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Predicting in vivo response to antineoplastics remains an elusive challenge. We performed a first-of-kind evaluation of two transcriptome-based precision cancer medicine methodologies to predict tumor sensitivity to a comprehensive repertoire of clinically relevant oncology drugs, whose mechanism of action we experimentally assessed in cognate cell lines. We enrolled patients with histologically distinct, poor-prognosis malignancies who had progressed on multiple therapies, and developed low-passage, patient-derived xenograft models that were used to validate 35 patient-specific drug predictions. Both OncoTarget, which identifies high-affinity inhibitors of individual master regulator (MR) proteins, and OncoTreat, which identifies drugs that invert the transcriptional activity of hyperconnected MR modules, produced highly significant 30-day disease control rates (68% and 91%, respectively). Moreover, of 18 OncoTreat-predicted drugs, 15 induced the predicted MR-module activity inversion in vivo. Predicted drugs significantly outperformed antineoplastic drugs selected as unpredicted controls, suggesting these methods may substantively complement existing precision cancer medicine approaches, as also illustrated by a case study. Significance: Complementary precision cancer medicine paradigms are needed to broaden the clinical benefit realized through genetic profiling and immunotherapy. In this first-in-class application, we introduce two transcriptome-based tumor-agnostic systems biology tools to predict drug response in vivo. OncoTarget and OncoTreat are scalable for the design of basket and umbrella clinical trials.

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Background: Predicting in vivo response to antineoplastics remains an elusive challenge. We evaluated two novel transcriptomic-based, tumor-agnostic systems biology tools, OncoTarget and OncoTreat. Both methodologies predict tumor response to a diverse repertoire of clinically relevant oncology drugs, based on tumor Master Regulator (MR) analysis and de novo, experimentally-assessed drug mechanism-of-action in cognate cell lines. Specifically, OncoTarget identifies high-affinity inhibitors of individual MR proteins, while OncoTreat identifies drugs that invert the transcriptional activity of hyper-connected MR modules. Methods: We enrolled patients with several distinct, poor prognosis malignancies that had progressed on multiple standard therapies, and developed low-passage, patient-derived xenograft (PDX) models. We assessed in vivo tumor response to 35 predicted patient-drug pairings in the first seven successfully engrafted models, including three basal-like breast cancers, a pancreatic ductal carcinoma, a KIT/PDGFR wildtype gastrointestinal stromal tumor, a colon cancer, and a recurrent atypical meningioma. Results: Both OncoTarget and OncoTreat produced highly significant 30-day disease control rates (68% and 91%, respectively) and markedly delayed time to treatment failure versus vehicle control by Kaplan-Meier analysis (p < 10−4, log-rank test). Predicted drugs significantly outperformed randomly selected antineoplastic drugs that were not predicted by either methodology. Importantly, of 18 OncoTreat-predicted drugs, 15 showed MR-module inversion in vivo, demonstrating conservation of drug effect on patient tumor MR modules between carefully selected cognate cell lines and PDXs. Further, extensive benchmarking of OncoTarget and OncoTreat on TCGA tumor cohorts and prospectively profiled tumors demonstrates that candidate drugs can be identified for the vast majority of patients. Conclusions: Complementary precision cancer medicine paradigms are needed to broaden the clinical benefit realized through genetic profiling and immunotherapy. We present extensive preclinical validation of two transcriptomic-based approaches. Our results suggest OncoTarget and OncoTreat may substantively complement existing PCM approaches. Importantly, as RNA-based prediction tools are relatively affordable and allow initial predictions within two weeks of receipt of specimen, OncoTarget and OncoTreat are readily scalable for the design of basket and umbrella clinical trials that would enroll diverse patient populations. Citation Format: Prabhjot S. Mundi, Filemon S. Dela Cruz, Michael V. Ortiz, Adina Grunn, Daniel Diolaiti, Mariano J. Alvarez, Andrew L. Kung, Andrea Califano. Evaluation of a transcriptomics-based precision oncology platform for patient-therapy alignment in treatment resistant malignancies [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 849.

