Pediatric Patient-derived Xenografts Research Articles

Abstract 146: Development of a novel pediatric patient-derived xenograft model with THPO and NDUFB3 mutations

Abstract Familial myeloproliferative neoplasms (MPN) of childhood are extremely rare and have propensity towards malignant transformation such as leukemia and lymphoma. Due to the rarity of these cases, few cellular and mouse models are available for understanding the tumor biology. Therefore, the molecular mechanisms of malignant transformation remain poorly understood limiting the therapeutic progress to treat these conditions. Here, we report the development of a novel pediatric patient-derived xenograft (PDX) mouse model harboring mutations in the THPO and mitochondrial subunit NDUFB3 genes and characterization of their functions. THPO is a regulator of hematopoiesis and induces trilineage hematopoiesis through HSPC proliferation while NDUFB3 is a subunit of the mitochondrial complex 1. We isolated malignant cells from an enlarged spleen sample and blood sample from a pediatric patient with TPO mediated MPN and established a PDX model (PDX-2333) that recapitulates the patient’s disease. Short tandem repeat analyses confirmed an exact match between the PDX-2333 tumors and patient samples. Exome and Sanger sequencing analyses were performed to identify mutations. Immunohistochemistry staining was used for tumor characterization. Wildtype (WT) and mutant forms of NDUFB3 genes were cloned into expression plasmids with a puromycin selection marker and stably transfected in HEK-293 cells. Stable clones of HEK-293 overexpressing WT and NDUFB3 mutants were tested for growth and sensitivity to anti-cancer therapies. We established a novel PDX model of lymphomagenesis harboring unique mutations in THPO and NDUFB3 genes. The THPO (c.-52C>T) mutation in upstream open reading frame causes loss of inhibition of THPO mRNA expression. The NDUFB3 (c.23 A>G) mutation results in a change of amino acid from glutamine 8 (E8) to glycine 8 (G8) (Ndufb3_E8) and is highly conserved in many vertebrate species. HEK-293 cell lines stably overexpressing WT or NDUFB3 mutants were tested for growth and their sensitivity to ruxolitinib, a Jak2 inhibitor and doxorubicin, an FDA-approved treatment for leukemia and lymphoma. Our data showed that cells overexpressing Ndufb3_E8 have increased proliferation and are highly resistant to ruxolitinib and doxorubicin in comparison to the WT NDUFB3. Developing a novel pediatric PDX model and exome sequencing allow us to identify novel genes involved in malignant transformation leading to lymphoma development. Our data demonstrated that mutations in the THPO (c.-52C>T) and NDUFB3 (c.23 A>G) genes play a key role in the pathogenesis of a lymphoma and in regulating drug sensitivity. Citation Format: Jonathan Shilyansky, Casandro J. Chan, Kristen L. Coleman, Mariah Leidinger, Sharathkumar Bhagavathi, Anjali Sharathkumar, Po Hien Ear. Development of a novel pediatric patient-derived xenograft model with THPO and NDUFB3 mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 146.

Bone Marrow Microenvironment-Induced Chemoprotection in KMT2A Rearranged Pediatric AML Is Overcome by Azacitidine–Panobinostat Combination

Advances in therapies of pediatric acute myeloid leukemia (AML) have been minimal in recent decades. Although 82% of patients will have an initial remission after intensive therapy, approximately 40% will relapse. KMT2A is the most common chromosomal translocation in AML and has a poor prognosis resulting in high relapse rates and low chemotherapy efficacy. Novel targeted approaches are needed to increase sensitivity to chemotherapy. Recent studies have shown how interactions within the bone marrow (BM) microenvironment help AML cells evade chemotherapy and contribute to relapse by promoting leukemic blast survival. This study investigates how DNA hypomethylating agent azacitidine and histone deacetylase inhibitor panobinostat synergistically overcome BM niche-induced chemoprotection modulated by stromal, endothelial, and mesenchymal stem cells and the extracellular matrix (ECM). We show that direct contact between AML cells and BM components mediates chemoprotection. We demonstrate that azacitidine and panobinostat synergistically sensitize MV4;11 cells and KMT2A rearranged pediatric patient-derived xenograft lines to cytarabine in multicell coculture. Treatment with the epigenetic drug combination reduced leukemic cell association with multicell monolayer and ECM in vitro and increased mobilization of leukemic cells from the BM in vivo. Finally, we show that pretreatment with the epigenetic drug combination improves the efficacy of chemotherapy in vivo.

