Treatment Of Patient-derived Xenografts Research Articles

Abstract 1789: Pre-clinical studies to advance anti-ROR1 CAR-T cell therapy for metastatic prostate cancer

Abstract Background: Alterations in WNT signaling are frequently associated with tumorigenesis and metastasis in many cancers including prostate cancer (PCa). The Wnt ligand, WNT5A, is required for normal prostate gland development and is increased in bone metastatic, castration resistant PCa (CRPC) patients. WNT5A signaling is mediated in part through ROR1, a non-canonical Wnt receptor and fetal oncoprotein for which the therapeutic inhibitory antibody, Zilovertamab, has been developed. Its safety has been clinically proven in trials for chronic lymphocytic leukemia (CLL) and metastatic breast cancer. We sought to investigate Zilovertamab-based anti-ROR1 therapies for metastatic prostate cancer.Our Hypothesis is that WNT5A may activate a stem-cell-like program via ROR1 which leads to therapy resistance in metastatic prostate cancer. The anti-ROR1 biologic, Zilovertamab, may inhibit this mechanism of resistance. The expression of ROR1 on CRPC and NEPC tumors and its lack of expression on normal adult tissues makes it a promising CART cell target. Methods: We used the patient-derived xenograft (PDX), PCSD13, small cell bone metastatic prostate cancer model and the neuroendocrine PCa cell lines, PC3 and DU145, to test the effect of Zilovertamab and Zilovertamab-CART cells in vitro using the real time cell viability, proliferation, and cell cycle tracking assays in an Incucyte S3. We used in vivo bioluminescence and tumor caliper measurements to monitor effects in vivo. Results: Studies using RNASeq, qRT-PCR, FACS and Westerns showed high expression of ROR1 in PC3, DU145, and in PCSD13. CRISPR-Cas9 Knock out of ROR1 in PC3 and DU145 cells showed increased inhibition of proliferation at lower docetaxel concentrations. Treatment of PCSD13 PDX in vivo with Zilovertamab increased docetaxel-mediated tumor growth inhibition. Mice bearing PC3 xenografts injected intravenously with Zilovertamab anti-ROR1 CAR-T cells showed durable, tumor ablation in 67% of mice compared to 22% of mice injected with activated T cells from the same donor and 0% of untreated mice. Survival of mice at Day 70 injected with CART was 78% compared to 0% of mice injected with control donor T cells and 0% of untreated mice. Zilovertamab antibody could inhibit Zilovertamab CART cell tumor cell killing. Conclusions: ROR1 was expressed at high levels on castration resistant small cell PCa and neuroendocrine PCa cell lines and PDX models. Zilovertamab synergized with docetaxel to inhibit tumor growth in patient derived xenograft in vivo and in organoid cultures. Zilovertamab-based CART cells durably eradicated ROR1+ prostate cancer xenograft tumors. These studies supported the recently launched Phase 1b clinical trial of Zilovertamab plus docetaxel in metastatic CRPC patients. Clinical development of GMP Zilovertamab CART cells for a clinical trial in CLL is in progress and may lead to rapid progression to a clinical trial for metastatic CRPC and NEPC. Citation Format: Christina A.M. Jamieson, Jamillah Murtadha, Christopher S. Oh, Michelle Muldong, Evodie Koutouan, JOngwook Kim, Niloofar Etemadfard, Hae Soo Choo, Navyaa Sinha, Sanghee Lee, Christina Wu, Gabriel Pineda, Kathleen Lennon, Karl Willert, Catriona H. Jamieson, Terry Gaasterland, Rana Mckay, Christopher J. Kane, Thomas J. Kipps, Anna A. Kulidjian, Nicholas A. Cacalano, Charles Prussak. Pre-clinical studies to advance anti-ROR1 CAR-T cell therapy for metastatic prostate cancer [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 1789.

Abstract 3521: CCND2 promotes proliferation in Drosha and DICER1 driven tumors

Abstract Mutations in microRNA processing genes such as DROSHA and DICER1 consist of 1.5-2% of pediatric cancers including a brain tumor called pineoblastoma. We developed a genetically engineered mouse model of Drosha- and Dicer1- driven pineal tumors. These tumors develop without microRNAs and express early pineal developmental markers. Gene set enrichment analysis revealed E2F target genes to be highly expressed in these tumors. One of the most upregulated genes in these tumors is cyclin D2 (Ccnd2). Additionally, these tumors stain positively for Rb phosphorylation. In a Wilms tumor cell line (WiT49) with Drosha knocked down, RNA sequencing revealed that CCND2 was the most upregulated gene. Similarly, in patient-derived xenografts (PDXs) of DICER1-related tumors such as pleuropulmonary blastoma (PPB), we observed high expression of CCND2 and positive staining for Rb phosphorylation. To test the dependence of Rb phosphorylation in Drosha and Dicer tumors, we next treated a Drosha pineal tumor allograft with a CDK4/6 inhibitor, palbociclib, and observed tumor growth suppression. Additionally, treatment of DICER1 mutant PPB PDXs with palbociclib had similar results. These experiments suggest that CDK4/6 is important for tumor proliferation in Drosha and DICER1 driven cancers. Citation Format: Claudette R. Fraire, Uma Obalapuram, Patricia D. Tiburcio, Kavita Desai, Kenneth S. Chen. CCND2 promotes proliferation in Drosha and DICER1 driven tumors. [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 3521.

PD04-10 PRE-CLINICAL STUDIES TO ADVANCE ZILOVERTAMAB-BASED ANTI-ROR1 DUAL SPECIFICITY CAR T-CELL THERAPY FOR METASTATIC PROSTATE CANCER.

PD04-10 PRE-CLINICAL STUDIES TO ADVANCE ZILOVERTAMAB-BASED ANTI-ROR1 DUAL SPECIFICITY CAR T-CELL THERAPY FOR METASTATIC PROSTATE CANCER.

