Primary Patient-derived Xenograft Research Articles

Abstract 816: Biological evaluation of novel stearoyl gemcitabine nanoparticles in primary pancreatic cancer cells and PDX mice tumor models

Abstract Purpose: This study aimed to improve the systemic stability and enhance the anticancer activity of gemcitabine (Gem) by modifying Gem with stearic acid to form 4-N-stearoylGem (4NSG). The 4NSG compound was formulated into solid lipid nanoparticles and tested against patient-derived pancreatic cancer (PCa) cell lines and patient-derived xenograft (PDX) mice bearing subcutaneous tumors. Methods: Gem was modified by linking the 4-amino group of Gem and stearoyl acyl derivative to form 4-(N)-stearoyl-gemcitabine (4NSG). 4NSG was characterized using high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and elemental analysis. 4NSG was developed into solid lipid nanoparticles (4NSG-SLN) and characterized using a particle size analyzer. Patient-derived primary PCa cells (PPCL-46 and PPCL-68) were treated with GemHCl and 4NSG-SLN. Cytotoxicity, cell migration, and cell-cycle studies were performed to determine the effectiveness of 4NSG-SLN against primary PCa cell lines. We performed antitumor efficacy testing using GemHCl and 4NSG-SLN in PDX mice bearing subcutaneous pancreatic tumors. Results: Analysis of the H-NMR spectra displayed an amide bond at 10.88 ppm, confirming the conjugated bond between the 4-amino group of Gem and stearic acid. The purity of 4NSG was 99.8%. The hydrodynamic diameter (particle size) of 4NSG-SLN was 82±2.3nm, while the blank nanoparticle (SLN) was found to be 35 ± 4.3 nm. The half-maximal inhibitory concentration of 4NSG-SLN-treated PPCL-46 (IC50 =12 ± 2.1µM) and PPCL-68 cultures (IC50 =22 ± 2.6µM) showed higher cytotoxic activity compared with GemHCl-treated PPCL-46 and PPCL-68 cultures respectively (IC50 = 56 ± 2.4 µM, IC50 = 57 ± 1.5 µM, p-value: p<0.001). Our migration study showed 4NSG-SLN treated PPCL-46 cells at 5μΜ concentrations significantly reduced cell mobility towards the wound area with (28 ± 3.5) number of cells migrated compared to GemHCl treatment which showed (120 ± 3.8). The cell-cycle analysis showed that 4NSG-SLN treated PPCL-46 cells at 5μΜ concentration had a higher G1 population (78.25%) than GemHCl (72.75%). 4NSG-SLN may have triggered apoptosis in PPCL-46 cells (2.45%) at 20μΜ in the S and G2/M phases compared to GemHCl treatments which showed a higher cell population of (9.54%).4NSG-SLN treated PDX mice exhibited a two-fold decrease in tumor growth compared to GemHCl-treated PDX mice bearing tumors. Conclusion: This study reveals that 4NSG-SLNs may potentially prolong Gem's systemic circulation, increase its bioavailability, and, most importantly, enhance the therapeutic efficacy of Gem in the treatment of PCa. Citation Format: Esther K. Frimpong, Andriana Inkoom, Nkafu B. Ndemazie, Raviteja Bulusu, Joy C. Okoro, Bo Han, Jose Trevino, Edward Agyare. Biological evaluation of novel stearoyl gemcitabine nanoparticles in primary pancreatic cancer cells and PDX mice tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 816.

Abstract 171: Prostate cancer organoids from KUCaP patient-derived xenograft(PDX) series enables us to genetic engineering human-derived cellular models

Abstract Introduction and Objectives Cancer organoids are expected to be valuable models that recapitulate intratumor heterogeneity and the phenotypic diversity of the disease ex vivo. However, the establishment rate of prostate cancer (PCa) organoids directly from clinical specimens are still low, and we need to improve. Patient-derived xenograft model (PDX) is widely used as a human-derived in vivo model, but genetic engineering is impossible, limiting its use. We aimed to develop a method to stably grow prostate cancer organoids from PDX and evaluate the feasibility of gene engineering using PDX-derived organoids. Materials and Methods KUCaP PDX series, which were established from castration-resistant prostate cancer patient tissues, were used to establish organoids. Eight organoids from eight models of KUCaP PDX were established and maintained for at least three passages. Organoids were reinjected into immunodeficient mice to establish organoid-derived xenograft (ODX) with 100% efficacy. We compared histopathological and molecular characteristics of primary patient tumors, PDX tumors, organoids, and ODX tumors. We conducted whole exome sequencing and RNA sequencing to assess the genomic profiles across the models. Firefly luciferase was introduced into organoids to show the feasibility of genetic engineering. Results Pathological findings were similar across the models derived from the same patient. WES analysis revealed most of the mutations detected in the matched patient tumors and organoids. Hierarchical clustering of RNA sequencing showed that the primary tumors, PDX tumors, organoids, and ODX tumors derived from the same patient cluster, which indicates that even after clonal selection during the establishment of organoids, the basic molecular feature of the model is maintained (Figure). Firefly luciferase was successfully introduced in seven out of seven tested organoids and imaged the ODX tumors from these organoids in vivo. Conclusions Organoids were established with a high success rate using PDX. Firefly luciferase was successfully introduced, showed the feasibility of genetic engineering using prostate cancer organoids. Matched PDX and organoids of PCa recapitulate the basic molecular features of primary human PCa. PDX-derived organoids of PCa, which enables genetic engineering of human-derived cellular models, which could be a valuable tool to enhance PCa research. Citation Format: Takuro Sunada, Takayuki Goto, Takayuki Sumiyoshi, Akamatsu Shusuke, Takashi Kobayashi. Prostate cancer organoids from KUCaP patient-derived xenograft(PDX) series enables us to genetic engineering human-derived cellular models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 171.

