PDX Tumors Research Articles

Abstract 3577: The role of PLXND1 in lineage plasticity and progression to neuroendocrine prostate cancer

Abstract Background: Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer and is commonly associated with therapy resistance and poor prognosis. Treatment-induced NEPC (t-NEPC) predominantly develops by lineage plasticity from adenocarcinoma in response to androgen receptor signaling inhibitors, such as enzalutamide. However, the mechanisms underlying the lineage plasticity and progression to NEPC are unclear. PLXND1 (PlexinD1) is a cellular receptor of the semaphorin family and has important functions in modulating the cytoskeleton and cell adhesion. However, the role of PLXND1 in lineage plasticity to NEPC development and progression is not well established. Methods: We performed transcriptomic analysis on NEPC patients’ cohorts and the enzalutamide-resistant prostate cancer cell lines. Correlation of PLXND1 with neuroendocrine feature genes and AR-targeting genes was determined. Kaplan-Meier method and the Cox proportional hazards model was used to estimate the correlation between PLXND1 expression level and patients’ survival. Immunohistochemistry was used to analyze PLXND1 expression in prostate adenocarcinoma and NEPC. Cell viability was determined in C4-2B MDVR, CWR22Rv1, and H660 cells after knocking down PLXND1 using siRNA or knocking out PLXND1 using CRISPR/CAS9. Organoids viability of patient-derived xenografts was measured after knocking down or knocking out PLXND1. Results: Our analysis on different patient cohorts suggests that PLXND1 is highly expressed in NEPC patients, and the expression of PLXND1 is positively correlated with neuroendocrine feature genes (CHGA, ENO2, SYP, and NCAM1). Kaplan-Meier survival analysis shows that high expression of PLXND1 is significantly associated with the poor prognosis of prostate cancer patients. PLXND1 is overexpressed and negatively regulated by androgen receptor signaling in enzalutamide-resistant cells and prostate cancer patients’ cohorts. Furthermore, downregulation of PLXND1 expression suppressed proliferation of the NEPC cells and viability of the PDX tumor organoids. Conclusions: PLXND1 expression is elevated in NEPC and correlated with the poor survival of prostate cancer patients, suggesting it is a potential molecular indicator and therapy target for NEPC. Citation Format: Bo Chen, Pengfei Xu, Joy C. Yang, Shu Ning, Leyi Wang, Christopher Nip, Allen C. Gao, Chengfei Liu. The role of PLXND1 in lineage plasticity and progression to neuroendocrine prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3577.

Abstract 597: Vebreltinib: A novel brain-penetrating MET kinase inhibitor demonstrates the mechanism of action and pharmacological anti-tumor activity in diverse patient-derived MET-dysregulated tumor models at clinically relevant drug levels

Abstract Background: Dysregulation of the MET signaling pathway drives oncogenesis in many human cancers, the majority of which have not been addressed by a MET targeted therapy albeit the emerging therapies for MET exon 14 skipping (MET ex14) NSCLC. We have developed a novel highly selective MET kinase inhibitor vebreltinib (APL-101, PLB1001, bozitinib) to address unmet medical need. Vebreltinib is being jointly investigated in ongoing pivotal Phase 2 and 2/3 studies in MET-dysregulated NSCLC, primary CNS cancers and basket solid tumors in China (as PLB-1001) and globally ex-China (as APL-101). The Phase 1 portion of both studies have been concluded with the same RP2D (200 mg BID by oral administration). Vebreltinib demonstrated blood-brain barrier permeability in PLB-1001 Phase 1 glioblastoma study as previously reported. Methods: MET enzyme kinetics and vebreltinib intrinsic potency were studied by a radiometric MET kinase assay. Selectivity was profiled by in vitro KINOME. Vebreltinib cellular activity on MET signaling and in vivo anti-tumor activity were assessed in tumor cell lines and patient-derived tumors (PDX) carrying MET alterations. Results: Vebreltinib is an ATP-competitive inhibitor with Ki of approximately 2.2 nM for recombinant MET kinase and inhibited the native form of activated MET with 0.52 nM IC50. Vebreltinib demonstrated exquisite selectivity in KINOMEscan selectivity panel. Vebreltinib inhibited HGF-dependent and -independent MET signaling in KP4 tumor cell lines with nanomolar concentrations. Vebreltinib inhibited IL-3 independent Ba/F3 cell lines carrying exogenous MET ex14 or with secondary mutations induced by other MET TKIs. Vebreltinib inhibited MKN45 gastric tumor xenograft in vivo with ED90 of 6 mg/kg PO/QD. Furthermore, Vebreltinib demonstrated strong anti-tumor activity in a variety of PDX tumor types carrying MET ex14, MET fusions, and MET amplification. In these preclinical studies, the total and unbound vebreltinib plasma concentration observed at steady state trough of RP2D was used as a clinically relevant drug level to interpret Vebreltinib efficacy in blocking oncogenic MET signaling, proliferation of Ba/F3 cell lines with a broad spectrum of MET mutations and in vivo tumor growth in PDXs carrying diverse MET alterations. We demonstrated the rationale for Vebreltinib to be an efficacious single-agent MET inhibitor in treating tumors carrying MET driver alterations beyond MET ex14 NSCLC. Conclusion: Vebreltinib is a novel potent MET kinase inhibitor showing promising preclinical activity against PDXs from diverse organs sites and genomic alterations such as MET ex14, MET fusion, MET amplification, or HGF over-expression at clinically relevant drug levels, providing proof-of-concepts for continued clinical development. Citation Format: Xiaoling Zhang, Elaine Liu, Yan Song, Peony Yu, Sanjeev Redkar, Guo-Liang Yu. Vebreltinib: A novel brain-penetrating MET kinase inhibitor demonstrates the mechanism of action and pharmacological anti-tumor activity in diverse patient-derived MET-dysregulated tumor models at clinically relevant drug levels [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 597.