BMI1 nuclear location is critical for RAD51-dependent response to replication stress and drives chemoresistance in breast cancer stem cells

Replication stress (RS) has a pivotal role in tumor initiation, progression, or therapeutic resistance. In this study, we depicted the mechanism of breast cancer stem cells’ (bCSCs) response to RS and its clinical implication. We demonstrated that bCSCs present a limited level of RS compared with non-bCSCs in patient samples. We described for the first time that the spatial nuclear location of BMI1 protein triggers RS response in breast cancers. Hence, in bCSCs, BMI1 is rapidly located to stalled replication forks to recruit RAD51 and activate homologous-recombination machinery, whereas in non-bCSCs BMI1 is trapped on demethylated 1q12 megasatellites precluding effective RS response. We further demonstrated that BMI1/RAD51 axis activation is necessary to prevent cisplatin-induced DNA damage and that treatment of patient-derived xenografts with a RAD51 inhibitor sensitizes tumor-initiating cells to cisplatin. The comprehensive view of replicative-stress response in bCSC has profound implications for understanding and improving therapeutic resistance.

Extracellular Vesicles from Pancreatic Cancer Stem Cells Lead an Intratumor Communication Network (EVNet) to fuel tumour progression

ObjectiveIntratumor heterogeneity drives cancer progression and therapy resistance. However, it has yet to be determined whether and how subpopulations of cancer cells interact and how this interaction affects the tumour.DesignWe...

Abstract 2463: Enhancing the antileukemic activity of venetoclax against leukemia stem cells by targeting oxidative phosphorylation through dual inhibition of PI3K and HDAC

Abstract Over 40% of acute myeloid leukemia (AML) patients experience relapse after remission from intensive chemotherapy and stem cell transplant. Therefore, novel therapies are in demand to target the root cause of relapse, leukemia stem cells (LSCs). Recently, a selective inhibitor of anti-apoptotic Bcl-2, venetoclax, was FDA approved for treating AML in combination with less intensive therapies for patients with comorbidities. However, anti-apoptotic Mcl-1 overexpression and pro-apoptotic Bim downregulation manifests venetoclax resistance. Our previous work demonstrated that a novel dual inhibitor of PI3K and HDAC, CUDC-907, synergizes with venetoclax by overcoming these resistance mechanisms in bulk AML cells and suppressing c-Myc expression and mTOR activity. Previous literature has reported that Bcl-2 inhibition suppresses oxidative phosphorylation (OXPHOS), which is what LSCs rely on for energy production. This is in contrast to normal HSCs that can readily shift their reliance to glycolysis for ATP production if OXPHOS is inhibited. Since c-Myc and mTOR signaling are also known to support mitochondrial respiration, we hypothesize that the combination of CUDC-907 and venetoclax can selectively target LSCs. Metabolomics analyses revealed that pre-treatment of CUDC-907 followed by venetoclax affects the abundance of intermediates in the TCA cycle of AML cells, prior to apoptosis. RNAseq analyses with the same treatment regimen found that the transcription of the TCA cycle regulator pyruvate dehydrogenase kinase 2 (PDK2) is upregulated, and the transcription of pyruvate carboxylase (PC), aconitase 2 (ACO2) and the glutamine transporter SLC1A5 is downregulated, some of which are known cMyc target genes. These findings were correlated with a reduced oxygen consumption rate, indicating the suppression of OXPHOS. Treatment of patient-derived xenograft (PDX) mice with venetoclax and CUDC-907 reduced primary AML cell engraftment, suggesting both bulk AML cell and LSC suppression. These results suggest that the combination of CUDC-907 with venetoclax may target LSCs by interfering with the TCA cycle. To further establish our hypothesis, C13 glucose flux analysis will assist in following the effected metabolic pathways. Rescue experiments supplementing metabolites that were downregulated in treated AML cells and/or inhibition of PDK2 will confirm the mitochondrial respiration based mechanism of AML cell death by the combination of CUDC-907 and venetoclax. Finally we will confirm that these two drugs when combined, reduce the number of LSCs in primary patient samples with a PDX model and flow cytometry for known LSC markers, CD38, CD34 & CD123. The results of this study will form a solid foundation for the clinical evaluation of this promising combination therapy for the treatment of AML and potentially reduce the incidence of relapse. Citation Format: Katie Hege, Hasini Kalpage, Maik Hüttemann, Holly Pitman, Jeffrey W. Taub, Lisa Polin, Jing Li, Yubin Ge. Enhancing the antileukemic activity of venetoclax against leukemia stem cells by targeting oxidative phosphorylation through dual inhibition of PI3K and HDAC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2463.

Developing a novel biologic for the treatment of high-risk B cell acute lymphoblastic leukemia

Abstract The subtype of B cell acute lymphoblastic leukemia (B-ALL) at highest risk for relapse overexpresses surface CRLF2 (CRLF2 B-ALL) and disproportionately affects Hispanic children and adults. CRLF2 partners with the IL-7Ra to form a receptor complex activated by the cytokine, Thymic Stromal Lymphopoietin (TSLP). TSLP activates the JAK/STAT and PI3/AKT/mTOR pathways which are known to promote cell survival and proliferation. Our in vitro studies of CRLF2 B-ALL cells showed that supra-physiological levels of TSLP (1 ng/ml) induced a complete shutdown of TSLP receptor signaling, following the initial activation event. This effect was dose dependent and did not occur at physiological levels (20 pg/ml) of TSLP. This signal shutdown was correlated with the loss of surface IL-7Ra. Using a novel patient-derived xenograft (PDX) model that expresses human TSLP (hTSLP, mouse TSLP does not activate the human TSLP receptor) created in our lab, we showed that high dose hTSLP virtually eliminated human CRLF2 B-ALL cells in vivo. The goal of work presented here was to test whether treatment of CRLF2 B-ALL PDX with high doses of recombinant hTSLP (rhTSLP) could re-capitulate anti-tumor effects of hTSLP observed in our engineered mice and to evaluate its effects on prolonged survival. After 24 days of treatment, rhTSLP reduced the leukemia cell burden by 90% in treated PDX as compared to control PDX that received physiological levels of rhTSLP. Similarly, survival was prolonged in PDX mice receiving high-dose rhTSLP as compared to controls: all 11 control mice died before any of the 11 treated mice died. Taken together, these data suggest that TSLP has promise as a biologic for the treatment of CRLF2 B-ALL.