Abstract 1747: Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy

Abstract Treatment options for glioblastoma (GBM) are limited. Prognosis remains dismal, with an 18 month on average survival rate following diagnosis due to treatment resistance and disease recurrence. The goal of this project is to investigate hallmarks of cancer progression that contribute to temozolomide (TMZ) resistance, a first tine treatment for GBM. Two signaling pathways were investigated in TMZ-sensitive and -resistant GBM cell lines and in primary and recurrent patient-derived xenograft (PDX) tumor cells by genetically and pharmacologically inhibiting methionine adenosyltransferase 2A (MAT2A) and adenosylhomocysteinase (AHCY). Cell growth and survival were assessed by measuring protein expression of proliferation, oxidative stress and cell cycle arrest markers. EPIC array analysis and targeted bisulfite sequencing were conducted to identify changes in genome-wide and specific CpG island methylation. The Seahorse XF Analyzer measured mitochondrial respiratory capacity and oxidative metabolism. Induced pluripotent stem cell organoids were co-cultured with PDX tumor cells to determine if treatments mitigate tumor cell invasiveness. Compared to parental cells (PC), MAT2A gene expression was increased by 1.7-fold in acquired resistant and de novo resistant GBM cells (RC) [(transcript per million): PC, 7386 ± 412; RC, 12941 ± 1023; n=2; p=2.10e-8].Compared to TMZ-sensitive cells (TS), TMZ-resistant cells (TR) demonstrated a 56% increase in baseline oxygen consumption rate [(pmol/min): TS, 179 ± 6.7; TR, 279 ± 13; n=18; p=.0012] and 64% increase in maximal respiratory capacity [(pmol/min): TS, 403 ± 29; TR, 659 ± 35; n=6; p<.0001]. Both primary and recurrent GBM cells were subject to select MAT2A and AHCY inhibitor compounds and EC50s were determined. Recurrent cells demonstrated a vulnerability to TMZ-generated cell death both by genetically knocking down and pharmacologically inhibiting MAT2A and AHCY. MAT2A and AHCY contribute to TMZ resistance and recurrence by dysregulating methylation programs and upregulating antioxidant programs, respectively. These findings provide a foundation for developing novel combinatory therapeutic strategies and inform clinical studies intended to augment the practically negligible rate of remission for GBM and improve treatment outcomes for cancer patients. Citation Format: Emma Rowland, Jordan Walter, Anna Jermakowicz, Robert Suter, Rebecca Riggins, Nagi Ayad. Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1747.

337 Targeting metabolic and epigenetic programs to re-sensitize glioblastoma to chemotherapy

OBJECTIVES/GOALS: Treatment options for glioblastoma (GBM) are limited. Prognosis remains dismal, with an 18 month on average survival rate following diagnosis due to treatment resistance and disease recurrence. The goal of this project is to investigate hallmarks of cancer progression that contribute to temozolomide (TMZ) resistance, a first tine treatment for GBM. METHODS/STUDY POPULATION: Two signaling pathways were investigated in TMZ-sensitive and -resistant GBM cell lines and in primary and recurrent patient-derived xenograft (PDX) tumor cells by genetically and pharmacologically inhibiting methionine adenosyltransferase 2A (MAT2A) and adenosylhomocysteinase (AHCY). Cell growth and survival were assessed by measuring protein expression of proliferation, oxidative stress and cell cycle arrest markers. EPIC array analysis and targeted bisulfite sequencing were conducted to identify changes in genome-wide and specific CpG island methylation. The Seahorse XF Analyzer measured mitochondrial respiratory capacity and oxidative metabolism. Induced pluripotent stem cell organoids were co-cultured with PDX tumor cells to determine if treatments mitigate tumor cell invasiveness. RESULTS/ANTICIPATED RESULTS: Compared to parental cells (PC), MAT2A gene expression was increased by 1.7-fold in acquired resistant and de novo resistant GBM cells (RC) [(transcript per million): PC, 7386 ± 0.012; RC, 12925 ± 0.023; n=2; p=2.10e-8]. Compared to TMZ-sensitive cells (TS), TMZ-resistant cells (TR) demonstrated a 56% increase in baseline oxygen consumption rate [(pmol/min): TS, 179 ± 6.7; TR, 279 ± 13; n=18; p=.0012] and 64% increase in maximal respiratory capacity [(pmol/min): TS, 403 ± 29; TR, 659 ± 35; n=6; p DISCUSSION/SIGNIFICANCE: MAT2A and AHCY contribute to TMZ resistance and recurrence by dysregulating methylation programs and upregulating antioxidant programs, respectively. These findings provide a foundation for developing novel combinatory therapeutic strategies and inform clinical studies intended to increase remission and reduce recurrence for GBM patients.

Preparation and application of patient-derived xenograft mice model of colorectal cancer.