Malignant rhabdoid tumors (MRTs) and Wilms' tumors (WTs) are rare and aggressive renal tumors of infants and young children comprising ∼5% of all pediatric cancers. MRTs are among the most genomically stable cancers, and although WTs are genomically heterogeneous, both generally lack therapeutically targetable genetic mutations.Comparative protein activity analysis of MRTs (n = 68) and WTs (n = 132) across TCGA and TARGET cohorts, using metaVIPER, revealed elevated exportin 1 (XPO1) inferred activity. In vitro studies were performed on a panel of MRT and WT cell lines to evaluate effects on proliferation and cell-cycle progression following treatment with the selective XPO1 inhibitor selinexor. In vivo anti-tumor activity was assessed in patient-derived xenograft (PDX) models of MRTs and WTs.metaVIPER analysis identified markedly aberrant activation of XPO1 in MRTs and WTs compared with other tumor types. All MRT and most WT cell lines demonstrated baseline, aberrant XPO1 activity with in vitro sensitivity to selinexor via cell-cycle arrest and induction of apoptosis. In vivo, XPO1 inhibitors significantly abrogated tumor growth in PDX models, inducing effective disease control with sustained treatment. Corroborating human relevance, we present a case report of a child with multiply relapsed WTs with prolonged disease control on selinexor.We report on a novel systems-biology-based comparative framework to identify non-genetically encoded vulnerabilities in genomically quiescent pediatric cancers. These results have provided preclinical rationale for investigation of XPO1 inhibitors in an upcoming investigator-initiated clinical trial of selinexor in children with MRTs and WTs and offer opportunities for exploration of inferred XPO1 activity as a potential predictive biomarker for response.This work was funded by CureSearch for Children's Cancer, Alan B. Slifka Foundation, NIH (U01 CA217858, S10 OD012351, and S10 OD021764), Michael's Miracle Cure, Hyundai Hope on Wheels, Cannonball Kids Cancer, Conquer Cancer the ASCO Foundation, Cycle for Survival, Paulie Strong Foundation, and the Grayson Fund.

Predicting tumor sensitivity to antineoplastics remains an elusive challenge, with no methods demonstrating predictive power. Joint analysis of tumors—from patients with distinct malignancies who had progressed on multiple lines of therapy—and drug perturbation transcriptional profiles predicted sensitivity to 28 of 350 drugs, 26 of which (93%) were confirmed in low-passage, patient-derived xenograft (PDX) models. Drugs were prioritized based on their ability to either invert the activity of individual Master Regulator proteins, with available high-affinity inhibitors, or of the modules they comprise (Tumor-Checkpoints), based on de novo mechanism of action analysis. Of 138 PDX mice enrolled in 16 single and 18 multi-protein treatment arms, a disease control rate (DCR) of 68% and 91 %, and an objective response rate (ORR) of 12% and 17%, were achieved respectively, compared to 6% and 0% in the negative controls arm, with multi-protein drugs achieving significantly more durable responses. Thus, these approaches may effectively complement and expand current precision oncology approaches, as also illustrated by a case study.

Malignant rhabdoid tumors (MRTs) are rare, aggressive pediatric solid tumors, characterized by a 22q11 deletion that inactivates the SMARCB1 gene. Outcomes remain poor despite multimodality treatment. MRTs are among the most genomically stable cancers and lack therapeutically targetable genetic mutations. We utilized metaVIPER, an extension of the Virtual Inference of Protein-activity by Enriched Regulon (VIPER) algorithm, to computationally infer activated druggable proteins in the tumor of an eight month old patient and then expanded the analysis to TCGA and TARGET cohorts.In vitrostudies were performed on a panel of MRT and atypical teratoid/rhabdoid tumor cell lines. Two patient-derived xenograft (PDX) mouse models of MRT were used forin vivoefficacy studies. MetaVIPER analysis from the patient’s tumor identified significantly high inferred activity of nuclear export protein Exportin-1 (XPO1). Expanded metaVIPER analysis of TCGA and TARGET cohorts revealed consistent elevations in XPO1 inferred activity in MRTs compared to other cancer types. All MRT cell lines demonstrated baseline activation of XPO1. MRT cell lines demonstratedin vitrosensitivity to the XPO1 inhibitor, selinexor which led to cell cycle arrest and induction of apoptosis. Targeted inhibition of XPO1 in patient-derived xenograft models of MRT using selinexor resulted in abrogation of tumor growth. Selinexor demonstrates efficacy in preclinical models of MRT. These results support investigation of selinexor in a phase II study in children with MRT and illustrate the importance of an N-of-1 approach in driving discovery beyond the single patient.Statement of Translational RelevanceWe describe the patient-driven discovery of XPO1 activation as a non-genetically encoded vulnerability in MRTs. The application of metaVIPER analysis to tumors lacking actionable oncogenic alterations represents a novel approach for identifying potential therapeutic targets and biomarkers of response. Our preclinical validation of selinexor confirms XPO1 inhibition as a promising therapeutic strategy for the treatment of MRT.

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.