Abstract 5498: SHP2 inhibition enhances antitumor effect of mirdametinib in a pediatric brain tumor model bearing CDC42SE2BRAF fusion by rewiring the proteome and phosphoproteome landscape

Abstract Pediatric gliomas are the most common type of pediatric brain tumors representing wide range of molecularly and clinically heterogenous subtypes. The hyperactivity of mitogen-activated protein kinases (MAPK) pathway has been identified in the majority of pediatric glioma suggesting its therapeutic potential. However, pharmacologic targeting single MAPK pathway’s component is limited due to the development of drug resistance and differential response associated with tumor molecular landscape. Therefore, effective combination strategy in the framework of precision medicine is needed. Here we report combination benefit and molecular underpinning therapeutic response of brain penetrant MEK inhibitor (mirdametinib) and SHP2 inhibitor (SHP099) in a pediatric patient derived xenograft (PDX) and xenoline developed at our institution. Our model was derived from a pediatric patient who was diagnosed with rare high-grade subtype of glioma, anaplastic pleomorphic xanthoastrocytoma, and did not respond to MEK inhibitor, trametinib. Integrative multi-omics revealed molecular fidelity between our model and its patient tumor counterpart including the presence of 7q35 fusion, CDC52SE2-BRAF, CDKN2A/B loss, and MAPK pathway hyperactivation. In vitro studies using our xenoline IU-X128 demonstrated synergy between SHP099 and mirdametinib to curtail cell proliferation (p<0.05). Moreover, this combination was well tolerated in our PDX, IU-RHT128, and potentiated anti-tumor effect of the single agent within clinically achievable doses. Reverse Phase Proteome Array (RPPA) identified MAPK reactivation via Mushasi RNA binding protein-PI3K-AKT crosstalk as a potential innate resistance mechanism to single agent MEK inhibitor in the PDX tumor. Further, tandem mass tags (TMT)-LC-MS/MS profiling on tumor treated with single agent SHP099 or mirdametinib and their combination revealed that combination therapy does not only revert certain proteome and phosphoproteome reprogramming from single agent treatment but also created a novel landscape which can be associated with anti-tumor effect. In this case, kinase network reprograming leading to MAPK reactivation was identified in mirdametinib treated tumor which was attenuated in the combination treatment. In summary, our results demonstrated that combination SHP099 and mirdametinib is superior to single agent alone in the pediatric A-PXA brain tumor model with proteome and phosphoproteome reprogramming of multiple networks as potential molecular mechanisms underlying therapeutic benefit of combination therapy. Ultimately, clinical translation of this finding will potentially benefit patient of this malignant rare pediatric glioma subset which currently does not have standard therapy. Citation Format: Nur P. Damayanti, Anthony Alfonso, Josue D. Ordaz, Erika Dobrota, M. Reza Saadatzadeh, Pankita Pandya, Barbara J. Bailey, Khadijeh Bijangi-Vishehsaraei, Harlan E. Shannon, Kathy Coy, Melissa Trowbridge, Anthony L. Sinn, Rosa Gallager, Julia Wulfkuhle, Emanuel Petricoin, Amber Mosley, Mark S. Marshall, Alex Lion, Michael J. Fergusson, Karl Balsara, Karen E. Pollok. SHP2 inhibition enhances antitumor effect of mirdametinib in a pediatric brain tumor model bearing CDC42SE2BRAF fusion by rewiring the proteome and phosphoproteome landscape. [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 5498.

Abstract 1197: B7-H3-CAR T cells for the treatment of pediatric adrenocortical carcinoma