Patient-derived xenograft (PDX) model becomes a more and more important tool for tumor research. This study aimed to establish a colorectal cancer PDX model and verify its applicability. Fresh human colorectal cancer tissue was surgically removed and subcutaneously inoculated into immunodeficient mice to establish the PDX model. Hematoxylin and eosin (HE) staining and immunohistochemical staining were used to evaluate the model. The successful PDX model was selected to study the efficacy of capecitabine in treating colorectal cancer. HE staining showed that the PDX mice model of colorectal cancer could preserve the histological characteristics of the primary tumor. Immunohistochemistry staining showed α-fetoprotein (AFP), carcinoembryonic antigen (CEA), and E-cadherin were strongly positively expressed in primary human and PDX tumor tissues, with a high degree of similarity. Capecitabine significantly inhibited PDX tumor growth and reduced the expression of AFP and CEA proteins in the tumor tissues (all P s<0.05). We successfully established a colorectal cancer PDX model, and the PDX model could retain the histological and biological characteristics of the primary tumor. Using this PDX model, we revealed that capecitabine at a dose of 300-400 mg/kg can effectively treat colorectal cancer, and no significant difference in toxicity was found among different dose groups. The current work provides a feasible framework for establishing and validating the PDX tumor model to better facilitate the evaluation of drug efficacy and safety.

Establishment and Characterization of Patient-Derived Xenograft Model of Non-Small-Cell Lung Cancer Derived from Malignant Pleural Effusions.

Non-small-cell lung cancer (NSCLC) comprises approximately 80% of all lung malignancies. The 5-year survival rate of patients with advanced lung cancer who lost their chances of surgery is approximately 15%. Suitable animal models are important in screening individualized treatment plans for patients with lung cancer, evaluating the pre-clinical efficacy of new drugs, and conducting basic research. In this study, we collected malignant pleural effusion (MPE) samples from 31 patients with NSCLC, successfully constructed 11 NSCLC patient-derived xenografts (PDXs), and analyzed the factors affecting their successful establishment. Primary PDX tumors were characterized using histological analysis, immunohistochemistry, short tandem repeat (STR) profiling, and cytogenetic analysis. The PDXs preserved the histopathology and protein expression pattern of parental tumors. STR analysis revealed the PDX tissue and a tumor tissue of the same individual origin. Statistical analysis showed that the survival time of patients reflected the malignant degree of MPEs to a certain extent, thus affecting the establishment of PDXs. However, the age, gender, and clinical and biochemical indicators of the patients did not affect the establishment of PDX models. These data suggest that the established NSCLC PDXs preserved the molecular characteristics of primary lung cancer and can serve as a new tool to elucidate the pathogenesis of tumors, explore new treatment methods, and conduct the research and development of new drugs for tumors.

PDX Models: A Versatile Tool for Studying the Role of Myeloid-Derived Suppressor Cells in Breast Cancer.

The pivotal role of myeloid-derived suppressive cells (MDSCs) in cancer has become increasingly apparent over the past few years. However, to fully understand how MDSCs can promote human tumor progression and to develop strategies to target this cell type, relevant models that closely resemble the clinical complexity of human tumors are needed. Here, we show that mouse MDSCs of both the monocytic (M-MDCS) and the granulocytic (PMN-MDSC) lineages are recruited to human breast cancer patient-derived xenograft (PDX) tumors in mice. Transcriptomic analysis of FACS-sorted MDSC-subpopulations from the PDX tumors demonstrated the expression of several MDSC genes associated with both their mobilization and immunosuppressive function, including S100A8/9, Ptgs2, Stat3, and Cxcr2, confirming the functional identity of these cells. By combining FACS analysis, RNA sequencing, and immune florescence, we show that the extent and type of MDSC infiltration depend on PDX model intrinsic factors such as the expression of chemokines involved in mobilizing and recruiting tumor-promoting MDSCs. Interestingly, MDSCs have been shown to play a prominent role in breast cancer metastasis, and in this context, we demonstrate increased recruitment of MDSCs in spontaneous PDX lung metastases compared to the corresponding primary PDX tumors. We also demonstrate that T cell-induced inflammation enhances the recruitment of MDSC in experimental breast cancer metastases. In conclusion, breast cancer PDX models represent a versatile tool for studying molecular mechanisms that drive myeloid cell recruitment to primary and metastatic tumors and facilitate the development of innovative therapeutic strategies targeting these cells.

A Phase 1, Open-Label, Safety, Tolerability, and Efficacy Study of NC525 in Subjects with Advanced Myeloid Neoplasms

