Lineage Leukemia 1 and menin. NUP98-rearranged (NUP98-r) leukemia cells also rely on specific kinases, including CDK6 and/or FLT3, suggesting that simultaneous targeting of these kinases and menin could overcome limited sensitivity to single agents. Here, we found that combinations of menin inhibitor, MI-3454, with kinase inhibitors targeting either CDK6 (Palbociclib) or FLT3 (Gilteritinib) strongly enhance the anti-leukemic effect of menin inhibition in NUP98-r leukemia models. We found strong synergistic effects of both combinations on cell growth, colony formation and differentiation in patient samples with NUP98 translocations. These combinations also markedly augmented anti-leukemic efficacy of menin inhibitor in Patient Derived Xenograft models of NUP98-r leukemia. Despite inhibiting two unrelated kinases, when Palbociclib or Gilteritinib were combined with the menin inhibitor, they affected similar pathways relevant to leukemogenesis, including cell cycle regulation, cell proliferation and differentiation. This study provides strong rationale for clinical translation of the combination of menin and kinase inhibitors as novel treatments for NUP98-r leukemia, supporting the unexplored combinations of epigenetic drugs with kinase inhibitors.

DIPG-24. COMBINED EPIGENETIC THERAPY WITH FACT AND BET INHIBITORS REMODELS CHROMATIN AND DISRUPTS ONCOGENIC TRANSCRIPTION IN DIFFUSE MIDLINE GLIOMA

Abstract BACKGROUND Aberrant epigenetic regulation is a hallmark of Diffuse Midline Glioma (DMG), an incurable pediatric brain tumor. The H3K27M driver histone mutation leads to transcriptional dysregulation, indicating that targeting the epigenome and transcription may be a key therapeutic strategy against this aggressive cancer. One such target is the Facilitates Chromatin Transcription (FACT) histone chaperone. FACT is targeted by the curaxin compound CBL0137 which is currently in Phase I/II Children’s Oncology Group clinical trial as a single agent for pediatric cancer patients (NCT04870944). METHODS We applied a multi-omics approach by integrating epigenetic profiling with transcriptomics (CUT&RUN, ATAC-seq and RNA-seq) to interrogate the role of FACT in DMG. We then employed in vitro cytotoxicity assays and in vivo orthotopic patient-derived xenograft (PDX) pre-clinical models to test a novel, mechanistic-anchored epigenetic combination therapy. RESULTS We found FACT to be enriched at developmental gene promoters, coinciding with regions of open chromatin and binding motifs of core DMG regulatory transcription factors (OLIG2, TCF12, and SNAI1). FACT interacted and co-localized with the Bromodomain and Extra-Terminal Domain (BET) protein BRD4 at active promoters and enhancers, suggesting functional cooperation between FACT and BRD4 in DMG. Investigating the combination of CBL0137 with two BET inhibitors (JQ1 and trotabresib) revealed potent cytotoxicity in vitro, particularly against H3K27M-mutant cells. Furthermore, this approach was recapitulated in vivo, significantly extending the survival of three independent orthotopic PDX models of DMG. Mechanistically, CBL0137 decreased chromatin accessibility, synergising with BET inhibition to disrupt transcription, leading to transcriptional silencing of several key oncogenes (MYC, PDGFRA and MDM4) as well as causing alterations to the splicing landscape. CONCLUSIONS This study highlights the therapeutic promise of simultaneously targeting FACT and BRD4 in DMG, and provides the rationale for a combination clinical trial, currently in development.