Abstract Outcomes for pediatric patients with unresectable, metastatic, or relapsed adrenocortical carcinoma (ACC) are dismal despite aggressive management with multimodality therapies. CAR T cell therapy (CAR T) is a promising approach to improve outcomes for patients with this disease. However, progress is hindered by a lack of targetable surface antigens and limited availability of relevant preclinical models. B7-H3 is a promising immunotherapeutic target but little is known about its expression in pediatric ACC. Thus, we sought to i) evaluate B7-H3 expression in primary pediatric ACC samples and ii) assess B7-H3-CAR T activity in two novel pediatric patient-derived xenograft (PDX) models. First, we assessed B7-H3 expression in 44 pediatric adrenocortical tumors (ACTs) using a CLIA-approved immunohistochemistry (IHC) assay. ACTs had a range of B7-H3 positivity, including 24 (55%) with expression above the threshold for eligibility on our active B7-H3-CAR T clinical trial (H-score ≥ 100; NCT04897321). Univariate analyses demonstrated B7-H3 expression was correlated with the presence of germline TP53 mutations and poor prognostic factors including the inability to achieve complete resection, Cushing phenotype, and the absence of TP53 by IHC. Given a dearth of in vivo models, we next established two clinically relevant pediatric ACC PDX models. SJ-ACC4 was derived from a metastatic tumor and had somatic mutations in TP53 (p.C135Lfs*14) and CTNNB1 (p.S45F). SJACT030812_X1 (www.stjude.org/cstn) was derived from a primary tumor and had a germline TP53 mutation (p.R110Pfs*39). Both had partial ATRX deletions (exons 11-30; 3-28), uniparental disomy at chromosome 11p15, and were B7-H3-positive. Notably, SJ-ACC4 was resistant to a battery of chemotherapeutic agents. To evaluate B7-H3-CAR T anti-ACC activity in vivo, SJ-ACC4 tumor fragments were implanted subcutaneously in NOD SCID gamma (NSG) mice and treated with B7-H3-CAR or control T cells. Strikingly, B7-H3-CAR T eradicated 100% of SJ-ACC4 tumors and mice survived long term compared to controls that met endpoints within 35 days (n=8 per group, p< 0.0001). Finally, we asked if B7-H3-CAR T had antitumor activity in an orthotopic pediatric ACC PDX model. SJACT030812_X1 cells were injected into the para-adrenal region of NSG mice under ultrasound guidance and monitored by serial volumetric ultrasonography. B7-H3-CAR T again had significant antitumor activity, and mice survived long-term compared to controls that met endpoints within 42 days (n=5 per group, p< 0.01). In conclusion, B7-H3 is expressed in a majority of pediatric ACTs and B7-H3-CAR T have potent anti-ACC activity. These findings provide a strong rationale for clinically evaluating B7-H3-CAR T for pediatric patients with chemotherapy-resistant ACC. Additionally, the newly established pediatric ACC PDX models provide a valuable resource for investigating different aspects of this rare and aggressive disease. Citation Format: Rebecca Epperly, Emilia M. Pinto, Teresa Santiago, Heather Sheppard, Michael R. Clay, Christopher Morton, Phuong Nguyen, Yinmei Zhou, Mary Woolard, Carla O'Reilly, Julie Justice, Walter Akers, Melissa Johnson, Asa Karlstrom, Cheng Cheng, Gerard Zambetti, Andrew Davidoff, Raul Ribeiro, Christopher DeRenzo. B7-H3-CAR T cells for the treatment of pediatric adrenocortical carcinoma [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 1197.

Abstract 704: Development of a patient-derived xenograft (PDX) modeling program to enable pediatric precision medicine