Background With the exception of certain select subtypes, Acute Myeloid Leukemia (AML) remains incurable for a majority of patients with current therapies. Despite recent drug approvals targeting actionable molecular mutations and BCL-2, relapse remains inevitable for most patients with AML due to leukemia-initiating cells and progenitors {i.e., leukemic stem cells (LSCs)} which are resistant to conventional cytotoxic drugs. Novel agents that preferentially target LSCs to lessen or eliminate the risk for relapse while avoiding significant myelosuppression by sparing normal hematopoietic stem cells (HSCs), are highly sought-after anti-leukemia therapeutics. Leukocyte Associated Immunoglobulin-like Receptor 1 (LAIR-1) is an immune inhibitory receptor that is present on most immune cell subsets, including T cells, NK cells, monocytes, macrophages, dendritic cells, and granulocytes. LAIR-1, a surface leukocyte receptor containing two immune receptor tyrosine-based inhibitory motifs (ITIM), binds to endogenous collagen ligands and plays an important role in inducing an immunosuppressive microenvironment. In AML, the engagement of LAIR-1 inhibits intracellular downstream survival signals, leading to decreased GM-CSF-mediated survival and proliferation of AML blasts. LAIR-1 is expressed on AML blasts and LSCs regardless of FAB subtypes, cytogenetics or molecular mutations. Recent work demonstrates that myeloid leukemia cell lines, primary AML blasts, monoblasts and monocytes, and MDS-EB-2 blasts all express LAIR-1. Critically, while high expression of LAIR-1 is characteristic of LSCs, this receptor is minimally detectable on normal HSCs, making it an ideal anti-leukemic target. NC525 is a humanized immunoglobulin gamma 1, kappa (IgG1κ) monoclonal antibody that targets LAIR-1-expressing AML blasts and LSCs. Extensive IND-enabling preclinical studies have shown the following features of NC525: (i) Inhibition of colony formation of AML CD34+ bone marrow cells; (ii) sparing of colony formation and differentiation ability of healthy CD34+ HSCs; (iii) direct ex vivo killing of AML blasts without killing healthy leukocytes; (iv) suppression of AML in both MV4-11 and THP-1 CDX models; (v) restriction of MV4-11 in a humanized murine model, without depletion or suppression of human T cells; (vi) restriction of MV4-11 tumor growth in murine blood, spleen, and bone marrow; and (vii) reduction of AML disease burden in primary patient-derived AML xenograft (PDX) models. Finally, NC525 completely inhibits leukemia propagation in secondary transplant models, suggesting that NC525 is capable of eradicating LSCs. Furthermore, NC525 destroys leukemic cells by ligation-mediated apoptosis of leukemic blasts and stem cells without evidence of an immunomodulatory effect on other immune subsets. Methods This is an open-label, non-randomized, Phase 1 dose escalation study to determine the safety and tolerability of NC525 in adult patients with advanced myeloid neoplasms. This study will also assess the clinical benefit of NC525. Eligible patients must have relapsed or refractory AML, myelodysplastic syndrome (MDS), or chronic myelomonocytic leukemia (CMML) that has progressed after standard therapy. Participants will receive NC525 by intravenous infusion on Days 1 and 15 of each 28-day cycle. During Phase 1, the safety and tolerability of NC525 will be evaluated by exploring escalating dose levels of NC525. In addition, the pharmacologically active dose (PAD), defined as the dose that provides a maximal biologic effect, such as achieving a complete remission (CR), CR with partial hematological recovery (CRh) or CR with incomplete hematological recovery (CRi), and the maximum tolerated dose (MTD) of NC525 will be determined. Safety and tolerability will be further assessed through the expansion of a few dose levels to determine an optimal recommended Phase 2 Dose (RP2D) and administration schedule of NC525, based on the PK/PD profiles. Taken together, preclinical data suggest that targeting LAIR-1 in AML and other myeloid malignancies may be an effective therapy for these diseases with the advantage of specifically eradicating leukemic stem cells and sparing normal HSCs.

A Micro-Scale Analytical Method for Determining Glycogen Turnover by NMR and FTMS.

Glycogen is a readily deployed intracellular energy storage macromolecule composed of branched chains of glucose anchored to the protein glycogenin. Although glycogen primarily occurs in the liver and muscle, it is found in most tissues, and its metabolism has been shown to be important in cancers and immune cells. Robust analysis of glycogen turnover requires stable isotope tracing plus a reliable means of quantifying total and labeled glycogen derived from precursors such as 13C6-glucose. Current methods for analyzing glycogen are time- and sample-consuming, at best semi-quantitative, and unable to measure stable isotope enrichment. Here we describe a microscale method for quantifying both intact and acid-hydrolyzed glycogen by ultra-high-resolution Fourier transform mass spectrometric (UHR-FTMS) and/or NMR analysis in stable isotope resolved metabolomics (SIRM) studies. Polar metabolites, including intact glycogen and their 13C positional isotopomer distributions, are first measured in crude biological extracts by high resolution NMR, followed by rapid and efficient acid hydrolysis to glucose under N2 in a focused beam microwave reactor, with subsequent analysis by UHR-FTMS and/or NMR. We optimized the microwave digestion time, temperature, and oxygen purging in terms of recovery versus degradation and found 10 min at 110–115 °C to give >90% recovery. The method was applied to track the fate of 13C6-glucose in primary human lung BEAS-2B cells, human macrophages, murine liver and patient-derived tumor xenograft (PDTX) in vivo, and the fate of 2H7-glucose in ex vivo lung organotypic tissue cultures of a lung cancer patient. We measured the incorporation of 13C6-glucose into glycogen and its metabolic intermediates, UDP-Glucose and glucose-1-phosphate, to demonstrate the utility of the method in tracing glycogen turnover in cells and tissues. The method offers a quantitative, sensitive, and convenient means to analyze glycogen turnover in mg amounts of complex biological materials.

Therapeutic targeting of ATR in alveolar rhabdomyosarcoma

Despite advances in multi-modal treatment approaches, clinical outcomes of patients suffering from PAX3-FOXO1 fusion oncogene-expressing alveolar rhabdomyosarcoma (ARMS) remain dismal. Here we show that PAX3-FOXO1-expressing ARMS cells are sensitive to pharmacological ataxia telangiectasia and Rad3 related protein (ATR) inhibition. Expression of PAX3-FOXO1 in muscle progenitor cells is not only sufficient to increase sensitivity to ATR inhibition, but PAX3-FOXO1-expressing rhabdomyosarcoma cells also exhibit increased sensitivity to structurally diverse inhibitors of ATR. Mechanistically, ATR inhibition leads to replication stress exacerbation, decreased BRCA1 phosphorylation and reduced homologous recombination-mediated DNA repair pathway activity. Consequently, ATR inhibitor treatment increases sensitivity of ARMS cells to PARP1 inhibition in vitro, and combined treatment with ATR and PARP1 inhibitors induces complete regression of primary patient-derived ARMS xenografts in vivo. Lastly, a genome-wide CRISPR activation screen (CRISPRa) in combination with transcriptional analyses of ATR inhibitor resistant ARMS cells identifies the RAS-MAPK pathway and its targets, the FOS gene family, as inducers of resistance to ATR inhibition. Our findings provide a rationale for upcoming biomarker-driven clinical trials of ATR inhibitors in patients suffering from ARMS.