A Subset of Colorectal Cancers with Cross-Sensitivity to Olaparib and Oxaliplatin.

Defects in the homologous recombination (HR) repair pathway are of clinical interest due to sensitivity of HR-deficient cells to PARP inhibitors. We were interested in defining PARP vulnerability in patients with metastatic colorectal cancer (mCRC) carrying KRAS and BRAF mutations who display poor prognosis, have limited therapeutic options, and represent an unmet clinical need. We tested colorectal cancer cell lines, patient-derived organoids (PDO), and patient-derived xenografts (PDX) enriched for KRAS and BRAF mutations for sensitivity to the PARP inhibitor olaparib, and the chemotherapeutic agents oxaliplatin and 5-fluorouracil (5-FU). Genomic profiles and DNA repair proficiency of colorectal cancer models were compared with pharmacologic response. Thirteen of 99 (around 13%) colorectal cancer cell lines were highly sensitive to clinically active concentrations of olaparib and displayed functional deficiency in HR. Response to PARP blockade was positively correlated with sensitivity to oxaliplatin in colorectal cancer cell lines as well as patient-derived organoids. Treatment of PDXs with olaparib impaired tumor growth and maintenance therapy with PARP blockade after initial oxaliplatin response delayed disease progression in mice. These results indicate that a colorectal cancer subset characterized by poor prognosis and limited therapeutic options is vulnerable to PARP inhibition and suggest that PDO-based drug-screening assays can be used to identify patients with colorectal cancer likely to benefit from olaparib. As patients with mCRC almost invariably receive therapies based on oxaliplatin, "maintenance" treatment with PARP inhibitors warrants further clinical investigation.

Small-Molecule Dual PLK1 and BRD4 Inhibitors are Active Against Preclinical Models of Pediatric Solid Tumors

Simultaneous inhibition of multiple molecular targets is an established strategy to improve the continuance of clinical response to therapy. Here, we screened 49 molecules with dual nanomolar inhibitory activity against BRD4 and PLK1, best classified as dual kinase-bromodomain inhibitors, in pediatric tumor cell lines for their antitumor activity. We identified two candidate dual kinase-bromodomain inhibitors with strong and tumor-specific activity against neuroblastoma, medulloblastoma, and rhabdomyosarcoma tumor cells. Dual PLK1 and BRD4 inhibitor treatment suppressed proliferation and induced apoptosis in pediatric tumor cell lines at low nanomolar concentrations. This was associated with reduced MYCN-driven gene expression as assessed by RNA sequencing. Treatment of patient-derived xenografts with dual inhibitor UMB103 led to significant tumor regression. We demonstrate that concurrent inhibition of two central regulators of MYC protein family of protooncogenes, BRD4, and PLK1, with single small molecules has strong and specific antitumor effects in preclinical pediatric cancer models.

Spleen tyrosine kinase expression is correlated with human papillomavirus in head and neck cancer.

Spleen tyrosine kinase (SYK) is a promoter of cell survival in a variety of cell types, including normal and cancerous epithelial cells. We hypothesized that SYK would an important therapeutic target to inhibit for the treatment of HNSCC. SYK protein abundance in patient tumours was evaluated. SYK protein and mRNA abundance was used to examine patient survival and human papillomavirus (HPV) status. Small-interfering RNAs and gene editing with CRISPR/Cas9 were used to evaluate SYK expression on proliferation in HNSCC cell lines. The potency of SYK inhibitor ER27319 maleate on cellular proliferation was tested using a panel of 28 HNSCC cell lines and in vivo in HNSCC patient-derived xenograft (PDX) models. Moderate to high protein expression of SYK was observed in 24% of patient tumors and high SYK expression was exclusively observed in HPV-positive samples (p<0.001). SYK inhibition with RNA interference, gene editing or a SYK inhibitor (ER27319) decreased cell proliferation and migration. Treatment of PDXs with ER27319 maleate was observed to reduce tumour burden in vivo in two of three models. HPV-positive HNSCC harbours high SYK protein levels. We demonstrate that proliferation, migration and overall burden of these tumours can be reduced by genetic or pharmacologic inhibition of SYK. Taken together, these data establish SYK as a therapeutic target for HNSCC.