Abstract Background: Recapitulation of the full spectrum of genomic changes driving patient tumors have resulted in increased use of patient-derived xenograft (PDX) models in studies of basic cancer biology and preclinical drug development. Given the translational potential of PDXs and limited availability of pediatric cancer models, we established a PDX program to expand the existing collection of pediatric PDXs in the community and enable pre- and post-clinical studies. Methods: PDX generation requests were integrated into clinical workflows to maximize identification of eligible patients for informed consent and tissue collection at Memorial Sloan Kettering Cancer Center. Methodologies for tissue procurement and cryopreservation were optimized to facilitate implantation into host immunodeficient mice and enable multi-institutional tissue exchange for model building. A bioinformatics pipeline was established to allow molecular validation of engrafted PDXs using a next-generation targeted gene panel (MSK-IMPACT) evaluating concordance based on acquired mutations, copy number alterations and clonal structure. Results: Between November 2016 - October 2021, 379 PDX models were developed (265 distinct models) representing 69 discrete diagnoses. Sarcoma represents the most common model type (50 discrete osteosarcoma, 20 desmoplastic small round cell tumor, 14 Ewing sarcoma, 24 rhabdomyosarcoma, 2 CIC/DUX4 and 2 BCOR-rearranged sarcoma) followed by neuroblastoma (n=35), leukemia (n=44), and Wilms tumor (n=15). While the majority of PDXs were established from recurrent or metastatic tissue, 7 paired diagnostic/pre-therapy and post-therapy or relapse models were generated. Genomic characterization of PDXs demonstrate excellent concordance and recapitulation of single nucleotide variants (90%), structural (88%) and copy number variants (94%) between patient tumor and matched PDX. Discrepancies between matched patient/PDX pairs are due to sub-clonal heterogeneity in source tumors with clonal outgrowth in the PDX. Analysis of serial PDX passages also demonstrate stable recapitulation of the genomic profile. Establishment of a diverse PDX collection allowed preclinical evaluation of 10 targeted agents across a spectrum of pediatric tumors and provided the preclinical rationale for 3 investigator-initiated pediatric clinical trials. Conclusions: Investment in the development of a phenotypically diverse and biologically faithful collection of pediatric PDX models enables the goals of precision medicine. Optimization of PDX workflows and methods has also enabled the development of a pediatric PDX consortium (PROXC - Pediatric Research in Oncology Xenografting Consortium) to further support the development of pre- and post-clinical studies for pediatric cancer. Citation Format: Filemon S. Dela Cruz, Joseph G. McCarter, Daoqi You, Nancy Bouvier, Xinyi Wang, Kristina C. Guillan, Armaan H. Siddiquee, Katie B. Souto, Hongyan Li, Teng Gao, Dominik Glodzik, Daniel Diolaiti, Neerav N. Shukla, Joachim Silber, Umeshkumar K. Bhanot, Faruk Erdem Kombak, Diego F. Coutinho, Shanita Li, Juan E. Arango Ossa, Juan S. Medina-Martinez, Michael V. Ortiz, Emily K. Slotkin, Michael D. Kinnaman, Sameer F. Sait, Tara J. O'Donohue, Marissa Mattar, Maximiliano Meneses, Michael P. LaQuaglia, Todd E. Heaton, Justin T. Gerstle, Nicola Fabbri, Chelsey M. Burke, Irene M. Rodriquez-Sanchez, Christine A. Iacobuzio-Donahue, Julia L. Glade Bender, Ryan D. Roberts, Jason T. Yustein, Nino C. Rainusso, Brian D. Crompton, Elizabeth Stewart, Alejandro Sweet-Cordero, Leanne C. Sayles, Andrika D. Thomas, Michael H. Roehrl, Elisa de Stanchina, Elli Papaemmanuil, Andrew L. Kung. Development of a patient-derived xenograft (PDX) modeling program to enable pediatric precision medicine [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 704.

HGG-12. HUMAN IPSC-DERIVED H3.3K27M NEUROSPHERES: A NOVEL MODEL FOR INVESTIGATING DIPG PATHOGENESIS AND DRUG RESPONSE

Diffuse intrinsic pontine glioma (DIPG) is a subset of high-grade glioma that occurs predominantly in children and has no cure. Up to 80% of DIPG harbor a heterozygous point mutation that results in a lysine 27 to methionine substitution in histone variant H3.3 (H3.3K27M). Existing DIPG models have provided insight into the role of H3.3K27M but have limitations: genetically engineered murine models often rely on overexpression of the mutant histone to form tumors; patient-derived xenografts (PDX) are more genetically faithful but preclude examination of the effect of individual mutations on pathogenesis. To address these shortcomings and better recapitulate the genetics of human tumors, we designed a novel DIPG model based on human induced pluripotent stem cells (iPSC) edited via CRISPR to express heterozygous H3.3K27M. Edited iPSC were chemically differentiated into neural progenitor cells, which upon implantation into the brainstems of immunodeficient mice formed diffusely invasive tumors that were histologically consistent with high-grade glioma. Further, neurospheres cultured from primary tumors formed secondary tumors upon reimplantation with more diffuse invasion, suggesting in vivo evolution. To validate this model’s relevance to DIPG transcriptionally, we performed RNA-sequencing on a cohort of primary and secondary tumor neurospheres (termed primary and secondary iDIPG) and compared them to published RNA-seq data from pediatric PDX and patient tumor samples. Hierarchical clustering and principal component analysis on differentially expressed genes (P<0.05) showed that H3.3K27M iDIPG cluster with H3.3K27M PDX and patient tumors. Further, ssGSEA showed that H3.3K27M iDIPG are enriched for astrocytic and mesenchymal signature genes, a defining feature of H3.3K27M DIPG. Finally, we found that primary H3.3K27M iDIPG neurospheres are sensitive to panobinostat, an HDAC inhibitor shown to be effective against H3.3K27M DIPG cells in vitro. Overall, these data suggest that H3.3K27M iDIPG are a promising tool for investigating DIPG biology and new therapeutic strategies.