P344: THE DUAL BCL-2 AND BCL-XL INHIBITOR AZD4320 SHOWS ON-TARGET ACTIVITY IN ALL AND ACTS SYNERGISTICALLY WITH MCL-1 INHIBITION

Background: Targeting anti-apoptotic BCL-2 family proteins by BH3-mimetics has become a promising treatment strategy in acute lymphoblastic leukemia (ALL). Heterogeneous activity of the selective BCL-2 inhibitor venetoclax has been observed, but other anti-apoptotic proteins including BCL-XL promote venetoclax insensitivity. In clinical trials, targeting BCL-XL has previously resulted in a dose-limiting decrease of platelets. AZD4320 was developed as a dual inhibitor of BCL-2 and BCL-XL, and its dendrimer conjugate (AZD0466) was recently reported to demonstrate anti-tumor activity in hematological cancer models, while showing only a transient thrombocytopenia. Aims: In this study, the anti-leukemia activities of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of MCL-1-selective AZD5991 were evaluated and compared to the effects of other BH3-mimetics (BCL-2-selective venetoclax, BCL-XL-selective A-1331852 and MCL-1-selective S63845). The on-target activity of the inhibitors was functionally characterized and combination effects were analyzed. Methods: Cell viability assays were performed in ALL cell lines and patient-derived xenograft (PDX) samples analyzing half maximal effective concentrations (EC50). Protein complexes and expression of apoptosis regulators were analyzed by immunoprecipitation and immunoblotting. Dynamic BH3 profiling using synthetic BH3-peptides was performed to determine the dependency of ALL cells on BCL-2 family proteins. Combination effects were assessed by dose-response matrix analyses. Results: First, we determined the efficacy of the dual BCL-2 and BCL-XL inhibitor AZD4320 and of the MCL-1 inhibitor AZD5991 for cell death induction in seven B-cell precursor (BCP) ALL cell lines and in a series of 13 PDX samples, showing heterogeneous responses. Interestingly, sensitivities of individual samples to both compounds were not associated with each other. However, we found a significant correlation of sensitivity to the dual inhibitor (AZD4320) with BCL-2 inhibition (venetoclax; N=13; rs=0.56; p=0.049), but no association with BCL-XL inhibition (A-1331852). Analyzing activities of all five BH3-mimetics including venetoclax, we found lowest EC50 values for AZD4320, indicating particular sensitivity for this dual inhibitor as compared to the single inhibitors (p<0.001). Investigating dependencies of ALL cells on BCL-2 family proteins by dynamic BH3 profiling, we found a shift towards MCL-1-dependence upon exposure to AZD4320, while AZD5991 induced an increased combined dependence on BCL-2 and BCL-XL, indicating potential synergistic activity of triple inhibition. Using co-immunoprecipitation analyses, we found that the exposure of ALL cells to AZD4320 reduced binding of both BCL-2 and BCL-XL to BIM, confirming on-target activity. Moreover, AZD5991 reduced binding of BIM to MCL-1. Accordingly, combined treatment with both inhibitors results in the release of BIM and downstream apoptosis signaling. Assessing combinatorial treatment in a primary PDX sample using multi-dose matrix analyses of both inhibitors revealed a positive mean Bliss synergy score of +4.8 indicating synergistic activity. Importantly, the highest synergism was found at low concentrations of both inhibitors, suggesting efficacy at moderate concentrations, which could potentially be achieved in vivo. Summary/Conclusion: In summary, our study demonstrates sensitivity, on-target activity and synergism of the dual BCL-2 and BCL-XL inhibitor AZD4320 with inhibition of MCL-1, thereby providing strong evidence for further clinical evaluation in ALL.

CRISPR-mediated MECOM depletion retards tumor growth by reducing cancer stem cell properties in lung squamous cell carcinoma

Targeted therapy for lung squamous cell carcinoma (LUSC) remains a challenge due to the lack of robust targets. Here, we identified MECOM as a candidate of therapeutic target for LUSC by screening 38 genes that were commonly amplified in three pairs of primary tumors and patient-derived xenografts (PDXs) using a clustered regularly interspaced short palindromic repeats (CRISPR)-mediated approach. High MECOM expression levels were associated with poor prognosis. Forced expression of MECOM in LUSC cell lines promoted cancer stem cell (CSC) properties, and its knockout inhibited CSC phenotypes. Furthermore, systemic delivery of CRISPR-mediated MECOM depletion cassette using adenovirus with an adaptor, which is composed of a single-chain fragment variable (scFv) against epithelial cell adhesion molecules (EpCAM) fused to the ectodomain of coxsackievirus and adenovirus receptor, and a protector, which consists of the scFv connected to the hexon symmetry of the adenovirus, could specifically target subcutaneous and orthotopic LUSC and retard tumor growth. This study could provide a novel therapeutic strategy for LUSC with high efficacy and specificity.