S131 IL7R EXPRESSION PREDICTS T‐ALL SENSITIVITY TO JAK INHIBITORS REGARDLESS OF IL7R/JAK/STAT MUTATIONAL STATUS

Background:Normal T‐cell development is supported by cytokine signaling with IL7 as key player. This is highlighted by the absence of T‐cells in gamma‐chain SCID‐patients. T‐ALL represents a heterogeneous disease characterized by expansion of immature T‐cells blocked in their differentiation. Despite therapeutic improvements, it remains associated with a poor prognosis mainly due to relapses. This results in a medical need for new therapeutic approaches. Several studies reported the deregulation of IL7R signaling notably via genomic alterations accounting for ∼30% of T‐ALL. In keeping with this, preclinical models supported the possibility to target the IL7R‐pathway (IL7Rp) in IL7Rp‐mutated T‐ALL using pharmacological JAK inhibitors such as ruxolitinib. Moreover, ex vivo culture of primary T‐ALL samples requires addition of IL7 cytokine in culture medium to support leukemic cell survival. Altogether, this data supports a strong dependency of T‐ALL on IL7R signalling. However, the question of which T‐ALL patients may benefit from JAK inhibitors remains unclear.Aims:We aimed to investigate surface IL7R (sIL7R) expression and mutational status of IL7Rp in T‐ALL, and to identify a readout for patient JAK inhibitor sensitivity.Methods:159 T‐ALL samples were prospectively analyzed by FACS for IL7R expression. Genetic data of IL7R pathway genes (IL7R, JAK1/2, STAT5) were assessed on 87 adult T‐ALL enrolled in the ongoing GRAALL‐2014 trial by targeted‐sequencing. 21 patient derived xenograft (PDX) T‐ALL were generated for ex vivo studies including i) phosphoprotein flow analysis of pSTAT5 upon IL7 stimulation and JAK inhibition, ii) apoptosis assay by Annexin V‐PI staining, and iii) proliferation assay using Celltrace.Results:sIL7R was expressed at early stages of normal T‐cell development, downregulated during the cortical stage, and up‐regulated at the mature stage. In T‐ALL, sIL7R was expressed in 84/159 cases (53%). We found IL7Rp mutations in 29% of cases and all IL7Rmut cases expressed sIL7R defining 3 classes of T‐ALL depending on sIL7R expression and mutational status of IL7Rp: IL7R‐/WT, IL7R+/WT, IL7R+/mut. IL7R+ T‐ALLs were mainly of immature non‐ETP, cortical and TCRgd phenotype. These data support frequent and abnormal expression of sIL7R in T‐ALL as regard to normal expression pattern in thymocytes.sIL7R expression was always associated with functional signaling of the receptor: pSTAT5 was activated upon IL7 exposure and abrogated by the addition of ruxolitinib in both normal thymocytes and PDX T‐ALL. Constitutive STAT5 phosphorylation was observed in IL7R+/mut but not in IL7R+/WT PDX T‐ALL. However, pSTAT5 activation reached similar levels in both IL7R+/mut and IL7R+/WT PDX T‐ALL upon IL7 exposure regardless of their mutational status, which was completely reversed by ruxolitinib addition. Ex vivo treatment of PDX T‐ALL with increasing doses of ruxolitinib induced apoptosis and cytostatic effect in both IL7R+/WT and IL7R+/mut PDX T‐ALL.Summary/Conclusion:IL7Rp genes are mutated in 29% in T‐ALL and sIL7R is expressed in 53% of cases. Ruxolitinib induces apoptosis and impairs cell proliferation in sIL7R+ T‐ALL irrespective of IL7Rp mutational status. Our data shows that sensitivity to JAK inhibitors is determined by the expression of IL7R regardless of the IL7Rp genomic status. IL7R expression is a readout for JAK inhibitors sensitivity, and provides a fast and accurate companion theranostic tool using flow‐cytometry.

Ca2+ and CACNA1H mediate targeted suppression of breast cancer brain metastasis by AM RF EMF

BackgroundBrain metastases are a major cause of death in patients with metastatic breast cancer. While surgical resection and radiation therapy are effective treatment modalities, the majority of patients will succumb from disease progression. We have developed a novel therapy for brain metastases that delivers athermal radiofrequency electromagnetic fields that are amplitude-modulated at breast cancer specific frequencies (BCF). Methods27.12 MHz amplitude-modulated BCF were administered to a patient with a breast cancer brain metastasis by placing a spoon-shaped antenna on the anterior part of the tongue for three one-hour treatments every day. In preclinical models, a BCF dose, equivalent to that delivered to the patient's brain, was administered to animals implanted with either brain metastasis patient derived xenografts (PDXs) or brain-tropic cell lines. We also examined the efficacy of combining radiation therapy with BCF treatment. Additionally, the mechanistic underpinnings associated with cancer inhibition was identified using an agnostic approach. FindingsAnimal studies demonstrated a significant decrease in growth and metastases of brain-tropic cell lines. Moreover, BCF treatment of PDXs established from patients with brain metastases showed strong suppression of their growth ability. Importantly, BCF treatment led to significant and durable regression of brain metastasis of a patient with triple negative breast cancer. The tumour inhibitory effect was mediated by Ca2+ influx in cancer cells through CACNA1H T-type voltage-gated calcium channels, which, acting as the cellular antenna for BCF, activated CAMKII/p38 MAPK signalling and inhibited cancer stem cells through suppression of β-catenin/HMGA2 signalling. Furthermore, BCF treatment downregulated exosomal miR-1246 level, which in turn decreased angiogenesis in brain environment. Therefore, targeted growth inhibition of breast cancer metastases was achieved through CACNA1H. InterpretationWe demonstrate that BCF, as a single agent or in combination with radiation, is a novel treatment approach to the treatment of brain metastases. This paradigm shifting modality warrants further clinical trials for this unmet medical need.

Genetic Analysis of B-Cell Acute Lymphoblastic Leukemia Dissemination to the Central Nervous System Identifies Clonal Selection and Therapeutic Vulnerability