EPEN-24. 5FU SENSITIVITY IS MEDIATED THROUGH TRISOMY UCK2 EXPRESSION IN 1Q+ PFA EPN

Abstract BACKGROUND Nearly all children with gain of chromosome 1q (1q+) PFA ependymoma (EPN) will die, highlighting the urgent need to develop new treatment strategies for these patients. It has recently been shown that 1q+ creates a p53 mutant-like phenotype through trisomy expression of MDM4, a repressor of p53 signaling. This same study found toxic uracil analogs could reverse trisomy MDM4 effect on p53 and this was mediated through another 1q-trisomy gene UCK2. We and other have previously identified 5-fluorouracil (5FU) as an ependymoma specific inhibitor. In this study, we present the mechanism for 5FU sensitivity. METHODS In this study we utilize 1q+ PFA in vitro models to test the efficacy of combination radiation and 5FU in a preclinical setting. We measured p53 signaling through proteomic array and qRT-PCR for p21, a p53 response gene. RESULTS 5FU enhances radiotherapy in 1q+ PFA cell lines. Specifically, 5FU increases p53 activity mediated by the extra copy of UCK2 located on chromosome 1q in 1q+ PFA. Experimental down regulation of UCK2 resulted in decreased 5FU sensitivity in 1q+ PFA cells. Though not statistically significant, we did see prolong survival and decreased tumor burden in 1q+ PFA orthotopic patient-derived xenograft models treated with focal 2Gyx5days radiation and 5FU. CONCLUSION These results are the first to identify a 1q+ specific therapy approach in PFA EPN. Existing phase 1 studies in pediatrics have already established safety and dose of 5FU, providing a rapid translation into phase 2 trials for children both up front and at recurrence for 1q+ PFA.

Chip collection of hepatocellular carcinoma based on O2 heterogeneity from patient tissue

Hepatocellular carcinoma frequently recurs after surgery, necessitating personalized clinical approaches based on tumor avatar models. However, location-dependent oxygen concentrations resulting from the dual hepatic vascular supply drive the inherent heterogeneity of the tumor microenvironment, which presents challenges in developing an avatar model. In this study, tissue samples from 12 patients with hepatocellular carcinoma are cultured directly on a chip and separated based on preference of oxygen concentration. Establishing a dual gradient system with drug perfusion perpendicular to the oxygen gradient enables the simultaneous separation of cells and evaluation of drug responsiveness. The results are further cross-validated by implanting the chips into mice at various oxygen levels using a patient-derived xenograft model. Hepatocellular carcinoma cells exposed to hypoxia exhibit invasive and recurrent characteristics that mirror clinical outcomes. This chip provides valuable insights into treatment prognosis by identifying the dominant hepatocellular carcinoma type in each patient, potentially guiding personalized therapeutic interventions.

A positive feedback loop between PLD1 and NF-κB signaling promotes tumorigenesis of nasopharyngeal carcinoma

Phospholipase D (PLD) lipid-signaling enzyme superfamily has been widely implicated in various human malignancies, but its role and underlying mechanism remain unclear in nasopharyngeal carcinoma (NPC). Here, we analyze the expressions of 6 PLD family members between 87 NPC and 10 control samples through transcriptome analysis. Our findings reveal a notable upregulation of PLD1 in both NPC tumors and cell lines, correlating with worse disease-free and overall survival in NPC patients. Functional assays further elucidate the oncogenic role of PLD1, demonstrating its pivotal promotion of critical tumorigenic processes such as cell proliferation and migration in vitro, as well as tumor growth in vivo. Notably, our study uncovers a positive feedback loop between PLD1 and the NF-κB signaling pathway to render NPC progression. Specifically, PLD1 enhances NF-κB activity by facilitating the phosphorylation and nuclear translocation of RELA, which in turn binds to the promoter of PLD1, augmenting its expression. Moreover, RELA overexpression markedly rescues the inhibitory effects in PLD1-depleted NPC cells. Importantly, the application of the PLD1 inhibitor, VU0155069, substantially inhibits NPC tumorigenesis in a patient-derived xenograft model. Together, our findings identify PLD1/NF-κB signaling as a positive feedback loop with promising therapeutic and prognostic potential in NPC.

Knocking Out CD70 Rescues CD70-Specific NanoCAR T Cells from Antigen-Induced Exhaustion.