Glioblastoma heterogeneity and plasticity remain controversial, with proposed subtypes representing the average of highly heterogeneous admixtures of independent transcriptional states. Single-cell, protein-activity-based analysis allowed full quantification of >6,000 regulatory and signaling proteins, thus providing a previously unattainable single-cell characterization level. This helped identify four novel, molecularly distinct subtypes that successfully harmonize across multiple GBM datasets, including previously published bulk and single-cell profiles and single cell profiles from seven orthotopic PDX models, representative of prior subtype diversity. GBM is thus characterized by the plastic coexistence of single cells in two mutually-exclusive developmental lineages, with additional stratification provided by their proliferative potential. Consistently, all previous subtypes could be recapitulated by single-cell mixtures drawn from newly identified states. Critically, drug sensitivity was predicted and validated as highly state-dependent, both in single-cell assays from patient-derived explants and in PDX models, suggesting that successful treatment requires combinations of multiple drugs targeting these distinct tumor states.SignificanceWe propose a new, 4-subtype GBM classification, which harmonizes across bulk and single-cell datasets. Single-cell mixtures from these subtypes effectively recapitulate all prior classifications, suggesting that the latter are a byproduct of GBM heterogeneity. Finally, we predict single-cell level activity of three clinically-relevant drugs, and validate them in patient-derived explant.

Despite the important role of the PI3K/AKT/mTOR axis in the pathogenesis of cancer, to date there have been few functional oncogenic fusions identified involving the AKT genes. A 12-year-old female with a histopathologically indeterminate epithelioid neoplasm was found to harbor a novel fusion between the LAMTOR1 and AKT1 genes. Through expanded use access, she became the first pediatric patient to be treated with the oral ATP-competitive pan-AKT inhibitor ipatasertib. Treatment resulted in dramatic tumor regression, demonstrating through patient-driven discovery that the fusion resulted in activation of AKT1, was an oncogenic driver, and could be therapeutically targeted with clinical benefit. Post-clinical validation using patient-derived model systems corroborated these findings, confirmed a membrane-bound and constitutively active fusion protein, and identified potential mechanisms of resistance to single-agent treatment with ipatasertib. Significance: This study describes the patient-driven discovery of the first AKT1 fusion–driven cancer and its treatment with the AKT inhibitor ipatasertib. Patient-derived in vitro and in vivo model systems are used to confirm the LAMTOR1–AKT1 fusion as a tumorigenic driver and identify potential mechanisms of resistance to AKT inhibition. This article is highlighted in the In This Issue feature, p. 565

CD70 (CD27L) is a member of the tumor necrosis factor superfamily transiently expressed on activated adaptive and innate immune cells. Engagement of CD70 to the co-stimulatory receptor CD27, which is present on adaptive immune cells, promotes antigen-specific T cell responses. Both CD70 and CD27 have been reported to be aberrantly expressed on AML and MDS blasts, but are absent on normal hematopoietic progenitor cells, making them appealing therapeutic targets. CD70-CD27 interactions, working through auto- and/or paracrine signaling, can induce a stem-like signature and stimulate the proliferation of leukemic blasts. In addition, the CD70-CD27 interaction drives inflammatory cytokine production from both CD27 and CD70 pathways, resulting in cytokine production from leukemic blasts and the myeloid cells. Inflammatory signaling is a critical component of the crosstalk between hematopoietic stem and progenitor cells and their microenvironment during infection or hematopoietic stress. However, chronic inflammation can suppress normal hematopoietic stem cell function and eventually lead to bone marrow failure. Similarly, preleukemic and leukemic cell' interactions with the bone marrow microenvironment can induce secretion of pro-inflammatory factors, which in turn, could suppress normal blood cell formation. Therefore, blockade of the CD70-CD27 inflammatory signal pathway could help restore normal hematopoiesis, limit immune evasion, and increase the immune directed responses in MDS and AML. SEA-CD70 is a humanized, nonfucosylated monoclonal antibody targeting CD70. SEA-CD70 retains the ability to block CD27 mediated signaling and binds significantly better than the fucosylated parent to the FcγRIIIa receptor. SEA-CD70 mediates potent target killing through Fc-dependent antibody effector functions, including antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity (CDC). SEA-CD70 can also disrupt CD70-CD27 signaling and could block cellular proliferation and survival of blast cells in vitro. SEA-CD70 may also inhibit the inflammatory signals originating from the interaction between the tumor microenvironment and leukemic blasts. In vivo, SEA-CD70 delayed tumor growth and increased survival in subcutaneous AML xenograft mouse tumor models both as a single agent and when combined with standard of care (SOC) agents. Overall, our data highlight the therapeutic potential of SEA-CD70 for the treatment of AML and MDS patients. Disclosures Diolaiti: Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Haass:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Ulrich:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Chen:Seattlke Genetics:Current Employment, Current equity holder in publicly-traded company.Ho:Seattle Genetics:Current Employment, Current equity holder in publicly-traded company.Gardai:Seattle genetics:Current Employment, Current equity holder in publicly-traded company.