Without prophylactic therapy, B-cell Acute Lymphoblastic Leukemia (B-ALL) spreads to the leptomeninges of the central nervous system (CNS) in up to 70% of patients. CNS involvement is more common in certain high risk B-ALL subgroups, including patients with KMT2A (MLL)-translocations, and disease relapse in the CNS carries a poor prognosis. The genetic determinants and biology of B-ALL dissemination to the CNS are poorly defined and therefore therapies targeting the drivers of CNS disease are lacking. Whereas B-ALL exhibits significant subclonal diversity that contributes to functional heterogeneity and disease relapse, recent reports suggest similar clonal composition of bone marrow (BM) and CNS disease, with the potential for CNS dissemination being a universal property of B-ALL cells (Williams et al. 2016, Bartram et al. 2018). Furthermore, functional studies of leptomeningeal disease have focused on the invasion of B-ALL cells into the CNS but limited studies have addressed the selection of genetic clones with the ability to grow within the subarachnoid space.To better define the evolutionary history and biology of leptomeningeal B-ALL we performed targeted DNA, SNP copy number, RNA sequencing, and functional analysis on cells isolated from matched BM and CNS tissue of patient derived xenografts (PDX) generated from a cohort of paired diagnosis and relapse samples from 14 pediatric and adult B-ALL patients of varying cytogenetics. The majority of primary patient samples yielded CNS disease 20 weeks after intrafemoral injection into NSG mice. CNS disease burden was higher in PDXs derived from relapsed B-ALL samples. Human B-ALL cells isolated from the CNS of PDXs retained competence to repopulate disease in the BM, spleen, and CNS upon serial transplantation. Targeted DNA sequencing results analyzed using a Bayesian clustering method revealed different genetic clonal composition between matched BM and CNS cells in approximately half of the xenografts. PDXs from relapse samples were more likely to show genetic discordance between the BM and CNS. Copy number analysis also confirmed frequent genetic discordance between cells isolated from the BM and CNS from individual PDXs. Interestingly, in one patient all PDXs generated from the relapse sample displayed chromosome 6p and 17p hemi-deletions that were unique to the CNS. In total, PDXs from four patients showed recurrent enrichment of specific lesions in CNS-engrafting cells, suggesting that transit to and/or survival within the subarachnoid space can be the product of selection for genetic clones with increased CNS tropism.RNA-seq of matched BM and CNS cells derived from 45 of the primary PDXs demonstrated that CNS-isolated cells were transcriptionally distinct from their matched BM. These differences were most pronounced in samples from patients with MLL-AF4 translocations, whose CNS isolated cells grouped together in multi-dimensional scaling. Using GSEA, the most highly CNS-enriched gene sets in MLL samples were related to mRNA translation initiation and polypeptide elongation. Translation-related gene sets are similarly enriched in the blasts of MLL B-ALL patients with CNS disease in the COG 9906 study. CNS-isolated cells from PDXs of MLL patients exhibited altered rates of protein synthesis compared to matched BM-isolated cells. Treatment of PDXs with the clinically-approved translational inhibitor omacetaxine mepesuccinate (OMA) effectively decreased rates of translation in CNS-engrafting cells. Moreover, OMA reduced leukemia burden nearly 4-fold in PDXs bearing established CNS infiltration generated from two MLL patients.Our data represent an advance in the understanding of B-ALL CNS disease. We present a rich resource of genomic and transcriptomic data from xenografts spanning multiple B-ALL subgroups across diagnosis and relapse and have identified selection for genetically and biologically distinct clones in the CNS, contrary to the current model. Furthermore, we demonstrate that in MLL patients, dysregulation of protein synthesis occurs at CNS dissemination and targeting this process is a novel therapeutic paradigm that may benefit patients with CNS disease. DisclosuresMullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding.

Abstract 4636: The antibody-drug conjugate D3-GPC2-PBD potently eradicates neuroblastoma patient-derived xenografts

Abstract Background: We recently developed an antibody-drug conjugate (ADC; D3-GPC2-PBD) for neuroblastoma linking a specific glypican 2 (GPC2) targeting antibody (D3) to the potent DNA damaging pyrrolobenzodiazepine (PBD) dimers (Cancer Cell, 2017). Methods: To determine the spectrum of in vivo efficacy of the D3-GPC2-PBD ADC, we utilized a panel of highly annotated neuroblastoma patient-derived xenografts (PDXs) passaged in C.B-17 scid mice. Cohorts of mice (n = 9 - 10) were stratified to treatment with either vehicle or D3-GPC2-PBD at doses of 0.5 - 3 mg/kg given once or 1 mg/kg given twice weekly x 2 weeks when mean tumor volumes reached 200-300 mm3. Co-treatment with excess D3-GPC2-IgG1, the efficacy of free PBD dimers, re-treatment of the rare ADC treated tumors that regrew, treatment of larger PDXs, and the assessment of DNA damage and apoptosis by immunohistochemistry (IHC) were also explored. PDX GPC2 cell surface expression was quantified by flow cytometry. The binding of D3-GPC2-IgG1 to mouse Gpc2 was tested by flow cytometry and mouse normal tissue Gpc2 expression was profiled utilizing IHC. Results: We first showed D3-GPC2-IgG1 binds avidly to mouse Gpc2 by flow cytometry and that murine and human GPC2 expression is similarly restricted in normal tissues by IHC. To date, the D3-GPC2-PBD ADC was tested in three neuroblastoma PDXs with a range of GPC2 expression (Molecules of Equivalent Soluble Fluorochrome [MESF]) and genomic characteristics: CHLA79 (GPC2 MESF = 825, MYCN non-amplified, ALK wild-type, TP53 wild-type), COG-N-421x (GPC2 MESF = 3301, MYCN amplified, ALK wild-type, TP53 wild-type), and SKNAS (GPC2 MESF = 672, MYCN non-amplified, ALK wild-type, TP53 mutated). Treatment with D3-GPC2-PBD resulted in complete and sustained tumor regression up to 100 days in 61% (17/28) of mice treated with 1 mg/kg ADC, 96% (27/28) of mice treated with 3 mg/kg ADC, and 95% (18/19) of mice treated with 1 mg/kg ADC given twice weekly x 2 weeks. Treatment of the CHLA79 PDX with an equivalent dose of free PBD dimers had no effect on tumor growth and co-treatment with excess D3-GPC2-IgG1 (50x) abrogated a significant amount of the ADC induced tumor regression. The rare 1 mg/kg ADC treated CHLA79 PDXs that began to regrow after 100 days (n = 2) showed complete responses to re-dosing of the ADC. Additionally, the D3-GPC2-PBD ADC dosed at 1 mg/kg ADC given twice weekly x 2 weeks induced complete regression of larger COG-N-421x PDXs (tumor volumes of 600-1000 mm3). ADC treated tumors showed significant upregulation of DNA damage (γH2AX) and apoptosis (cleaved caspase 3 and cleaved PARP) by IHC. ADC treatment was well tolerated with no discernible drug-related toxicities. Conclusions: The D3-GPC2-PBD ADC is safe and potently efficacious in a genomically diverse panel of neuroblastoma PDXs with a range of GPC2 cell surface expression. These data support the development of a PBD dimer containing GPC2 directed ADC. Citation Format: Kristopher R. Bosse, Zhongyu Zhu, Maria Lane, Daniel Martinez, John DeLong, Doncho V. Zhelev, Yang Feng, Yanping Wang, Jennifer Hwang, Dimiter S. Dimitrov, John Maris. The antibody-drug conjugate D3-GPC2-PBD potently eradicates neuroblastoma patient-derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4636.