CD70 is an attractive target for chimeric antigen receptor (CAR) T-cell therapy for the treatment of both solid and liquid malignancies. However, the functionality of CD70-specific CAR T cells is modest. We optimized a CD70-specific VHH-based CAR (nanoCAR). We evaluated the nanoCARs in clinically relevant models in vitro, using co-cultures of CD70-specific nanoCAR T cells with malignant rhabdoid tumor organoids, and in vivo, using a diffuse large B-cell lymphoma patient-derived xenograft (PDX) model. Although the nanoCAR T cells were highly efficient in organoid co-cultures, they showed only modest efficacy in the PDX model. We determined that fratricide was not causing this loss in efficacy but rather CD70 interaction in cis with the nanoCAR-induced exhaustion. Knocking out CD70 in nanoCAR T cells using CRISPR/Cas9 resulted in dramatically enhanced functionality in the diffuse large B-cell lymphoma PDX model. Through single-cell transcriptomics, we obtained evidence that CD70 knockout CD70-specific nanoCAR T cells were protected from antigen-induced exhaustion. In addition, we demonstrated that wild-type CD70-specific nanoCAR T cells already exhibited signs of exhaustion shortly after production. Their gene signature strongly overlapped with gene signatures of exhausted CAR T cells. Conversely, the gene signature of knockout CD70-specific nanoCAR T cells overlapped with the gene signature of CAR T-cell infusion products leading to complete responses in chronic lymphatic leukemia patients. Our data show that CARs targeting endogenous T-cell antigens negatively affect CAR T-cell functionality by inducing an exhausted state, which can be overcome by knocking out the specific target.

Anti-tumor Effects of the eIF4A Inhibitor Didesmethylrocaglamide and Its Derivatives in Human and Canine Osteosarcomas.

Inhibition of translation initiation using eIF4A inhibitors like (-)-didesmethylrocaglamide [(-)-DDR] and (-)-rocaglamide [(-)-Roc] is a potential cancer treatment strategy as they simultaneously diminish multiple oncogenic drivers. We showed that human and dog osteosarcoma cells expressed high levels of eIF4A1/2, particularly eIF4A2. Genetic depletion of eIF4A1 and/or 2 slowed osteosarcoma cell growth. To advance preclinical development of eIF4A inhibitors, we demonstrated the importance of (-)-chirality in DDR for growth-inhibitory activity. Bromination of DDR at carbon-5 abolished growth-inhibitory activity, while acetylating DDR at carbon-1 was tolerated. Like DDR and Roc, DDR-acetate increased the γH2A.X levels and induced G2/M arrest and apoptosis. Consistent with translation inhibition, these rocaglates decreased the levels of several mitogenic kinases, the STAT3 transcription factor, and the stress-activated protein kinase p38. However, phosphorylated p38 was greatly enhanced in treated cells, suggesting activation of stress response pathways. RNA sequencing identified RHOB as a top upregulated gene in both DDR- and Roc-treated osteosarcoma cells, but the Rho inhibitor Rhosin did not enhance the growth-inhibitory activity of (-)-DDR or (-)-Roc. Nonetheless, these rocaglates potently suppressed tumor growth in a canine osteosarcoma patient-derived xenograft model. These results suggest that these eIF4A inhibitors can be leveraged to treat both human and dog osteosarcomas.

Activation of NOTCH signaling impedes cell proliferation and survival in acute megakaryoblastic leukemia

The genetic lesions that drive acute megakaryoblastic leukemia (AMKL) have not been fully elucidated. To search for genetic alterations in AMKL, we performed targeted deep sequencing in 34 AMKL patient samples and 8 AMKL cell lines and detected frequent genetic mutations in the NOTCH pathway in addition to previously reported alterations in GATA-1 and the JAK-STAT pathway. Pharmacological and genetic NOTCH activation, but not inhibition, significantly suppressed AMKL cell proliferation in both in vitro and in vivo assays employing a patient-derived xenograft model. These results suggest that NOTCH inactivation underlies AMKL leukemogenesis. and NOTCH activation holds the potential for therapeutic application in AMKL.

A Compound That Inhibits Glycolysis in Prostate Cancer Controls Growth of Advanced Prostate Cancer.