Abstract 5735: Efficient and safe delivery of doxorubicin by DNA fragments: A full preclinical study

Abstract The clinical uses of anthracyclines (e.g., doxorubicin) are hampered especially by dose-dependent cardiac toxicity, resulting in a narrow therapeutic window. Doxorubicin formulations have marginally improved its therapeutic efficacy while suffering from difficult and costly manufacturing, poor characterization and quality control, limited scalability in production, and disappointingly low drug loading. This study employs DNA fragments extracted from the salmon sperm cells, which is known for high biocompatibility in various biomedical application, to self-assemble via intercalation by doxorubicin. DNA/DOX nanocomplexes are easy in manufacturing, high scalability, straightforward characterization and quantification, and efficiency in drug loading. Doxorubicin and DNA fragments were dissolved in water and mixed at the optimized 1:6 weight ratio, followed by stirring and incubation for 30 min. DNA/DOX nanocomplexes were characterized for doxorubicin loading and release, size, surface charge, and morphology by UV/fluorescence spectroscopy, stable isotope tracer ultrafiltration assay (SITUA), dynamic light scattering particle analysis, and transmission electron microscopy. The anticancer efficacy of the DNA/DOX nanocomplexes were tested in vitro and in vivo using syngeneic and PDX tumor models. Along with PD/PK studies, DNA/DOX nanocomplexes were also evaluated for their systemic and cardiac toxicity. Characterization and quantification studies revealed DNA/DOX nanocomplexes to be relatively monodisperse with a size of 40 nm in diameter, efficient for drug encapsulation (~100%) and drug loading (14.3%, w/w), and stable for storage while readily releasing doxorubicin in a cell. The self-assembled nanocomplexes were formed when doxorubicin inter- and intra-molecularly chelated DNA fragments. DNA/DOX nanocomplexes were more efficient in killing cancer cells in vitro than both doxorubicin and the liposomal doxorubicin Doxil. PD/PK studies demonstrated that DNA/DOX nanocomplexes circulated longer with less accumulation in the heart than doxorubicin, which altogether lower toxicity. A repeated administration of the DNA/DOX nanocomplexes to Sprague-Dawley rats exhibited a substantially lower cardiotoxicity score than that of doxorubicin. The improved anti-tumor efficacy of the DNA/DOX nanocomplexes were finally confirmed using a syngeneic lymphoma mouse model and PDX mouse models of triple-negative breast and ovarian cancers. Complexing doxorubicin with DNA fragments addresses the current technological challenges in achieving an efficient and safe therapy for a broad range of cancers. DNA/DOX nanocomplexes are amenable to rigorous chemistry, manufacturing, and controls (CMC) evaluation with proven anti-cancer efficacy and improved safety profiles. Clinical trials on DNA/DOX nanocomplexes for cancer chemotherapy are warranted to fully validate their clinical utilities. Citation Format: Minhyeong Park, Youngwoo Kim, Hyunchu Cho, Saad Mohammad, Yeon Su Choi, Hyunjoo Lee, Seonah Lee, Edward Cedrone, Barry Neun, Maria Dobrovolskaia, David Fruman, Juwan Kim, Young Jik Kwon. Efficient and safe delivery of doxorubicin by DNA fragments: A full preclinical study [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 5735.

Abstract 6904: Model-derived progression-free survival (mPFS): A better metric for patient-derived xenograft studies

Abstract Patient-derived xenografts (PDX) are a powerful assay system for translational prediction of therapeutic potential for experimental therapeutics. The primary readout from PDX models is tumor shrinkage, measured by the ratio of treated/control tumor volume. This readout corresponds loosely to the Objective Response Rate (ORR) metric used in clinical trials, in that it is a readout of drug effect on the sensitive subclones within a tumor. In prior work, we have shown that there is a paradoxical relationship between short-term tumor response and progression-free survival (PFS). While ORR is the primary measure utilized in the Phase II to III transition in clinical development, approval hinges on survival metrics, and the disconnect between ORR and PFS creates a potential risk of late-stage failures for experimental anticancer therapeutics. Thus, it is of interest to understand whether there are alternative approaches to analyzing PDX data that can enable the assessment of therapeutic potential in terms of a projected PFS. In this simulation study, we will examine the accuracy of progression-free survival (PFS) estimates obtained directly from observation during a PDX study and compare their accuracy with the accuracy of PFS estimates derived using a mathematical model-based approach that relies on a kinetic model of tumor subclone growth (model-based PFS, or mPFS) fitted directly to PDX tumor growth and rebound kinetics under treatment. We generated our test dataset using a mathematical model of tumor kinetic growth to simulate xenograft data using fitted parameters derived from literature, adding in experimental error consistent with prior experimental measurement-derived variability. We used the simulated raw data to calculate an observed PFS as well as mPFS derived from fitting the tumor clonal kinetic model to the raw data in a blinded fashion. Our results show that the empirical PFS from PDX models is subject to overestimation, as a function of the observation time interval. On the other hand, mPFS is significantly less biased and more accurate, but is subject to some logistical constraints on sampling. Our work demonstrates the practical translational utility of a novel analysis method (mPFS) in deriving estimates of progression-free survival from PDX model data. Citation Format: Andrew Chen, Madison Stoddard, Lin Yuan, Debra Van Egeren, Arijit Chakravarty, Dean Bottino. Model-derived progression-free survival (mPFS): A better metric for patient-derived xenograft studies [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 6904.

Abstract 5334: Humanization of NOG mice and next-generation NOG strains to induce lineage-specific differentiation of immune cells for assessment of novel immune cell therapies, check point inhibitors, and immune cell engagers for translational immuno-oncology research