EPCT-22 SAFETY AND EFFICACY OF INTRAVENTRICULAR IMMUNOVIROTHERAPY WITH ONCOLYTIC HSV-1 G207 FOR TREATMENT OF LEPTOMENINGEAL DISEASE

Leptomeningeal metastatic disease (LMD) occurs in 30–50% of newly diagnosed and recurrent pediatric malignant cerebellar tumors and 20–45% of malignant supratentorial tumors. Radiation and chemotherapy often cause substantial long-term neurotoxicity and outcomes remain poor for patients with LMD. At recurrence, LMD is generally minimally responsive to conventional therapies. Immunovirotherapy with engineered oncolytic HSV-1 G207 has emerged as a promising treatment for children with high-grade brain tumors. G207 infects and kills tumor cells while sparing normal cells and stimulates a robust anti-tumor immune response. Intratumoral G207 inoculation demonstrated safety and preliminary efficacy in a pediatric Phase 1 trial in recurrent/progressive high-grade glioma (NCT02457845), and a Phase 2 trial (NCT04482933) is forthcoming. Additionally, a Phase 1 trial of intratumoral G207 in recurrent/progressive malignant pediatric cerebellar tumors is ongoing (NCT03911388). While intratumoral inoculation delivers G207 directly to a primary tumor, it requires neurosurgical procedures thereby limiting repeat doses. Thus, we sought to establish the safety and efficacy of intraventricular G207. Utilizing an immunocompetent, HSV-sensitive murine strain, we determined that a standard 1x107 plaque-forming units (PFU) dose of G207 resulted in damage to the ependymal lining. However, interferon induction with an intraventricular low-dose (1x104 PFU) of G207 or polyinosinic-polycytidylic acid (poly I:C), a toll-like receptor 3 agonist, three days prior to standard treatment dose protected the ependymal lining. This approach enabled safe delivery of multiple subsequent doses. Importantly, with these protective measures, G207 significantly prolonged survival in pediatric patient-derived xenograft models and an immunocompetent murine LMD model of group 3 medulloblastoma, the most aggressive and fatal subtype. Collectively, these data indicate that toxicity from intraventricular G207 can be safely mitigated prior to a therapeutic dose, and that intraventricular G207 effectively targets group 3 medulloblastoma including LMD. These findings provide support for clinical translation of intraventricular G207.

IMMU-06. T-CELL IMMUNOTHERAPY FOR PEDIATRIC BRAIN TUMORS: DIVERSITY IN CELL SURFACE ANTIGEN AND HLA EXPRESSION NECESSITATES A MULTI-PRONGED APPROACH

Cell surface or intracellular antigens expressed in pediatric brain tumors are potential targets for chimeric antigen receptor (CAR) or ab (T-cell receptor) TCR T-cell immunotherapy. At present it remains unknown what cell surface antigens are suitable CAR targets for pediatric brain tumors; in addition, cell surface expression of HLA class I, a molecule critical for ab TCR T-cell recognition, has not been systemically studied in these tumors. Therefore, we set out to assess expression of five CAR targets (IL13Ra2, HER2, EphA2, B7-H3, GD2) and HLA class I. We established and validated a flow cytometry-based method to profile CAR targets and HLA class I expression from pediatric patient-derived xenograft (PDX) samples. To date, we profiled 53 PDX samples, including medulloblastoma, HGG, DIPG, ATRT, and ependymoma. We found that antigen expression has high intra- and inter-PDX sample variability with B7-H3 and IL13Ra2 being most consistently expressed. We confirmed these findings using conventional IHC for B7-H3 with PDX samples and patient tissue microarrays. HLA class I was present on the cell surface of HGGs and DIPGs, however significantly down-regulated in 26 out of 36 other brain tumor types. Finally, matched fresh tissue and PDX sample analysis revealed that cells derived from PDX models are indeed representative of fresh tissue. Our results indicate that more than one antigen needs to be targeted to achieve a more complete tumor clearance. In addition, variable expression of HLA class I suggests that pediatric brain tumors have developed immune evasion strategies to prevent recognition by conventional T cells.