Abstract 3691: Dynamic BH3 profiling identifies novel combinations in malignant pleural mesothelioma with in vivo efficacy

Abstract Currently, malignant pleural mesothelioma (MPM) is mainly treated with conventional therapies such as surgery, chemotherapy and radiation, and patients often suffer from the lack of second line therapy options. In this study, we aimed to identify efficacious drug combinations in primary MPM patient samples and novel MPM patient-derived xenograft (PDX) models to provide better therapeutic options for MPM patients. BH3 profiling is a functional assay that measures mitochondrial priming of the cell. It uses BH3 peptides derived from the BH3 domain of pro-apoptotic BH3-only Bcl-2 family members to provoke a response from viable mitochondria. When tumor cells are treated with drugs prior to BH3 profiling, early changes in their apoptosis signaling can be captured before frank apoptosis occurs. This method is termed dynamic BH3 profiling (DBP), which has been applied in multiple cancer types to identify efficacious drugs. Here, we used high throughput dynamic BH3 profiling (HTDBP) to assess hundreds of drug treatments simultaneously to identify drug combinations that increased apoptotic priming in primary MPM cells ex vivo. Next, we created novel MPM PDX models and tested these drug combinations to determine the ones that primed MPM PDX cells. The HTDBP result from MPM PDX cells recapitulated the chemical vulnerabilities of the primary MPM cells. Moreover, one of the top combination candidates that comprised of navitoclax (BCL-xL, BCL-2 and BCL-w antagonist) and AZD8055 (mTORC1/2 inhibitor) was shown to be safe and efficacious in vivo in our CPDM_011x MPM PDX model. Finally, on the molecular level, we revealed that treatment with navitoclax drove the anti-apoptotic dependence of MPM cell lines towards MCL-1, whose level was downregulated by AZD8055 treatment. In this study, we identified a novel drug combination for MPM with HTDBP that consists of navitoclax and AZD8055. In addition, our results further corroborated that DBP, as a functional assay, can be used to rationally construct novel drug combinations to improve treatment outcomes for cancer patients. Citation Format: Danielle S. Potter, Ruochen Du, Kin-Hoe Chow, Keith L. Ligon, Raphael Bueno, Anthony Letai. Dynamic BH3 profiling identifies novel combinations in malignant pleural mesothelioma with in vivo efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3691.

Abstract 2324: Small-scale microwave-assisted acid hydrolysis method for glycogen determination and turnover in tumors using Stable Isotope Resolved Metabolomics

Abstract Introduction: Glycogen is a readily deployed intracellular energy storage macromolecule composed of branched chains of glucose. Although glycogen primarily occurs in the liver and muscle, it can be found in most tissues throughout the body, and its metabolism has been shown to be important in cancers and immune cells. Robust analysis of glycogen turnover requires stable isotope tracing plus a reliable means of quantifying total and labeled glycogen derived from precursors such as 13C6-glucose. Current methods for analyzing glycogen are time- and sample-consuming, at best semi-quantitative, and unable to measure stable isotope enrichment. Methods: We have developed a microscale method for quantifying both intact and acid-hydrolyzed glycogen by ultra-high-resolution Fourier transform mass spectrometric (UHR-FTMS) and/or NMR analysis in stable isotope resolved metabolomics (SIRM) studies. Polar metabolites, including intact glycogen and their 13C positional isotopomer distributions were first measured in crude biological extracts by high resolution NMR, followed by rapid and efficient acid hydrolysis to glucose in 1 N HCl for 10 minutes at 110 °C under a N2 atmosphere in a microwave-assisted synthesis reactor. The resulting glucose and its 13C isotopologues were then analyzed by UHR-FTMS and/or NMR. Results: We optimized the microwave digestion time, temperature, and oxygen purging in terms of recovery versus degradation and found 10 minutes at 110-115 °C to give &amp;gt; 90% recovery. The method was applied to track the fate of 13C6-glucose in primary human lung BEAS-2B cells, human macrophages, murine liver and patient-derived tumor xenograft (PDTX) in vivo, and the fate of 2H7-glucose in ex vivo lung organotypic tissue cultures of a lung cancer patient. We showed the incorporation of 13C6-glucose into glycogen and its metabolic intermediates, UDP-Glucose and glucose-1-phosphate, both in terms of the 13C levels and fractional enrichment, thereby demonstrating the utility of the method in tracing glycogen turnover in cells and tissues. Conclusions: The method offers a quantitative, sensitive, and convenient means to analyze glycogen turnover in mg amounts of complex biological materials. Keywords: glycogen turnover; 13C6-glucose, stable isotope resolved metabolomics (SIRM); microwave-assisted hydrolysis. Citation Format: Andrew N. Lane, Timothy L. Scott, Juan Zhu, Teresa A. Cassel, Sara Vicente-Munoz, Penghui Lin, Richard M. Higashi, Teresa W-M Fan. Small-scale microwave-assisted acid hydrolysis method for glycogen determination and turnover in tumors using Stable Isotope Resolved Metabolomics [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2324.