Abstract Background: The preclinical evaluation of novel immune therapies requires humanized immune system (HIS) mouse models. In previous studies we have demonstrated that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T and NK cells or CD34+ hematopoietic stem cells (HSC) can be used to establish a HIS model. With the development of next-generation NOG mice, lineage-specific differentiation of immune cell sub-populations of interest can be supported. Transplantation of cell line-derived (CDX) or patient-derived (PDX) tumor xenografts on HIS mice provides a full model for human tumors for the investigation of checkpoint inhibitors (CPI), as well as novel cell therapies and immune cell engagers. Methods: HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells to immunodeficient mice. For humanization, NOG mice and next-generation NOG strains were used: NOG, NOG-EXL, hIL-2 NOG, hIL-6 NOG and FcResolv™ NOG mice were compared to each other for lineage-specific differentiation using single donors or a mixed HSC donor pool. Engraftment of immune cells was monitored by FACS analysis of blood every four weeks. In humanized NOG mice, CDX and PDX from different entities were s.c. transplanted and used to evaluate CPI. Results: Humanized hIL-2 NOG mice showed significantly decreased survival after HSC transplantation in comparison to the other mouse strains and had to be sacrificed within the first 6-8 weeks after HSC transplantation. In the other mouse strains, transplanted HSCs engrafted and differentiated mainly into B and T cells. NOG-EXL mice displayed the highest engraftment, with up to 80% of human cells in the blood, including a higher portion of myeloid cells. In humanized hIL-6 NOG mice a higher portion of monocytes could be determined. FcResolv™ NOG mice lack murine Fc gamma receptors in order to reduce confounding interactions between residual murine immune cells and antibody-based therapies, and they engraft and differentiate human HSCs similarly to the parent NOG strain. In humanized NOG mice, selected CDX and PDX tumors successfully engrafted without significant differences in tumor growth compared to non-humanized mice. CPI treatments induced tumor growth delay in selected models. Conclusions: Next-generation NOG mouse strains are characterized by a lineage-specific differentiation of immune cells depending on integrated human cytokines. Furthermore, we established a human tumor-immune-cell model for NOG mice using different entities of CDX or PDX in combination with CPI. Our human tumor-immune cell models allow preclinical translational studies on tumor immune biology as well as evaluation of new therapies, drug combinations and biomarker identification and validation. Citation Format: Maria Stecklum, Louise Baskin, Jens Hoffmann. Humanization of NOG mice and next-generation NOG strains to induce lineage-specific differentiation of immune cells for assessment of novel immune cell therapies, check point inhibitors, and immune cell engagers for translational immuno-oncology research [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 5334.

Fumarate induces LncRNA-MIR4435-2HG to regulate glutamine metabolism remodeling and promote the development of FH-deficient renal cell carcinoma

Fumarate hydratase (FH) deficient renal cell carcinoma (RCC) is a type of tumor with definite metabolic disorder, but the mechanism of metabolic remodeling is still unclear. LncRNA was reported to closely correlate with cancer metabolism, however the biological role of LncRNA in the development of progression of FH-deficent RCC was not well studied either. FH-deficient RCC samples were collected in my hospital and used for RNA-sequencing and Mass spectrometry analysis. FH-deficient RCC cell line UOK262 and control pFH cells were used for in vitro experiments, including proliferation assay, transwell assay, western-blot, mass spectrometry and so on. PDX mouse model was used for further drug inhibition experiments in vivo. In this study, we analyzed the profiles of LncRNA and mRNA in FH-deficienct RCC samples, and we found that the LncRNA-MIR4435-2GH was specifically highly expressed in FH-deficient RCC compared with ccRCC. In vitro experiments demonstrated that MIR4435-2HG was regulated by Fumarate through histone demethylation, and the deletion of this gene could inhibit glutamine metabolism. RNA-pulldown experiments showed that MIR4435-2HG specifically binds to STAT1, which can transcriptionally activate GLS1. GLS1 inhibitor CB-839 could significantly suppress tumor growth in PDX tumor models. This study analyzed the molecular mechanism of MIR4435-2HG in regulating metabolic remodeling of FH-deficient RCC in clinical samples, cells and animal models by combining transcriptional and metabolic methods. We found that that GLS1 was a therapeutic target for this tumor, and MIR4435-2HG can be used as a drug sensitivity marker.

Functional Heterogeneity in MET Pathway Activation in PDX Models of Osimertinib-resistant EGFR-driven Lung Cancer.

Using a novel cohort of in vivo PDX models of MET pathway activation with acquired resistance to osimertinib in EGFR-mutant lung cancer, we demonstrate that phospho-MET may be a clinically relevant assay to guide treatment selection with osimertinib and savolitinib combination. In addition, our work shows that patients with MET polysomy tumors may have subclonal MET amplification and therefore require close follow up for the use of osimertinib and savolitinib combination.

Cytoskeleton remodeling induced by SMYD2 methyltransferase drives breast cancer metastasis

Malignant forms of breast cancer refractory to existing therapies remain a major unmet health issue, primarily due to metastatic spread. A better understanding of the mechanisms at play will provide better insights for alternative treatments to prevent breast cancer cell dispersion. Here, we identify the lysine methyltransferase SMYD2 as a clinically actionable master regulator of breast cancer metastasis. While SMYD2 is overexpressed in aggressive breast cancers, we notice that it is not required for primary tumor growth. However, mammary-epithelium specific SMYD2 ablation increases mouse overall survival by blocking the primary tumor cell ability to metastasize. Mechanistically, we identify BCAR3 as a genuine physiological substrate of SMYD2 in breast cancer cells. BCAR3 monomethylated at lysine K334 (K334me1) is recognized by a novel methyl-binding domain present in FMNLs proteins. These actin cytoskeleton regulators are recruited at the cell edges by the SMYD2 methylation signaling and modulate lamellipodia properties. Breast cancer cells with impaired BCAR3 methylation lose migration and invasiveness capacity in vitro and are ineffective in promoting metastases in vivo. Remarkably, SMYD2 pharmacologic inhibition efficiently impairs the metastatic spread of breast cancer cells, PDX and aggressive mammary tumors from genetically engineered mice. This study provides a rationale for innovative therapeutic prevention of malignant breast cancer metastatic progression by targeting the SMYD2-BCAR3-FMNL axis.