Abstract C001: Prospective use of the single mouse experimental design for evaluation of PLX038A against pediatric solid tumor models

Abstract Purpose: In a previous analysis of &amp;gt;2100 in vivo pediatric xenograft tumor/drug studies (Murphy et al. Cancer Research, 2016), we reported that results from one mouse/treatment group gave essentially similar results for 67 drugs as 10 mice (solid tumor models) or 8 mice (leukemia models). The use of fewer animals per treatment group allows for inclusion of more cancer models that more accurately represent genetic diversity within an histology or between histology’s. Further, this increase in genetic diversity allows for tumor sensitivity biomarker identification, either within a tumor type (e.g. neuroblastoma) or independent of tumor lineage. Here we have used the single mouse design to evaluate a nanoformulated camptothecin analog, PLX038A, where SN-38 (the active metabolite of irinotecan) is released at a controlled rate. Additional objectives of the study were to determine the relationship between initial tumor volume regression and Event-Free Survival (EFS), and to mine genomics data on each tumor model with a goal to identify potential biomarkers that relate to drug sensitivity. Experimental Procedures: Pediatric patient derived xenografts (PDX) were grown subcutaneously, and treatment was administered when tumors were ~300mm3(mean 297 ±34 mm3; range 260-370 mm3). Models tested included rhabdomyosarcoma (Rh10, Rh18, Rh28, Rh30, Rh30R, Rh41, Rh65), Wilms tumors (KT-5, KT-10, KT-11, KT-13); and non-CNS rhabdoid tumors (KT-12, KT-14, KT-16, RBD1, RBD2) and cell line-derived xenografts (Ewing sarcoma lines ES-1, ES-4, ES-5, ES-6, EW-8, CHLA258, TC-71, SK-NEP-1). New models include RBD1 an atypical teratoid rhabdoid tumor established from a metastatic lung lesion, NCH-EWS-1, a Ewing sarcoma from a lung lesion, and S12-6321 was established from a patient with metastatic pleiomorphic xanthoastrocytoma. All tumors were used at low passage and authenticated by STR analysis against reference profiles developed by this group. Tumor models were selected for testing without reference to genetic or molecular characteristics, or sensitivity to irinotecan in previous testing. PLX038A was administered one time at a dose of 120 umol/kg by intraperitoneal injection. For 13 models, with data for the single agent irinotecan (2.5 mg/kg daily x 5 with cycle repeated at day 21), the correlation between tumor responsiveness to PX038A and irinotecan was examined. Results: The activity of single-dose PLX038A was evaluable in 30/31 models. PLX038A induced &amp;gt;50% volume regressions in 25 models (83%). EFS varied from 30 to &amp;gt;120 days dependent on the xenograft model. For 13 tumor models having complete regression, EFS times varied from 38 to &amp;gt;120 days, hence initial tumor volume regression correlated only modestly with EFS (r2= 0.453). Sensitivity to PLX038A was highly correlated with response to irinotecan (r2=0.729). Mutations in TP53BP1 were enriched in 6 sensitive tumor models compared to 4 resistant models. Conclusions: PLX038A was highly active in most xenograft models, and tumor sensitivity to PLX038A was highly correlated with sensitivity to irinotecan. Biomarkers that correlated with model sensitivity included wild type TP53, or mutant TP53 but with a mutation in TP53BP1, thus a defect in DNA damage response. These results support the value of the single mouse experimental design, and also support further development of PLX038 for pediatric clinical evaluation. Support: UO1CA199297, CA169368 and CA165995 (PJH) from NCI and CPRIT RR170055 (SZ). Citation Format: Samson Ghilu, Qilin Li, Shaun D Fontaine, Daniel V. Santi, Raushan T. Kurmasheva, Siyuan Zheng, Peter J. Houghton. Prospective use of the single mouse experimental design for evaluation of PLX038A against pediatric solid tumor models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C001. doi:10.1158/1535-7163.TARG-19-C001