Abstract 3491: Heterogeneous extrachromosomal amplification of mutant PDGFRA is associated with an aggressive phenotype in glioblastoma

Abstract Background &amp; Objective: Receptor tyrosine kinase (RTK) signaling is altered in over 80% of glioblastoma (GBM) cases, frequently through gene amplification. About 14% of GBMs carry amplification in the gene coding for platelet-derived growth factor receptor A (PDGFRA), according to TCGA. PDGFRA plays a key role in brain development and is associated with GBM proneural (PN) subtype. Despite the frequency of oncogenic RTK signaling in GBMs, RTK inhibitors have not yet achieved sufficient efficacy in clinical trials to earn FDA approval. Imatinib, a multi-kinase inhibitor that targets PDGFRA, has not shown efficacy in earlier clinical trials for GBM, in which amplification status was not an inclusion criteria. It has been reported that treatment of GBMs carrying extrachromosomal (ecDNA) amplification with EGFR inhibitors leads to a decrease in ecDNA copy number as a mechanism of resistance. Here, our objective was to assess the heterogeneity of ecDNA PDGFRA amplification in a newly diagnosed PN GBM tumor (HF3253), and to evaluate whether similar modulation of ecDNA copy number could be attained by Imatinib treatment of patient-derived xenografts (PDX). Experimental Approaches &amp; Results: PDGFRA amplification was detected by low pass whole genome sequencing and confirmed to be extrachromosomal by fluorescent in situ hybridization (FISH) in metaphase spreads using PDGFRA and centromere 4 labeled DNA probes. The amplified PDGFRA also harbored a novel missense mutation corresponding to the extracellular domain. PDGFRA FISH signals/number ranged from 3 to 100 in the HF3253 samples, with high signal (&amp;gt; 20) evident in 67%, 39%, and 43% and low amplification signal (6 &amp;lt; x &amp;lt; 9) evident in only 0%, 6%, and 19% of nuclei from biopsy, neurosphere, and xenograft respectively. HF3253 neurospheres were orthotopically implanted into the brains of immunocompromised nude mice. Imatinib mesylate was administered by oral gavage at a 75 mg/kg/day dosage in 5-day cycles with 2-day drug holiday intervals, starting 2 weeks post implant. Control mice received vehicle gavage under the same schedule. Mice were monitored daily and sacrificed on the basis of weight loss or neurological symptoms. HF3253 PDX treated with Imatinib mesylate did not have a significant survival advantage (median survival: 47.2 days) relative to control mice (median survival: 45.8 days; log rank test p value = 0.7825). The untreated and treated PDXs exhibited high levels of phospho-PDGFRA by IHC. Two independent in vitro dose response assays showed no difference in the IC50 for Imatinib between PDGFRA positive and negative GBM neurospheres: (9.447 µM, 8.384 µM) and (8.972 µM, 6.896 µM). Conclusions: We have developed a patient derived model of glioblastoma that retains ecDNA PDGFRA amplification and high levels of expression and RTK activation. Although PDGFRA is a driver of malignancy, our results show that better inhibitors are needed. Citation Format: Artem D. Berezovsky, Andrea D. Transou, Susan M. Irtenkauf, Laura A. Hasselbach, Julie Koeman, Hoon Kim, Roel G. Verhaak, Tom Mikkelsen, Laila M. Poisson, Ana C. deCarvalho. Heterogeneous extrachromosomal amplification of mutant PDGFRA is associated with an aggressive phenotype in glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3491.

Capecitabine Efficacy Is Correlated with TYMP and RB1 Expression in PDX Established from Triple-Negative Breast Cancers.

Purpose: Triple-negative breast cancer (TNBC) patients with residual disease after neoadjuvant chemotherapy have a poor outcome. We developed patient-derived xenografts (PDX) from residual tumors to identify efficient chemotherapies and predictive biomarkers in a context of resistance to anthracyclines- and taxanes-based treatments.Experimental Design: PDX were established from residual tumors of primary breast cancer patients treated in neoadjuvant setting. TNBC PDX were treated by anthracyclines, taxanes, platins, and capecitabine. Predictive biomarkers were identified by transcriptomic and immunohistologic analysis. Downregulation of RB1 was performed by siRNA in a cell line established from a PDX.Results: Residual TNBC PDX were characterized by a high tumor take, a short latency, and a poor prognosis of the corresponding patients. With the exception of BRCA1/2-mutated models, residual PDX were resistant to anthracyclines, taxanes, and platins. Capecitabine, the oral prodrug of 5-FU, was highly efficient in 60% of PDX, with two models showing complete responses. Prior treatment of a responder PDX with 5-FU increased expression of thymidylate synthase and decreased efficacy of capecitabine. Transcriptomic and IHC analyses of 32 TNBC PDX, including both residual tumors and treatment-naïve derived tumors, identified RB1 and TYMP proteins as predictive biomarkers for capecitabine response. Finally, RB1 knockdown in a cell line established from a capecitabine-responder PDX decreased sensitivity to 5-FU treatment.Conclusions: We identified capecitabine as efficient chemotherapy in TNBC PDX models established from residual disease and resistant to anthracyclines, taxanes, and platins. RB1 positivity and high expression of TYMP were significantly associated with capecitabine response. Clin Cancer Res; 24(11); 2605-15. ©2018 AACR.