Abstract B163: Discovery and characterization of OKI-219, an orally bioavailable H1047R-mutant-selective inhibitor of PI3Kα

Abstract Mutations in PI3Kα lead to constitutive activation of the PI3K/AKT/mTOR pathway and are found in approximately 13% of human cancers, with the PI3KαH1047R mutation being the most common. Targeting PI3Kα in cancer is a therapeutically proven strategy, with the currently approved drug alpelisib showing clinical efficacy alone or in combination with other therapies. However, treatment with non-mutant selective PI3Kα inhibitors, such as alpelisib, is associated with significant toxicities such as hyperglycemia due to on-target inhibition of the wild-type enzyme, which often leads to dose modification or discontinuation. Therefore, there is a significant need to develop new PI3Kα-targeted therapies that can avoid or minimize on-target toxicity and improve the safety and clinical benefit for patients. OKI-219 is a PI3KαH1047R mutant-selective inhibitor, with ~100-fold cellular selectivity for the H1047R mutation over wild-type and biochemical selectivity across a 412 kinase panel, with no other kinases showing >30% inhibition at 1 µM. In vitro, OKI-219 drives decreased phosphorylated AKT (pAKT) and decreased proliferation selectively in PI3KαH1047R mutant cell lines across several tumor types. In vivo, as a monotherapy, OKI-219 demonstrates dose-dependent antitumor activity in multiple human CDX and PDX tumor xenograft models, with regression rates up to 100% in PI3KαH1047R heterozygous (T47D) and homozygous (SUM185PE) CDX models, at doses that show no sign of the metabolic dysfunction (increased glucose or insulin) that is associated with wild-type protein inhibition. The in vivo antitumor activity correlates with pathway inhibition, measured by decreased pAKT in tumors. In addition to the monotherapy efficacy observed, OKI-219 in combination with the selective estrogen receptor degrader (SERD) fulvestrant showed significant combination benefit leading to tumor regressions in the H1047R-mutated ER+HER2- breast cancer model xxT47D, at doses where no regressions were observed with single agent treatment. These data support the hypothesis that OKI-219 will offer improved efficacy and a wider therapeutic window compared to non-mutant selective PI3Kα inhibitors. This hypothesis will be tested, as OKI-219 is moving into clinical investigation. Citation Format: Molly A Taylor, Qian Zhao, David Mareska, Maria Hoh, Yevgeniy Izrayelit, Kevin Litwiler, Mark L Boys, Rich Woessner, Duncan Walker, Jennifer Diamond, James D Winkler. Discovery and characterization of OKI-219, an orally bioavailable H1047R-mutant-selective inhibitor of PI3Kα [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 B163.

Abstract B177: The discovery and preclinical characterization of AMG 193, a first-in-class MTA-cooperative PRMT5 inhibitor with broad activity against MTAP-null cancers

Abstract Homozygous deletion of the methylthioadenosine phosphorylase (MTAP) gene occurs in approximately 15% of all cancers due to its proximity to the commonly deleted tumor suppressor gene CDKN2A. Elevated methylthioadenosine (MTA) levels, driven by loss of MTAP, compete with the methyl donor S-adenosylmethionine (SAM) for binding to protein arginine N-methyltransferase 5 (PRMT5), placing the methyltransferase in a hypomorphic state, and vulnerable to further PRMT5 inhibition. The screening of a DNA-encoded library, designed to identify small molecules that preferentially bind to PRMT5 in the presence of MTA, resulted in the identification of AM-9934 as a promising hit. Using structure-based drug design, potency and cooperativity were enhanced to deliver AM-9747, an MTA-cooperative PRMT5 inhibitor suitable for in vivo proof-of-concept studies. Further optimization led to AMG 193, an orally bioavailable and MTA-cooperative PRMT5 inhibitor with potent biochemical and cellular activity, and an improved pharmacokinetic profile. Insights into the binding mode of AMG 193 were elucidated by the crystal structure of AMG 193 bound to the MTA-PRMT5 complex. AMG 193 activity is enriched in MTAP-null cells in vitro, as illustrated in the HCT116 isogenic pair; AMG 193 is ~40X selective for MTAP-null cells (IC50 = 0.1 mM) over MTAP WT cells (IC50 > 4 mM) in viability assays and >100X selective in an assay evaluating symmetric dimethylarginine (SDMA) levels. AMG 193 was profiled in a panel of >850 cancer cell lines, and response demonstrated preferential sensitivity in MTAP-null cells compared to WT across multiple cancer lineages, including pancreatic, lung, biliary tract, and lymphoma. Response strongly correlated with both MTAP and CDKN2A copy number loss, and cell lines that were dependent on PRMT5, as shown by RNAi and CRISPR knockdown, were most sensitive to AMG 193 treatment. In vitro mechanism-of-action studies demonstrated that PRMT5 inhibition induced DNA damage, cell cycle arrest in G2/M, and an increase in alternative mRNA splicing in MTAP-null cells. In mice oral, once-daily administration of AMG 193 selectively inhibited SDMA in tumor versus normal tissue and was well tolerated with no hematologic perturbations. Importantly, AMG 193 demonstrated robust anti-tumor activity across a broad range of MTAP-null CDX and PDX tumor models derived from NSCLC, pancreatic, and esophageal cancers. In vitro synergy was observed with the combination of AMG 193 and several chemotherapeutic agents, including carboplatin and paclitaxel, or the KRASG12C inhibitor, sotorasib. In vivo, these combinations led to enhanced anti-tumor activity relative to single agent in MTAP-null NSCLC tumor models. In February 2022, AMG 193 became the first MTA-cooperative PRMT5 inhibitor to enter clinical development and is currently being evaluated in subjects with advanced MTAP-null solid tumors in the ongoing FIH study (NCT05094336). Citation Format: Brian Belmontes, Katherine Slemmons, Siyuan Liu, Antonia Policheni, Jodi Moriguchi, Hong Tan, Fang Xie, Famke Aeffner, Matthew G Rees, Melissa M Ronan, Jennifer A Roth, Mikkel Vestergaard, Sanne Cowland, Jan Andersson, Ian Sarvary, Patricia Lopez, Nuria Tamayo, Liping H Pettus, Susmith Mukund, Jennifer R Allen, Sanne Glad, Matthew P Bourbeau. The discovery and preclinical characterization of AMG 193, a first-in-class MTA-cooperative PRMT5 inhibitor with broad activity against MTAP-null cancers [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 B177.