Modeling Effective AML Therapies - Comparative Anti-Leukemic Synergy of BCL2 and MCL1 Inhibition Combined and with Chemotherapy

Background: Resistance to chemotherapy-induced apoptotic death is a major mechanism responsible for the failure of AML therapies. Levels of anti-apoptotic proteins BCL2 and MCL1 are increased in relapsed AML samples. Venetoclax, a BH3 mimetic that binds to BCL-2, was recently granted accelerated approval for use in AML in combination with cytotoxic chemotherapy. However, MCL-1 and BCL-XL are known pathways of resistance to BCL-2 directed therapy. AMG-176 is a first-in-class MCL-1 specific inhibitor that induces specific and significant apoptosis-mediated toxicity against leukemia cell lines, tumor xenograft models, and primary patient samples. We compared anti-leukemic synergy of Venetoclax and AMG-176 together and individually with cytotoxic chemotherapy used in AML. We sought to identify optimal combinations for efficacy against varying molecular signatures of AML. Methods: To examine the potential of pairing Venetoclax with AMG-176 we performed a series of in vitro 8x8 dose response matrices on a panel of 11 leukemias spanning AML and Acute Lymphocytic Leukemia (ALL) (Table). This included 3 relapsed pediatric Patient Derived Xenograft (PDX) AML lines propagated in the presence of cytokines to better model inflammation, stem cell function, and niche derived support of leukemic blasts. An additional 8 established cell lines representing key AML &amp; ALL molecular classes were employed. We used the Delta Bliss (DB) method for scoring matrices and report the DBsum, the summation of all of the synergistic(-) and antagonistic(+) well scores in an 8x8 matrix. Drugs tested in combinations with Venetoclax and/or AMG-176 included cytarabine and daunorubicin. Results: The Figure shows average DBSum for the Venetoclax+AMG-176 combination was -3.41 in the 8 commercial lines, whereas combining the anthracycline Daunorubicin+AMG-176 combination yielded a DBsum score of -0.34, with the score being skewed by a marked antagonism observed with the t(8;21) AML line Kasumi-1 with a DBSum score of 2.13. The 3 relapsed pediatric PDX derived AML lines continued the trend of strong synergy with the Venetoclax+AMG-176 combination with an average score of -3.68 with no observed antagonistic interactions. For context, a synergistic DBSum score of -2.33 would be considered noteworthy from a combination dataset of over &gt;4000 discreet combinations. The Venetoclax+AMG-176 combination exhibited the highest degree of synergy broadly across AML and ALL molecular subgroups. Different patterns of cytotoxicity of combination of chemotherapy with BCL2 or MCL1 inhibition were observed in leukemias with varying molecular backgrounds and histology, including patterns of strong synergy, additivity or antagonism. However, leukemias carrying KMT2A rearrangements exhibited strong synergy or additivity of BCL2 or MCL1 combinations with either daunorubicin or cytarabine, regardless of histology. Conclusions: We identified broad synergy in high risk genetic subtypes of AML and ALL with the combination of BCL-2 and MCL-1 inhibition, and the effect was superior to either agent combined with chemotherapy individually. We identified potential genetic signatures associated with response to chemotherapy cytotoxicity with either Venetoclax or AMG-176. In vivo experiments are in progress to evaluate the efficacy of this combination and better determine genetic subtypes who would most benefit from this treatment approach. Figure Disclosures Perentesis: Kurome Therapeutics: Consultancy.

Abstract LB-B14: Pediatric Preclinical Testing Consortium evaluation of the second-generation selective inhibitor of nuclear export (SINE) compound KPT-8602