Abstract 131: The role of p66ShcA in the melanoma oncogenesis process

Abstract Background: The identification of crucial driver mutations that affect the MAPK signaling pathway (BRAF, NRAS, NF1) in melanomas, has allowed for the development of targeted therapies and shed light on the process of melanocyte transformation. Gain of function mutations in proteins within the Ras/MAPK pathway in melanocytes leads to the formation of benign neoplasms, or nevi. Multiple mechanisms prevent the transformation of nevi into invasive cancer, namely, oncogene-induced senescence (OIS) and immunosurveillance. Of interest to the melanomagenesis process is the p66ShcA redox protein. We have found that compared to other solid cancers, the majority of primary human melanomas, patient-derived xenografts and melanoma cell lines strongly overexpress p66ShcA, an adaptor protein that induces production of reactive oxygen species (ROS) in response to stress stimuli. Indeed, UV light, increases p66ShcA levels and p66ShcA-induced oxidative stress, which is relevant to this disease as sun exposure contributes significantly to melanoma development. We hypothesized that p66ShcA may play a crucial role in the early steps of melanocyte transformation, possibly by contributing to the overriding of OIS and immune surveillance. Methods: Melanoma initiation and progression will be examined in a known transgenic mouse model (Tyr::CRE/brafCA/ptenlox/lox) either in the presence of endogenous levels of p66ShcA or in an inducible p66ShcA over-expression setting. The necessity of p66ShcA-high expression for melanocyte BRAFV600E-transformation will be tested in immortalized melanocytes. Lastly, to explore the potential selection advantage conferred by p66ShcA, its expression will be silenced in a panel of known melanoma cell lines. Clonogenic assays as well as mouse xenografts will be performed. Results: TCGA melanoma datasets, immunoblot analysis of human melanoma cell lines (n&amp;gt;10), and a collection of metastatic patient-derived xenografts (PDX) (n=6) indicate uniformly elevated p66ShcA levels compared to other cancer types. Knock-down of p66ShcA in multiple melanoma cell lines reduces their clonogenic potential. This reduction in colony formation is independent of driver mutation (e.g., BRAF, NRAS, NF1). Suggesting that p66ShcA may sustain melanoma proliferation. Furthermore, xenografts of the murine YUMM1.7 cell line (braf mutant, cdkn2a null and pten null) in immune-deficient and immune-competent mice indicate that loss of p66ShcA (YUMM1.7 p66ShcA-KO) delays tumor formation, specifically in mice with an intact immune system. Citation Format: Eduardo Cepeda Canedo, Sonia Del Rincon, Peter Siegel, Michael Witcher, Josie Ursini-Siegel. The role of p66ShcA in the melanoma oncogenesis process [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 131.

Abstract 267: Xenograft models of non-metastatic non-small cell lung cancer reveals micrometastasis-associated single cell composition

Abstract Background: Circulating tumor cells (CTCs) represent micrometastatic disease and may offer unique insights into future recurrences in lethal malignancies, including non-small cell lung cancer (NSCLC). Due to CTC rarity and limited stability, no CTC-derived xenograft (CDX) models have ever been generated from non-metastatic NSCLC patients directly. Alternative strategies are needed to molecularly characterize CTCs in this potentially curable patient group. Methods: Surgically resected NSCLC primary tumor tissues were implanted in immunodeficient mice to establish ten patient-derived xenografts (PDXs). CTCs from 2/10 PDX models led to generation of two stable metastatic models that were studied by single cell sequencing. Results: Single cell analysis revealed an additional alveolar epithelial type II (AT2) population in metastatic tumors, besides a common AT2 cluster in PDX/metastatic tumors. This was consistent with an external validation set analysis in primary and metastatic NSCLC patient tumors. Further, AT2 clusters of metastatic tumors expressed higher cancer stemness genes versus primary PDX tumor that was recapitulated in patients primary and metastatic tumors. Conclusions: Stable metastatic models from early stage NSCLC patients can be generated with CTCs from PDX models. The distinct AT2 population identified in CDX tumors with cancer stemness features might be critical mediator of metastasis that deserves further study to discover personalized strategies against NSCLC micrometastases. Citation Format: Kanve Nagaraj Suvilesh, Yulia I. Nussbaum, Vijay Radhakrishnan, Yariswamy Manjunath, Diego M. Avella, Kevin F. Staveley-O’Carroll, Eric T. Kimchi, Aadel A. Chaudhuri, Chi-Ren Shyu, Guangfu Li, Klaus Pantel, Wesley C. Warren, Jonathan B. Mitchem, Jussuf T. Kaifi. Xenograft models of non-metastatic non-small cell lung cancer reveals micrometastasis-associated single cell composition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 267.

EPEN-08. Single-cell transcriptome analysis defines a tumor-supportive microenvironment and tumor-stroma crosstalk in pediatric ependymoma and medulloblastoma.

Abstract Brain tumors are the leading cause of disease-related death in childhood and strong efforts are required to develop innovative and efficient therapeutic strategies for patients with high-risk disease. Key critical factor of pediatric brain tumors is their molecular heterogeneity; one of the aspects that contributes to such heterogeneity is the intrinsic capacity of the tumor cells to organize, shape and exploit the surrounding brain tumor microenvironment (TME) to sustain tumor growth and malignant progression. TME was proved to play a crucial role in several malignancies, but in pediatric brain malignancies this has not been fully elucidated yet. Here, we aimed at characterizing the TME cell populations and their contributions in ependymoma and medulloblastoma, two of the most common pediatric brain tumor entities. Single-cell transcriptome analysis (n=65 ependymomas; n=39 medulloblastomas) of publicly available tumor datasets, as well as newly generated data of primary tumors and matching patient-derived tumor xenografts (PDX), showed an extensive heterogeneity of TME cell types with distinctive expression signatures. Amongst the identified TME populations, analysis revealed pro-inflammatory and proliferating myeloid cells, tumor-infiltrating lymphocytes, active regulatory T cells and vascular progenitor cells. Comparative analysis between primary and PDX tumors showed that tumor cells stimulated the host microenvironment, which in turn exhibited tumor-associated stromal signatures. Applying a deconvolution method, using our single cell data as reference, on a bulk tumor cohort including PFA ependymomas with different degree of immune infiltration, we observed an enrichment of polarized macrophages and microglia cells in tumor with high infiltration. Next, we identified TME markers involved in tumor-supportive functions, such as immune suppression (TIGIT, FOXP3, BCL2, CD19, ICAM2), pro-inflammatory stimuli (CCL3, CCL4, IL1B, GPNMB), extracellular matrix remodeling (COL4A1) and angiogenic processes (ANGPT2). TME markers with emerging role in ependymoma and medulloblastoma can be considered as possible targets for tailored and more effective anti-tumor therapeutic strategies.