Abstract A005: Preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers in humanized mouse models

Abstract Background The preclinical evaluation of novel immune therapies requires humanized mouse models with functional human immune cells. In previous studies we have demonstrated, that either peripheral blood mononuclear cells (PBMC), subsets of PBMCs like T- and NK-cells or hematopoietic stem cells (HSC) can be used to establish a humanized immune system with functional T-, B-, and NK cells, as well as monocytes and dendritic cells in immunodeficient mice. By xenotransplantation of cell-line-derived (CDX) or patient-derived (PDX) tumors on humanized mice, we successfully generated a full human tumor-immune-cell model for different disease entities. Finally, we validated the functionality of these models using checkpoint inhibitors like Ipilimumab (Ipi), Nivolumab (Nivo), Pembrolizumab (Pembro), cell therapies, and immune cell engagers. Methods HSC-humanized mice were generated by i.v. transplantation of CD34+ stem cells to immunodeficient 1st and 2nd generation NOG mice. Engraftment of immune cells was monitored by FACS analysis of blood samples. PBMC or isolated T- or NK-cell preparations were used to humanize mice by single or multiple i.v. injections. CDX and PDX from different entities were s.c. (i.e. lymphoma) and/or i.v. (leukemia) transplanted on those humanized mice. Tumor growth of orthotopic models was followed up by BLI measurement. Blood and tumor samples were analysed by for immune cell infiltration and activation. Results The transplanted HSCs engrafted in mice and established a functional human immune system with proliferation and differentiation. The 2nd generation NOG mice are showing an increased total engraftment and lineage specific differentiation of cells. The selected CDX and PDX tumors successfully engrafted on humanized mice without significant differences in tumor growth compared to non-humanized mice. Check point inhibitor treatments (Ipi, Nivo, or Pembro) induced tumor growth delay in selected models. BLI measurement is a suitable method to follow up systemic disease models and treatment effects over time. We identified a set of CDX and PDX models without interference with parallel injection of PBMC, T- or NK-cell preparations for the evaluation of immune cell engagers and other cell therapies. Conclusions We established human tumor-immune-cell models of different entities using CDX or PDX in combination with different donor derived immune cell subsets as effector cells. We demonstrated successful engraftment of HSC on different immunodeficient mouse strains, generating mice with a functional human hematopoiesis. These models have been successfully used for preclinical evaluation of novel check point inhibitors, cell therapies and immune cell engagers. Our human tumor-immune-cell models allow preclinical translational research on tumor immune biology as well as evaluation of new therapies, drug combinations, as well as biomarker identification and validation in subcutaneous and orthotopic models. Citation Format: Maria Stecklum, Annika Wulf-Goldenberg, Bernadette Brzezicha, Joshua Alcaniz, Glenn Smits, Wolfgang Walther, Jens Hoffmann. Preclinical evaluation of novel immune cell therapies, check point inhibitors, and immune cell engagers in humanized mouse 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 A005.

Abstract A045: EZH2 inhibition re-sensitizes drug resistant triple-negative breast cancer PDX models to Eribulin

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer predominantly affecting younger women. The mainstay of treatment is chemotherapy, to which most patients eventually stop responding. After demonstrating improved overall survival in the metastatic setting, eribulin, a microtubule inhibitor, was approved for the treatment of late-stage, heavily pretreated breast cancer. While frequently used in the clinic, most patients either do not respond initially or they stop responding to eribulin. Considering that there is a lack of oncology drugs including targeted therapies to treat TNBCs, patients are therefore left with few therapeutic options after eribulin. There is a paucity of data on mechanisms of acquired resistance to eribulin, and few potential uncovered targetable alterations. The goal of our study is to determine mechanisms of acquired resistance to Eribulin using isogenic acquired-resistant PDX models and to attempt to reverse this resistance with novel combinations. Methods: PDX models are grown as mammary fat pad engraftments of patient tumours in immuno-compromised (NGS) mice. To determine drug response, we treat tumour-bearing mice with weekly eribulin, measure the tumor volume once per week, then classify response based on mRECIST 1.1 criteria. Acquired resistance was generated in vivo in TNBC PDXs via the continuous or interrupted treatment of Eribulin in sensitive models that regressed on Eribulin. RNA-Seq analysis was performed on isogenic pairs of Eribulin sensitive and resistant PDX tumors and analyzed for differential gene expression and pathway enrichments. Drug combinations targeting candidate regulators of enriched pathways across our models were assessed in vivo in resistant PDXs. Drug trials were performed with n=5-7 mice per treatment arm until endpoint (tumour volume reaches 2000mm^3 or BW loss of 20%). Statistical analysis included t-tests for tumour volume and Wilcoxon test for survival. Results: 6 isogenic pairs of Eribulin acquired resistant TNBC PDX models were generated in vivo and molecular analysis was performed by comparing transcriptomic profiles of sensitive and resistant tumours. Pathway analysis revealed that, across 5 of 6 acquired resistant models, differentially expressed genes were significantly enriched for targets of SUZ12 (BH p-value<0.01). As SUZ12 makes up a core component of the PRC2 complex, we attempted to target EZH2, the catalytic component of PRC2, in vivo. Combining Tazemetostat, an FDA-approved EZH2 inhibitor, with Eribulin in one PDX model, resulted in tumor regression and improved overall survival (p<0.005) in one model. We will be testing this combination in more models, and performing differential chromatin accessibility studies to identify candidate driver genes in these regions. Conclusion: Our results suggest that combining an approved EZH2 inhibitor, Tazemetostat, with Eribulin can re-sensitize acquired-resistant TNBC PDXs to Eribulin. This approach could have significant clinical benefit in late stage hard to treat TNBCs. Citation Format: Kathryn Bozek, Cedric Darini, Cathy Lan, Marguerite Buchanan, Catherine Chabot, Josiane Lafleur, Juliet Guay, Eva Filosa, Adriana Aguilar-Mahecha, Mark Basik. EZH2 inhibition re-sensitizes drug resistant triple-negative breast cancer PDX models to Eribulin [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 A045.