Abstract Introduction: KPT-8602 is a second-generation SINE compound that specifically blocks exportin-1 (XPO1) cargo interactions. XPO1 is a nuclear export receptor, a critical regulator of cell proliferation and survival, that is characterized as the sole transporter of several tumor suppressor and growth regulatory proteins, including p53, p21, p27, BCR-ABL, FOXO and STAT3. Overexpression of XPO1 has been correlated with high-risk disease and poor survival in multiple pediatric cancers, including leukemia and neuroblastoma (NB). Our prior work demonstrated that selinexor, a closely-related SINE compound, induced complete remission (CR) or maintained CR (MCR) in 2 of 8 ALL patient-derived xenografts (PDXs) tested while it did not induce tumor regressions in 6 neuroblastoma models (Attiyeh EF, et al. Pediatr Blood Cancer. 2016;63:276-86). While selinexor has shown activity in a variety of adult malignancies and is being tested in pediatric phase 1 trials, KPT-8602 shows similar pharmacokinetic properties to selinexor and improved tolerability in preclinical models, and thus it was tested against ALL and NB models by the PPTC. Methods: KPT-8602 was tested against 6 NB and 12 ALL pediatric PDX models using 10 and 8 mice/arm, respectively. Based on tolerability, a dose of 12.5 mg/kg was selected and administered once daily via oral gavage 5 days per week for 4 weeks. Events were defined as quadrupling of tumor volume from day 0 (for NB) or as the proportion of human CD45+ cells in the peripheral blood (%huCD45+) ≥25% (for ALL). The Kaplan-Meier method was used to compare time-to-event between treated and control groups. Objective response categories were assigned as progressive disease (PD, which is subdivided into progressive disease without or with growth delay, PD1 or PD2, respectively), stable disease (SD), partial response (PR), CR, and MCR [Houghton PJ, et al. Pediatr Blood Cancer, 2007]. KPT-8602 was developed and provided for testing by Karyopharm Therapeutics, Inc. Results: KPT-8602 was generally well tolerated, but with weight loss during treatment of approximately 15% and 2% for the neuroblastoma and ALL models, respectively. KPT-8602 induced significant differences in event free survival (EFS) distribution compared to control in 16 of the 18 (88.9%) evaluable xenograft lines studied. Four NB and 10 ALL PDX models showed a &amp;gt;2-fold extension in median EFS for treated compared to control animals. For the 6 NB PDX models, 3 had a minimum relative tumor volume (minRTV) of &amp;lt;1, and 2 achieved PR while 1 was PD2. For the ALL panel, 3 achieved a CR and 3 more achieved a MCR. Among the ALL PDXs achieving CR/MCR were 4 B-cell ALL (including 2 Ph-like ALL) and 2 T-ALL. The neuroblastoma PDX showing the most favorable response to selinexor showed a PR to KPT-8602, and the 2 ALL PDXs with CR/MCR responses to selinexor showed MCR responses to KPT-8602. Conclusions: These data indicate that KPT-8602 demonstrates significant anti-cancer efficacy and tolerability in preclinical models of childhood ALL and NB. Our results support further preclinical testing of KPT-8602 in combination with other targeted therapies for ALL and NB, as well as testing against additional pediatric cancer models in the PPTC. (Supported by NCI Grants: CA199222; CA199000; CA199287) Citation Format: Kateryna Krytska, Kathryn Evans, Tara Pritchard, Matthew Tsang, Raymond Yung, Yael P. Mosse, Stephen W. Erickson, Yuelong Guo, Erkan Baloglu, Yosef Landesman, Kyle A. Jensen, William Senapedis, Beverly A. Teicher, Malcolm A. Smith, Richard B. Lock, John M. Maris. Pediatric Preclinical Testing Consortium evaluation of the second-generation selective inhibitor of nuclear export (SINE) compound KPT-8602 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B14.

SN-38-loaded nanofiber matrices for local control of pediatric solid tumors after subtotal resection surgery

In addition to surgery, local tumor control in pediatric oncology requires new treatments as an alternative to radiotherapy. SN-38 is an anticancer drug with proved activity against several pediatric solid tumors including neuroblastoma, rhabdomyosarcoma and Ewing sarcoma. Taking advantage of the extremely low aqueous solubility of SN-38, we have developed a novel drug delivery system (DDS) consisting of matrices made of poly(lactic acid) electrospun polymer nanofibers loaded with SN-38 microcrystals for local release in difficult-to-treat pediatric solid tumors. To model the clinical scenario, we conducted extensive preclinical experiments to characterize the biodistribution of the released SN-38 using microdialysis sampling in vivo. We observed that the drug achieves high concentrations in the virtual space of the surgical bed and penetrates a maximum distance of 2 mm within the tumor bulk. Subsequently, we developed a model of subtotal tumor resection in clinically relevant pediatric patient-derived xenografts and used such models to provide evidence of the activity of the SN-38 DDS to inhibit tumor regrowth. We propose that this novel DDS could represent a potential future strategy to avoid harmful radiation therapy as a primary tumor control together with surgery.