Abstract 2888: Dopamine signaling and therapeutic resistance in GBM

Abstract Glioblastoma multiforme (GBM), the most common and aggressive primary brain tumor in adults, is characterized by aggressive recurrence after conventional treatment, which include surgery followed by radiation and chemotherapy. This recurrence reilies on GBM cells’ ability to promote therapeutic resistance. The recently developed Cancer Stem Cell (CSC) hypothesis argues that GBMs are driven by a rare subset of cells called Glioma Stem Cells (GSCs). A significant corollary of the CSC hypothesis is that GSCs have the ability to survive intensive radio- and chemotherapy and give rise to recurrent disease. Recent studies from our laboratory and others have shown a high degree of plasticity in GBM cells that indicated normal GBM cells acquire a stem-like state during anti-glioma therapy. Termed conversion, this molecular process increases the frequency of therapy resistant GSCs and promotes disease recurrence. We hypothesize that a drug capable of blocking tumor cell plasticity would prevent the generation of therapy-resistant GSCs, thereby block GBM recurrence. Based on this concept, we have developed a plasticity-based high-throughput drug screening method for GSCs. Our initial screening has identified several novel FDA-approved compounds that act as antagonists of neurotransmitter receptors. Further investigation revealed that treatment of patient-derived xenograft (PDX) GBM lines with temozolomide (TMZ) led to the formation of a population of cells that express both CD133, a marker of CSCs, and dopamine receptors 2 and 3 (DRDs) (control mean CD133+/DRD2+: 19.7%, TMZ mean: 83.8%, p=.0001). PDX lines treated with a dopamine receptor agonist exhibited increased expression of SOX2, OCT4 and c-Myc, GSC-markers. However, this treatment did not increase the expression of Ki67 in the GSC compartment, a marker for cell proliferation (mean DMSO: 63.8%, mean TMZ: 57.4%). Thus, DRD signaling may influence the plasticity driven GSC niche. Utilizing a CD133-reporter cell line, we found that two anti-psychotics, chlorprothioxene and loxapine, prevented the induction of CD133 expression caused by treatment with TMZ. Finally, we treated PDX lines with chlorpromazine, a newer version chlorprothioxene, in conjunction with TMZ, which resulted in increased therapeutic effecacy (p&amp;lt;.0001 by MTT assay). Overall, these data suggest that dopamine receptor signaling and stress-induced cellular plasticity interact in GBM. We proposed that dopamine receptors represent a strategy by which specific molecular changes induces by canonical chemotherapeutic agents can be inhibited to prevent the induction of resistance in surviving cells. Citation Format: Seamus P. Caragher, Cheol H. Park, Fatemeh Atashi, Shivani Baisiwala, Atique U. Ahmed. Dopamine signaling and therapeutic resistance in GBM [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2888. doi:10.1158/1538-7445.AM2017-2888

Genome-wide methylation profiling of ovarian cancer patient-derived xenografts treated with the demethylating agent decitabine identifies novel epigenetically regulated genes and pathways.

BackgroundIn high-grade serous ovarian cancer (HGSOC), intrinsic and/or acquired resistance against platinum-containing chemotherapy is a major obstacle for successful treatment. A low frequency of somatic mutations but frequent epigenetic alterations, including DNA methylation in HGSOC tumors, presents the cancer epigenome as a relevant target for innovative therapy. Patient-derived xenografts (PDXs) supposedly are good preclinical models for identifying novel drug targets. However, the representativeness of global methylation status of HGSOC PDXs compared to their original tumors has not been evaluated so far. Aims of this study were to explore how representative HGSOC PDXs are of their corresponding patient tumor methylome and to evaluate the effect of epigenetic therapy and cisplatin on putative epigenetically regulated genes and their related pathways in PDXs.MethodsGenome-wide analysis of the DNA methylome of HGSOC patients with their corresponding PDXs, from different generations, was performed using Infinium 450 K methylation arrays. Furthermore, we analyzed global methylome changes after treatment of HGSOC PDXs with the FDA approved demethylating agent decitabine and cisplatin. Findings were validated by bisulfite pyrosequencing with subsequent pathway analysis. Publicly available datasets comprising HGSOC patients were used to analyze the prognostic value of the identified genes.ResultsOnly 0.6–1.0 % of all analyzed CpGs (388,696 CpGs) changed significantly (p < 0.01) during propagation, showing that HGSOC PDXs were epigenetically stable. Treatment of F3 PDXs with decitabine caused a significant reduction in methylation in 10.6 % of CpG sites in comparison to untreated PDXs (p < 0.01, false discovery rate <10 %). Cisplatin treatment had a marginal effect on the PDX methylome. Pathway analysis of decitabine-treated PDX tumors revealed several putative epigenetically regulated pathways (e.g., the Src family kinase pathway). In particular, the C-terminal Src kinase (CSK) gene was successfully validated for epigenetic regulation in different PDX models and ovarian cancer cell lines. Low CSK methylation and high CSK expression were both significantly associated (p < 0.05) with improved progression-free survival and overall survival in HGSOC patients.ConclusionsHGSOC PDXs resemble the global epigenome of patients over many generations and can be modulated by epigenetic drugs. Novel epigenetically regulated genes such as CSK and related pathways were identified in HGSOC. Our observations encourage future application of PDXs for cancer epigenome studies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-016-0361-5) contains supplementary material, which is available to authorized users.