Abstract B012: Validation of the OncoSignature assay, an ACR-368-tailored response-predictive quantitative multiplexed immunofluorescent assay for prediction of sensitivity to the CHK1/2 inhibitor ACR-368 in individual patients with cancer

Abstract Background: The CHK1/2 kinase inhibitor ACR-368 (Prexasertib) demonstrated durable single-agent efficacy in a subset of patients with high grade serous ovarian, anal and head and neck cancers in past trials. Using a quantitative phospho-proteomics approach, we identified three orthogonal candidate biomarkers tailored to predict tumor response to ACR-368 treatment. The OncoSignature® assay is based on immunofluorescent staining of biomarkers in FFPE tumor sections with image acquisition, analysis, and scoring of biomarkers. The sample is OncoSignature positive if all three biomarker scores are above their thresholds. We validated the OncoSignature® assay to predict response to ACR-368 in cancer cell lines and ovarian cancer PDX models. Here, we further validated OncoSignature® assay in novel cancer indications and in patient biopsy samples from ACR-368 clinical trials. Methods: Multiple cancer types were tested with the OncoSignature® assay for prevalence of potential ACR-368 responders. For validation, endometrial cancer PDX models were tested in anACR-368 efficacy study. Blinded untreated PDX tumors were analyzed in the OncoSignature® assay and results locked prior to unblinding and statistical analysis. Adapted [CS1] Oncosignature assay was applied to frozen pretreatment ovarian cancer biopsies from a single center ACR-368 monotherapy Ph2 trial (NCT02203513). OncoSignature® assay was also applied to FFPE pretreatment ovarian cancer biopsies from a multi-center ACR-368 monotherapy Ph 2 trial (NCT03414047) and from the NCT02203513 trial. After biomarker scores were generated and locked, patient treatment outcomes were unblinded and ACR-368 response was compared with OncoSignature prediction. Results: Evaluation of multiple cancer indications revealed a concordance between OncoSignature-predicted prevalence of ACR-368 responders and results of ACR-368 treatment observed in clinical trials. Metastatic ovarian cancer had 30% of predicted responders and 30% and 14% observed RECIST responses the above-cited trials. Squamous lung cancer had no predicted responders and no RECIST responses in a clinical trial. Endometrial and bladder cancer had a high prevalence of predicted responders with 40% and 36%, respectively. The endometrial cancer PDX efficacy study had multiple complete responses predicted by OncoSignature® assay with an AUC of 0.88. For frozen and FFPE patient biopsy samples from Phase 2 ACR-368 trials, OncoSignature positive groups had highly significant response enrichment to 47% and 57%, respectively, and significant increases in progression free survival time. Conclusions: Bladder and endometrial cancers were identified as novel indications with high prevalence of predicted ACR-368 responders. The OncoSignature® assay significantly predicted response in endometrial PDX models and in samples from ovarian cancer patients treated with ACR-368. OncoSignature® assay driven clinical trial (NCT05548296) in ovarian, endometrial and bladder cancers was initiated to test benefit to patients. Citation Format: Ayesha Murshid, Christina Noe, Kailash Singh, Sibgat Choudhury, Jayakumar Nair, James Dunyak, Michail Shipitsin, Jung-Min Lee, Peter Blume-Jensen. Validation of the OncoSignature assay, an ACR-368-tailored response-predictive quantitative multiplexed immunofluorescent assay for prediction of sensitivity to the CHK1/2 inhibitor ACR-368 in individual patients with cancer [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 B012.

The Mechanism of Therapy Resistance By Lineage Plasticity in AML and How to Overcome It

Acute myeloid leukemia (AML) is an aggressive blood cancer driven predominantly by mutations in epigenetic regulators. This characteristic allows AML to thrive in a hostile and rapidly changing environment where it needs to adapt to stressors both extrinsic (chemotherapy) and intrinsic (replicative and oxidative stress) to survive. Our study is the first to demonstrate the mechanism of how AML uses epigenetic reprogramming to adjust to these stressors via lineage plasticity. Venetoclax and Azacitidine (Ven/Aza) co-treatment is increasingly the front-line therapy for AML, functioning through BH3 inhibition and DNA hypomethylation. However, most patients will relapse with a highly aggressive refractory AML. Some studies suggest that monocytic character of the AML is responsible for relapse, but more recent evidence demonstrates that Ven/Aza treatment itself can induce a lineage switch to monocytic character. A potent selection mechanism facilitated by epigenetic dysregulation generating plastic clones is likely driving resistance, though the mechanism remains unclear and represents a significant barrier for AML treatment. Using a combination of single cell transcriptomics with surface immunophenotype sequencing in multiple human xenograft and primary patient samples, we discovered that this plasticity is widespread and mediated by epigenetic reprogramming of the tumor in direct response to the therapy. Broadly, we found that AML responds to Ven/Aza by transitioning into a reversible state of cell cycle arrest with transcriptional character similar to hematopoietic stem cells. This transient state protects AML from Azacitidine through replication arrest and protects from Venetoclax by upregulation of anti-apoptotic transcriptional programs. These protections operate simultaneously with reprogramming events driven by a switch from MYC-driven transcriptional programs to a PU.1-driven program. Reprogramming is both facilitated by and dependent on repression of the Chromatin Assembly Factor 1 (CAF1) complex, a chromatin-bound master epigenetic regulator responsible for maintaining lineage fidelity through transcriptional regulation via its role as a nucleosome assembly factor. Repressing CAF1 facilitates reprogramming where the output is an altered cell population that is distinct from the initial tumor both transcriptionally and by ATACseq. AML can repeatedly return to this plastic state, regardless of the number of cycles of Ven/Aza, and introducing other cytotoxic compounds does not remove this plastic stem-like population but instead initiates the iterative process of reprogramming anew until a different resistant population emerges. To find therapeutic vulnerabilities, we generated several PDX models of Ven/Aza resistance so that we could track the stages of acquired resistance in real time. When single cell data from these experiments were projected onto a reference map of normal hematopoiesis, we found that Ven/Aza resistance stems from a plastic population of cells that actively samples the niche by generating short-lived multi-lineage hybrid differentiated products. While the predominating output of this process was monocytic, we found that the induced plastic stem-like population was durably retained even after Ven/Aza withdrawal. To undermine this process, we took advantage of the plastic state to drive differentiation through an external stimulus. We expected this strategy to be particularly effective because CAF1 was repressed (CAF1 repression facilitates differentiation in every metazoan model) and, based on our single cell data, these plastic populations of AML cells express pro-differentiation surface receptors. In particular, Ven/Aza induced the expression of CSF3R (the G-CSF receptor) which when stimulated, drives neutrophil maturation through the JAK/STAT pathway. While G-CSF as a single agent or in sequence with Ven/Aza was unable to achieve maturation, when used concurrently with Ven/Aza (or recapitulated by CAF1 depletion using shRNA) we were able to drive neutrophil differentiation in vitro and fully resolve primary mouse and PDX tumors in vivo, even when using Ven/Aza resistant tumors as starting material. Together, these data demonstrate the possibility that Ven/Aza resistance can be completely subverted by taking advantage of the plastic state induced by the therapy to stimulate differentiation and resolve the disease.