Metastatic castration-resistant prostate cancer remains incurable regardless of recent therapeutic advances. Prostate cancer tumors display highly glycolytic phenotypes as the cancer progresses. Nonspecific inhibitors of glycolysis have not been utilized successfully for chemotherapy, because of their penchant to cause systemic toxicity. This study reports the preclinical activity, safety, and pharmacokinetics of a novel small-molecule preclinical candidate, BKIDC-1553, with antiglycolytic activity. We tested a large battery of prostate cancer cell lines for inhibition of cell proliferation, in vitro. Cell-cycle, metabolic, and enzymatic assays were used to demonstrate their mechanism of action. A human patient-derived xenograft model implanted in mice and a human organoid were studied for sensitivity to our BKIDC preclinical candidate. A battery of pharmacokinetic experiments, absorption, distribution, metabolism, and excretion experiments, and in vitro and in vivo toxicology experiments were carried out to assess readiness for clinical trials. We demonstrate a new class of small-molecule inhibitors where antiglycolytic activity in prostate cancer cell lines is mediated through inhibition of hexokinase 2. These compounds display selective growth inhibition across multiple prostate cancer models. We describe a lead BKIDC-1553 that demonstrates promising activity in a preclinical xenograft model of advanced prostate cancer, equivalent to that of enzalutamide. BKIDC-1553 demonstrates safety and pharmacologic properties consistent with a compound that can be taken into human studies with expectations of a good safety margin and predicted dosing for efficacy. This work supports testing BKIDC-1553 and its derivatives in clinical trials for patients with advanced prostate cancer.

Establishment and characterization of cMYB-expressing human salivary adenoid cystic carcinoma cell lines (UM-HACC-14, UM-HACC-6) and matching patient-derived xenograft model (UM-PDX-HACC-14)

Limited availability of authentic human adenoid cystic carcinoma (ACC) cell lines has hindered progress in understanding mechanisms underpinning the biology of this disease and the development of safe and effective therapies. Surgical human ACC specimens (UM-HACC-6, UM-HACC-14) were dissociated into single cell suspensions and cultured in fibronectin-coated flasks. Alternatively, tumor fragments were transplanted subcutaneously into female immunodeficient (SCID) mice to establish patient-derived xenograft tumors (PDX; UM-PDX-HACC-14). Both ACC cell lines showed continuous growth in monolayers for over 100 passages. Total RNA-Seq, RT-PCR, and FISH analysis revealed that both are MYB-NFIB fusion negative. Western blots revealed passage-dependent expression of E-Cadherin, PCNA, p63, phospho-c-MYB, and NFIB. Both, UM-HACC-14 and UM-HACC-6 cells exhibited tumorigenic potential when injected orthotopically into mouse submandibular glands. UM-HACC-14, patient-matching UM-PDX-HACC-14, and the UM-HACC-6 cell line are new, authenticated preclinical models of ACC that are well suited for mechanistic and developmental therapeutics studies.

Abstract IA003: Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase

Abstract The WRN helicase serves as a key target in the treatment of cancers characterized by microsatellite instability (MSI) because it plays a crucial role in resolving harmful non-canonical DNA structures that arise in cells with defective mismatch repair systems. Despite the critical functions of human DNA and RNA helicases, no drugs targeting these enzymes have been approved, largely due to the difficulties in identifying potent and selective inhibitors. In this study, we present the chemoproteomics-based identification of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This inhibitor specifically interacts with a cysteine residue (C727) within the helicase domain, which undergoes inter-domain movement during the DNA unwinding process. VVD-133214 reacts with the WRN protein in a nucleotide cooperative manner, promoting stable, compact structures that impede the enzyme's dynamic flexibility essential for its helicase activity. Inhibition of WRN by VVD-133214 leads to extensive double-stranded DNA breaks, nuclear enlargement, and ultimately cell death, specifically in MSI-high cells but not in microsatellite stable cells. The inhibitor demonstrated good tolerance in mice and significant tumor reduction in various MSI-high colorectal cancer cell lines and patient-derived xenograft models. Our findings highlight an allosteric strategy to inhibit WRN function that avoids interference from the endogenous ATP cofactor in cancer cells and positions VVD-133214 as a promising therapeutic candidate for patients with MSI-high cancers. Citation Format: Matthew P. Patricelli, Kristen A. Baltgalvis, Kelsey N. Lamb, Kent T. Symons, Chu-Chiao Wu, Melissa A. Hoffman, Aaron N. Snead, Xiaodan Song, Thomas Glaza, Shota Kikuchi, Jason C. Green, Donald C. Rogness, Betty Lam, Maria E. Rodriguez-Aguirre, David R. Woody, Christie L. Eissler, Socorro Rodilles, Seth M .Negron, Steffen M. Bernard, Eileen Tran, Jonathan Pollock, Ali Tabatabaei, Victor Contreras, Heather N Williams, Martha K. Pastuszka, John J. Sigler, Piergiorgio Pettazzoni, Markus G. Rudolph, Moritz Classen, Doris Brugger, Christopher Claiborne, Jean-Marc Plancher, Isabel Cuartas, Joan Seoane, Laurence E. Burgess, Robert T. Abraham, David S. Weinstein, Gabriel M. Simon, Todd M. Kinsella. Chemoproteomic discovery of a covalent allosteric inhibitor of WRN helicase [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA003.