Evaluation of Patient-Derived Xenografts for Modeling Outcome of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Introduction: Children with acute lymphoblastic leukemia (ALL) are stratified at diagnosis based on molecular/cytogenetic characteristics and their response to initial treatment to receive risk-adapted multi-agent chemotherapy. The majority of ALL patients are stratified as Intermediate Risk (IR) and present with moderate levels of minimal residual disease (MRD<5x104) after receiving induction therapy, although an unacceptably high proportion of these patients relapse. The lack of specific prognostic features makes it difficult to predict the response of IR patients to treatment. The early identification of patients who are destined to relapse would facilitate improvements in tailored treatments for IR ALL patients. Recent progress in the development of patient-derived xenografts (PDXs) in immune-deficient mice represents an opportunity to improve relapse prediction in ALL patients. The aims of this study were to: (1) optimize the engraftment conditions of IR pediatric ALL samples to predict patient response to treatment; and, (2) to assess the development and mechanisms of therapy-induced drug resistance.Methods: Two pairs of IR pediatric ALL patients were matched based on clinical and genetic features, except that one patient from each pair relapsed early while the other remains relapse-free (ALL-Rel and ALL-CR1, respectively). Three parameters were varied in establishing PDXs by inoculating one million bone marrow (BM) derived biopsy cells collected at diagnosis into groups of 4 mice: (1) mouse strain (NOD/SCID vs. NSG); (2) site of inoculation (intravenous vs. intra-femoral); and (3) early treatment of mice with a 2-week induction chemotherapy regimen of vincristine, dexamethasone, and L-asparaginase (VXL). Leukemia engraftment was monitored weekly based on the proportion of human versus mouse CD45+ cells in the murine PB, and the median times to engraftment were compared according to patient outcome. The median time to engraft was also compared between the VXL-treated and non-treated groups. PDXs harvested from mice were compared for ex vivo sensitivity to single agent vincristine, dexamethasone and L-asparaginase. PDX gene expression profiles were also compared to identify pathways associated with evasion of VXL treatment in vivo.Results: The efficiency of engraftment was greater for NSG mice (29/32 mice engrafted) versus NOD/SCID mice (20/32 mice), and primary ALL cells also engrafted significantly faster in NSG mice (median time to engraft 71.1 days) compared with NOD/SCID mice (83.5 days) (P < 0.01), with no apparent difference associated with clinical outcome. Intrafemoral inoculation did not improve the efficiency or speed of engraftment compared with intravenous inoculation, nor predicted clinical outcome. However, PDX responses to VXL induction chemotherapy reflected the clinical outcome of the patients from whom they were derived; those derived from the 2 ALL-Rel patients exhibited in vivo drug resistance (leukemia growth delay of 1 and 6.2 days) compared with those derived from the 2 ALL-CR1 patients (34.7 and >119.8 days). Further, ex vivo analysis showed that the PDXs derived from the ALL-Rel patients exhibited resistance to vincristine or L-asparaginase compared with those derived from the ALL-CR1 cases. Moreover, the in vivo VXL treatment of an ALL-CR1 PDX resulted in selection of cells that exhibited vincristine resistance. Gene expression profiling revealed significant up-regulation of microtubule associated proteins (MAPs) and tubulin isotypes (alpha and beta) in vincristine-resistant PDXs. Genes that were significantly upregulted in vincristine resistant PDXs with a false discovery rate (FDR) < 0.05 and P value < 0.02 include TUBB6, TUBA1A, TUBA1B, MAP1S, TUBA3D and TBCA. The increased expression of genes that affect microtubule functions suggest that changes in microtubule dynamics and/or stability led to decreased sensitivity to antimicrotubule agents.Conclusions: In vivo selection of PDXs with an induction-type regimen of chemotherapeutic drugs may lead to improved relapse prediction and identify novel mechanisms of drug resistance in IR pediatric ALL.Support: Steven Walter Foundation; NHMRC Australia, APP1057746 DisclosuresNo relevant conflicts of interest to declare.

Abstract C37: Molecular determinants of sensitivity and resistance to FGFR inhibition in FGFR2-amplified gastric cancer

Abstract Despite improvements in diagnostics and chemotherapy regiments in gastric cancer, there is still an urgent need for novel biomarkers and second-line treatment interventions. High level FGFR2 amplification is found in ∼5% gastric cancers, and responses in FGFR2-amplified gastric cancer have been observed in a phase II study of FGFR inhibitor AZD4547 (Smyth et al ASCO 2015, NCT01795768). Here we studied tumor progression biopsies and patient-derived xenografts (PDX) to understand the mechanisms of sensitivity and resistance in FGFR2-amplified gastric cancer. PDX models were generated from the baseline biopsies of two Caucasian patients with junctional FGFR2-amplified tumors who had durable responses to AZD4547 in the clinic. Both PDX recapitulated the histology of the original cancers, and whole exome sequencing demonstrated 85-90% agreement mutations between the patient biopsies and the PDX tumors. Similar to the patients, both models were highly sensitive to AZD4547, with regression of ∼60% seen after 10 days treatment, with subsequent stability on chronic dosing. To understand the molecular mechanisms of sensitivity to AZD4547, we profiled PDX tumors and PDX-derived spheroid cultures with phospho-RTK signaling arrays and by western blot. Although FGFR inhibition resulted in short-term suppression of PI3K-mTOR signaling, chronic exposure resulted in an increase in phospho-S6 and phospho-4EBP1 after 24 hours of treatment. Combination of AZD4547 and the catalytic mTOR inhibitor AZD2014 elicited a greater response in vitro than either drug alone. To examine adaptive response to AZD4547 we excised residual PDX tumors after 60 days treatment, demonstrating upregulated ERBB3 and insulin receptor phosphorylation in tumors adapted to long-term FGFR inhibition. A treatment break, which resumed tumor growth, re-sensitized to AZD4547, suggesting that ongoing FGFR inhibition was required to maintain the adaptive response. Finally we examined the mechanism of acquired resistance to AZD4547. Two responding patients had progression biopsies. Paired exome sequencing demonstrated an acquired KRAS amplification in one patient, though no evident onco-mutation was identified in patient two. In parallel, long-term treatment of PDX with AZD4547 gave rise to resistant PDX tumors, the results of which will be presented at the conference. In conclusion, we show that reactivation of mTOR signaling limits sensitivity of FGFR2-amplified tumors to AZD4547, identifying potential combination strategies to deepen response. Studies of acquired resistance are ongoing, with preliminary evidence suggesting acquisition of downstream genetic events. Citation Format: Irina Babina, Alex Pearson, Ros Cutts, Elizabeth Smyth, Jian Ning, Amanda Swain, David Cunningham, Nicholas C. Turner. Molecular determinants of sensitivity and resistance to FGFR inhibition in FGFR2-amplified gastric cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C37.