ATM-inhibitor AZD1390 is a radiosensitizer for breast cancer CNS metastasis.

Limited effective treatments are currently available for central nervous system (CNS) metastasis. This is largely driven by the inability of current therapeutics to penetrate the blood brain barrier (BBB) and the lack of preclinical models for testing new therapies. Here we study the efficacy of AZD1390, a BBB penetrating ataxia-telangiectasia mutated (ATM) inhibitor, as a radiosensitizer for breast cancer CNS metastasis treatment. Three PDX tumors including 2 HER2+ and 1 triple negative breast cancer (TNBC) harboring DNA damage response (DDR) gene mutations, were implanted subcutaneously in the flank of mice to assess tumor growth inhibition by AZD1390 combined with radiation. Animal survival was further assessed by implanting the best responding PDX model orthotopically in the brain. Pretreatment with AZD1390 followed by radiation therapy inhibited growth of PDX tumors implanted in the flank, and improved survival in orthotopic models with average survival of 222 days compared to 123 days in controls. Administration of AZD1390 post-treatment for 21 days had no further benefits. While the combination therapy resulted in sustained tumor inhibition, sporadic regrowth was observed in some mice 50-100 days post-treatment in all models. Gene expression comparing these tumors with complete responders demonstrated changes in upregulation of oncogenic proteins, which are potential drivers of tumor growth after treatment. Our results demonstrate that AZD1390 effectively sensitizes breast cancer CNS metastasis to radiation therapy in DDR mutant tumors. This study demonstrates the potential of using AZD1390 as a novel therapeutic agent for patients with breast cancer CNS metastasis.

Generation of Orthotopic Patient-Derived Xenografts in Humanized Mice for Evaluation of Emerging Targeted Therapies and Immunotherapy Combinations for Melanoma.

Current methodologies for developing PDX in humanized mice in preclinical trials with immune-based therapies are limited by GVHD. Here, we compared two approaches for establishing PDX tumors in humanized mice: (1) PDX are first established in immune-deficient mice; or (2) PDX are initially established in humanized mice; then established PDX are transplanted to a larger cohort of humanized mice for preclinical trials. With the first approach, there was rapid wasting of PDX-bearing humanized mice with high levels of activated T cells in the circulation and organs, indicating immune-mediated toxicity. In contrast, with the second approach, toxicity was less of an issue and long-term human melanoma tumor growth and maintenance of human chimerism was achieved. Preclinical trials from the second approach revealed that rigosertib, but not anti-PD-1, increased CD8/CD4 T cell ratios in spleen and blood and inhibited PDX tumor growth. Resistance to anti-PD-1 was associated with PDX tumors established from tumors with limited CD8+ T cell content. Our findings suggest that it is essential to carefully manage immune editing by first establishing PDX tumors in humanized mice before expanding PDX tumors into a larger cohort of humanized mice to evaluate therapy response.

Abstract A060: Genetic alterations induce distinct histone post-translational modifications during the transition to castration-resistant prostate cancer

Abstract Background: Epigenetic alterations can impact gene expression and have been implicated in tumorigenesis. One major process is the post-translational modification (PTM) of histone proteins governed by histone-modifying enzyme (HME) activity. There is growing evidence that the dysregulation of HMEs plays an essential role in cancer initiation and progression. In this study, we hypothesized that alterations in histone PTMs and their associated enzymes drive the development of castration-resistant prostate cancer (CRPC) from hormone-sensitive prostate cancer (HSPC). To address this, we performed an unbiased screen of histone PTMs in a series of prostate cancer (PC) PDXs before and after the transition to CRPC. Methods: Histone PTM levels were analyzed in 10 paired HSPC and CRPC PDXs using mass spectrometry. Histone PTM patterns were subcategorized based on common genetic alterations found in PC. RNA-sequencing data for the PDXs and publicly available human PC datasets were used to further characterize HMEs. Co-expression of CHD1, NSD2, and other HMEs was examined in human PC tissue microarrays (TMAs). CHD1 KO clones were generated using CRISPR and levels of histone PTMs and HMEs were assessed and quantified. Gene set enrichment analysis (GSEA) was performed to determine the pathways altered with CHD1 in CRPC PDXs. Results: A comparison of all PDX tumors revealed increased H3K14 and H4 acetylation in CRPC tumors relative to paired HSPC derivatives. We also observed distinct histone PTM patterns associated with common genetic alterations. When compared to tumors with CHD1 WT, tumors with CHD1 loss develop reduced histone PTM H3.3K36me2. CHD1 is a chromatin remodeler commonly deleted in ~ 15% of primary PCs. Screening the differential expression and activity of HMEs in these PDXs reveals that HME NSD2 correlates with CHD1. The human CRPC SU2C/PCF dataset shows a strong positive correlation between CHD1 and NSD2 mRNA (r=0.44). Human CRPC TMAs show decreased protein expression of NSD2 and its histone mark H3K36me2 is associated with reduced CHD1 levels. CHD1 KO in DU145 results in a loss of H3.3K36me2 and a 0.5-fold decrease in NSD2 expression. GSEA revealed that a reduction in the DNA repair pathway is associated with CHD1 loss. The CRPC PDXs (n=24) and human dataset SU2C/PCF 2019 showed a strong positive correlation between CHD1, NSD2, and DNA damage repair genes. Conclusions: We have identified for the first time that genetic alterations alter histone marks during the transition to castration-resistant cancer. Reduced histone H3.3K36 methylation is associated with the genetic loss of CHD1 during the transition to CRPC. The expression of NSD2 is altered in the CRPC state in a CHD1 status-dependent manner. The CHD1-NSD2 phenotype may be the target of novel therapeutic drug combinations – the subject of ongoing research. Citation Format: Tanaya A. Purohit, Joseph Gawdzik, Bing Yang, Eric A. Armstrong, Peter W. Lewis, John M. Denu, David F. Jarrard. Genetic alterations induce distinct histone post-translational modifications during the transition to castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A060.