Abstract IA005: Therapeutic vulnerabilities of cohesin-mutant myeloid malignancies

Abstract Splicing modulation is a promising treatment strategy pursued to date only in splicing-factor mutant cancers; however, its therapeutic potential is poorly understood outside of this context. Like splicing factors, genes encoding components of the cohesin complex are frequently mutated in myeloid malignancies, including 15-20% of myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (AML), where they are associated with poor outcomes. I will discuss our recent findings identifying cohesin mutations as biomarkers of sensitivity to drugs targeting splicing-factor SF3B1 (H3B-8800 and E-7107) and describe the mechanism by which drug-induced alterations in splicing of DNA repair genes, such as BRCA1 and BRCA2, underlie this sensitivity. We have demonstrated that treatment of cohesin-mutant cells with SF3B1 modulators results in impaired DNA damage response, accumulation of DNA damage, and increased sensitivity to PARP inhibitors and a panel of chemotherapeutic agents in vitro and in vivo, using AML cell lines and patient-derived xenograft models. Furthermore, we identified RAD51 foci formation as a predictive biomarker of sensitivity to SF3B1 splicing modulation alone or followed by sequential treatment with PARP inhibition and chemotherapy, and have identified additional non-cohesin mutant subtypes of MDS/AML and ovarian and breast cancer which are sensitive to this therapeutic approach. Our findings expand the potential therapeutic benefits of SF3B1 splicing modulators to include cohesin-mutant MDS/AML and we propose this as broader strategy for therapeutic targeting of other DNA damage-repair deficient cancers. Citation Format: Zuzana Tothova. Therapeutic vulnerabilities of cohesin-mutant myeloid malignancies [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr IA005.

Abstract B020: Novel WRN helicase inhibitors selectively target microsatellite unstable cancer cells

Abstract Microsatellite-unstable (MSI) cancers require WRN helicase to resolve replication stress due to expanded DNA (TA)n-dinucleotide repeats. WRN is a promising synthetic lethal target for MSI tumours, and WRN inhibitors are in development. Here, we used CRISPR-Cas9 base editing to map WRN residues critical for MSI cells, validating the helicase domain as the primary drug target. Fragment-based screening led to the development of potent and highly selective WRN helicase covalent inhibitors. These compounds selectively suppressed MSI model growth In vitro and In vivo by mimicking WRN loss, inducing DNA double-strand breaks at expanded TA-repeats and DNA damage. Assessment of biomarkers in preclinical models linked TA-repeat expansions and mismatch repair (MMR) alterations to compound activity. Efficacy was confirmed in immunotherapy-resistant organoids and patient-derived xenograft (PDX) models. The discovery of potent, selective covalent WRN inhibitors provides proof of concept for synthetic-lethal targeting of WRN in MSI cancer and tools to dissect WRN biology. Citation Format: Gabriele Picco, Yanhua Rao, Angham Al Saedi, Yang Lee, Shriram Bhosle, Carmen Herranz-Ors, Samantha Walker, Kieron May, Matthew Coelho, Jon Houseley, Benjamin Schwartz, Mathew J. Garnett. Novel WRN helicase inhibitors selectively target microsatellite unstable cancer cells [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Expanding and Translating Cancer Synthetic Vulnerabilities; 2024 Jun 10-13; Montreal, Quebec, Canada. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(6 Suppl):Abstract nr B020.