Molecular parameters impacting the success rate of a lung cancer PDX model.

e20592 Background: Patient-derived xenograft (PDX) models can provide renewable cancer tissue resources, are being increasingly utilized for the molecular characterizations of cancers and preclinical studies on drug activities, and have the potential to advance biomarker identification. Previous research has demonstrated that PDX tumors preserve the histologic and morphologic characteristics and gene expression and mutation patterns of primary tumors, and correlate to patient survival. Tumor histological subtypes and differentiation grades are known to contribute to the success of PDX intake, but little is known about how molecular features associate with the success of PDX engraftment or whether PDX preserves subclonal architecture of primary tumors. Methods: To fill this void, we analyzed 252 primary tumor samples from the SPORE/ICON project at MD Anderson Cancer Center that were used for generating PDXs and had comprehensive genomic, transcriptomic, and immune profiling data available to evaluate the parameters impacting the intake success of lung cancer PDXs. In order to establish the impact of molecular features on PDX intake success we also assessed the fidelity of PDX in representing the molecular features of this cohort of primary tumors. Results: Only 36% of the primary tumors in this study successfully generated a PDX, with Squamous Cell Carcinoma PDX demonstrating an intake rate of 67% and Adenocarcinoma PDX demonstrating an intake rate of 21%. Genomic architecture derived from whole exome sequencing showed not only 43% (38-48%) of mutations shared, but also similar clonal architecture between primary tumors and PDX. Higher copy number aberration burden, high tumor purity and low immune infiltration in primary tumors were found to be associated with successful intake of PDX. Transcriptomics revealed 6,103 genes differentially expressed between primary tumors with successful PDX intake versus those which were unsuccessful. Pathway analysis of these genes indicated that inhibition of cell proliferation, signaling, and migration as well as DNA replication, recombination and repair were associated with PDX intake failure regardless of histology. Conclusions: These findings indicate that PDX intake success is regulated by molecular features and multiomic evaluation of lung cancer primary tumors can be used as a marker for determining which samples to use for PDX model generation.

Therapeutic targeting of LCK tyrosine kinase and mTOR signaling in T-cell acute lymphoblastic leukemia

AbstractRelapse and refractory T-cell acute lymphoblastic leukemia (T-ALL) has a poor prognosis, and new combination therapies are sorely needed. Here, we used an ex vivo high-throughput screening platform to identify drug combinations that kill zebrafish T-ALL and then validated top drug combinations for preclinical efficacy in human disease. This work uncovered potent drug synergies between AKT/mTORC1 (mammalian target of rapamycin complex 1) inhibitors and the general tyrosine kinase inhibitor dasatinib. Importantly, these same drug combinations effectively killed a subset of relapse and dexamethasone-resistant zebrafish T-ALL. Clinical trials are currently underway using the combination of mTORC1 inhibitor temsirolimus and dasatinib in other pediatric cancer indications, leading us to prioritize this therapy for preclinical testing. This combination effectively curbed T-ALL growth in human cell lines and primary human T-ALL and was well tolerated and effective in suppressing leukemia growth in patient-derived xenografts (PDX) grown in mice. Mechanistically, dasatinib inhibited phosphorylation and activation of the lymphocyte-specific protein tyrosine kinase (LCK) to blunt the T-cell receptor (TCR) signaling pathway, and when complexed with mTORC1 inhibition, induced potent T-ALL cell killing through reducing MCL-1 protein expression. In total, our work uncovered unexpected roles for the LCK kinase and its regulation of downstream TCR signaling in suppressing apoptosis and driving continued leukemia growth. Analysis of a wide array of primary human T-ALLs and PDXs grown in mice suggest that combination of temsirolimus and dasatinib treatment will be efficacious for a large fraction of human T-ALLs.

Endometrial Cancer Patient-Derived Xenograft Models: A Systematic Review.

Background: Because patient-derived xenograft (PDX) models resemble the original tumors, they can be used as platforms to find target agents for precision medicine and to study characteristics of tumor biology such as clonal evolution and microenvironment interactions. The aim of this review was to identify articles on endometrial cancer PDXs (EC-PDXs) and verify the methodology and outcomes. Methods: We used PubMed to research and identify articles on EC-PDX. The data were analyzed descriptively. Results: Post literature review, eight studies were selected for the systematic review. Eighty-five EC-PDXs were established from 173 patients with EC, with a total success rate of 49.1%. A 1–10 mm3 fragment was usually implanted. Fresh-fragment implantation had higher success rates than using overnight-stored or frozen fragments. Primary tumors were successfully established with subcutaneous implantation, but metastasis rarely occurred; orthotopic implantation via minced tumor cell injection was better for metastatic models. The success rate did not correspond to immunodeficiency grades, and PDXs using nude mice reduced costs. The tumor growth period ranged from 2 weeks to 13 months. Similar characteristics were observed between primary tumors and PDXs, including pathological findings, gene mutations, and gene expression. Conclusion: EC-PDXs are promising tools for translational research because they closely resemble the features of tumors in patients and retain molecular and histological features of the disease.