Targeting PELP1 oncogenic signaling in TNBC with the small molecule inhibitor SMIP34

PurposeTriple-negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. Oncogenic PELP1 is frequently overexpressed in TNBC, and it has been demonstrated that PELP1 signaling is essential for TNBC progression. The therapeutic utility of targeting PELP1 in TNBC, however, remains unknown. In this study, we investigated the effectiveness of SMIP34, a recently developed PELP1 inhibitor for the treatment of TNBC.MethodsTo ascertain the impact of SMIP34 treatment, we used seven different TNBC models for testing cell viability, colony formation, invasion, apoptosis, and cell cycle analysis. Western blotting and RT-qPCR were used to determine the mechanistic insights of SMIP34 action. Using xenograft and PDX tumors, the ability of SMIP34 in suppressing proliferation was examined both ex vivo and in vivo.ResultsTNBC cells’ viability, colony formation, and invasiveness were all decreased by SMIP34 in in vitro cell-based assays, while apoptosis was increased. SMIP34 treatment promoted the degradation of PELP1 through the proteasome pathway. RT-qPCR analyses confirmed that SMIP34 treatment downregulated PELP1 target genes. Further, SMIP34 treatment substantially downregulated PELP1 mediated extranuclear signaling including ERK, mTOR, S6 and 4EBP1. Mechanistic studies confirmed downregulation of PELP1 mediated ribosomal biogenesis functions including downregulation of cMyc and Rix complex proteins LAS1L, TEX-10, and SENP3. The proliferation of TNBC tumor tissues was decreased in explant experiments by SMIP34. Additionally, SMIP34 treatment markedly decreased tumor progression in both TNBC xenograft and PDX models.ConclusionsTogether, these findings from in vitro, ex vivo, and in vivo models show that SMIP34 may be a useful therapeutic agent for inhibiting PELP1 signaling in TNBC.

Abstract IA26: Exploiting altered metabolism as a therapeutic strategy for RAS-driven cancers

Abstract We recently determined that concurrent inhibition of autophagy, using the lysosomal inhibitor chloroquine (CQ), and of ERK, using a small molecule ERK inhibitor (ERKi), synergistically suppressed the growth of pancreatic ductal adenocarcinoma (PDAC) cell lines and patient xenograft-derived (PDX) organoids in vitro and PDX tumors in vivo (Bryant et al., 2019, Nat Med 25:628). Our findings, together with similar observations by McMahon, Kinsey and colleagues (Kinsey et al., 2019, Nat Med 25:620), provided the rationale for our initiation of Phase I and Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with hydroxychloroquine (HCQ) in PDAC. We have extended these findings and determined that combined inhibition of the ERK MAPK pathway and autophagy is effective in additional cancers driven by KRAS/NRAS, BRAF, or EGFR. Additionally, we have also shown that the combined inhibition of KRASG12C or KRASG12D and autophagy was effective in KRAS-mutant cancers. Our ongoing studies are centered on developing additional combinations for targeting metabolic resistance mechanisms to KRAS or ERK MAPK targerted therapies. First, we performed a CRISPR/Cas-9 mediated genetic loss-of-function screen in the presence of CQ to determine additional sensitizers as well as mediators of resistance to autophagy inhibition. Top sensitizers included multiple facilitators of the DNA damage response, mTOR pathway components, and genes involved in the upstream regulation of the autophagy pathway. Second, we determined that autophagic signaling is temporally regulated following RAS pathway inhibition and hypothesize that further dissection of this regulation will improve current anti-nutrient scavenging treatment strategies. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Jonathan M. DeLiberty, Ryan Robb, Claire E. Gates, Noah L. Pieper, Runying Yang, Clint A. Stalnecker, Kirsten L. Bryant. Exploiting altered metabolism as a therapeutic strategy for RAS-driven cancers [abstract]. In: Proceedings of the AACR Special Conference: Targeting RAS; 2023 Mar 5-8; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Res 2023;21(5_Suppl):Abstract nr IA26.

NSCLC patient-derived xenografts perpetuating tumor-associated, tumor-reactive T cells that respond to immunotherapy but do not cause GVHD in host mice

Abstract Non-small cell lung cancer (NSCLC) is the leading cause of malignancy-related deaths worldwide. We wished to overcome a current major limitation of PDX modeling, by providing autologous human T cells suitable for immunotherapy responses, but notably, without graft versus host disease (GVHD). We have developed PDXs from NSCLC by implanting surgically resected patient tumors subcutaneously in NOD scid gamma (NSG) mice. We used a metastatic PDX model to test potential therapeutic efficacy of a novel T cell molecule, anti-human-CD3ɛ monovalent Fab fragment (Mono-OKT3-Fab) and compared tumor growth results with anti-human PD-L1 therapy. Overall engraftment rate of unique, primary patient tumors was 52%. Over multiple passages in vivo, PDX tumors retained expression of primary tumor-matched NSCLC-diagnostic pathological markers. Moreover, primary-tumor-associated T cells were present and passaged in mice in vivo, without producing any signs of GVHD. Immunotherapy showed significant response from a PDL-1+ tumor, consistent with functional tumor-specific T cell passaging in our system. In contrast, control experiments using patient-matched human PBMCs showed vigorous GVHD in the presence or absence of co-transplanted PDX tumor; this demonstrated that non-tumor-associated lymphocytes from these patients were capable of xenoreaction, even though tumor-associated T cells were not. We observed that PDX-NSG mice treated with either modality showed significantly reduced tumor burden and increased survival over control-Ig-treated mice. Overall, our study shows that PDXs implanted in NSG mice from surgically resected-NSCLC retain T cells sufficient for preclinical evaluation of novel T cell-targeting immunotherapies. NIH, National Cancer Institute, IOTN(U01 CA244314) NIH, NIAID, R01 AI097187 Mizzou Start-up funds