Background Precision medicine approaches are ideally suited for rare tumors where comprehensive characterization may have diagnostic, prognostic, and therapeutic value. We describe the clinical case and molecular characterization of an adolescent with metastatic poorly differentiated carcinoma (PDC). Given the rarity and poor prognosis associated with PDC in children, we utilized genomic analysis and preclinical models to validate oncogenic drivers and identify molecular vulnerabilities. Methods We utilized whole exome sequencing (WES) and transcriptome analysis to identify germline and somatic alterations in the patient’s tumor. In silico and in vitro studies were used to determine the functional consequences of genomic alterations. Primary tumor was used to generate a patient-derived xenograft (PDX) model, which was used for in vivo assessment of predicted therapeutic options. Results WES revealed a novel germline frameshift variant (p.E1554fs) in APC, establishing a diagnosis of Gardner syndrome, along with a somatic nonsense (p.R790*) APC mutation in the tumor. Somatic mutations in TP53, MAX, BRAF, ROS1, and RPTOR were also identified and transcriptome and immunohistochemical analyses suggested hyperactivation of the Wnt/ß-catenin and AKT/mTOR pathways. In silico and biochemical assays demonstrated that the MAX p.R60Q and BRAF p.K483E mutations were activating mutations, whereas the ROS1 and RPTOR mutations were of lower utility for therapeutic targeting. Utilizing a patient-specific PDX model, we demonstrated in vivo activity of mTOR inhibition with temsirolimus and partial response to inhibition of MEK. Conclusions This clinical case illustrates the depth of investigation necessary to fully characterize the functional significance of the breadth of alterations identified through genomic analysis. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0366-0) contains supplementary material, which is available to authorized users.

Summary There is an unmet clinical need for improved tissue and liquid biopsy tools for cancer detection. We investigated the proteomic profile of extracellular vesicles and particles (EVPs) in 426 human samples from tissue explants (TEs), plasma, and other bodily fluids. Among traditional exosome markers, CD9, HSPA8, ALIX, and HSP90AB1 represent pan-EVP markers, while ACTB, MSN, and RAP1B are novel pan-EVP markers. To confirm that EVPs are ideal diagnostic tools, we analyzed proteomes of TE- (n = 151) and plasma-derived (n = 120) EVPs. Comparison of TE EVPs identified proteins (e.g., VCAN, TNC, and THBS2) that distinguish tumors from normal tissues with 90% sensitivity/94% specificity. Machine-learning classification of plasma-derived EVP cargo, including immunoglobulins, revealed 95% sensitivity/90% specificity in detecting cancer. Finally, we defined a panel of tumor-type-specific EVP proteins in TEs and plasma, which can classify tumors of unknown primary origin. Thus, EVP proteins can serve as reliable biomarkers for cancer detection and determining cancer type.

3580 Background: XPO1 is a nuclear export protein that selectively transports tumor and growth regulatory proteins out of the nucleus, thereby effectively inhibiting their function. We previously utilized the Virtual Inference of Protein-activity by Enriched Regulon analysis (VIPER) algorithm to discover that malignant rhabdoid tumors were dependent upon XPO1 inhibition and then evaluated a preclinical cohort using selinexor (KPT-330), the first-in-class selective inhibitor of nuclear export, to demonstrate that XPO1 inhibition was sufficient to cause cell cycle arrest, apoptosis, and disease control in multiple cell line and patient derived xenograft (PDXs) models. Our subsequent analysis revealed that the most common childhood kidney tumor, Wilms tumor, has even high higher inferred activity of XPO1 than rhabdoid tumors leading to our hypothesis that XPO1 inhibition is an effective therapeutic strategy to treat Wilms tumors. Methods: A panel of 9 Wilms tumor cell lines and 3 Wilms tumor PDXs were genomically characterized and tested to evaluate the pre-clinical efficacy of XPO1 inhibition in Wilms tumors. Results: Proliferation rate, increased XPO1 protein expression, and loss of function mutations in TP53 correlated with in vitro Wilms tumor cell line sensitivity to selinexor. Evaluation of co-segregation of all single nucleotide variant changes using with inferred activity of XPO1 on VIPER in all TGCA tumors demonstrates a strong association with TP53 alterations. XPO1 inhibition was effective in all Wilms tumor models tested, most significantly in MSKREN-57196, a favorable histology Wilms tumor PDX with somatic 1q gain as well as WTX and MYCN mutations, as well as in MSKREN-31827, a diffusely anaplastic TP53 mutant Wilms tumor PDX. Eltanexor (KPT-8602) is an XPO1 inhibitor with decreased CNS penetration and an improved toxicity profile; this drug was tested in these in vivo models and found to be at least as effective as selinexor. Conclusions: Somatic 1q gain in favorable histology Wilms tumors and TP53 mutations in diffusely anaplastic Wilms tumors have a particularly poor prognosis in the relapsed setting. Our study demonstrates that XPO1 inhibition may provide a rational therapeutic option to treat such high-risk Wilms tumors. Future clinical trials evaluating XPO1 inhibitors should evaluate its efficacy in children with relapsed Wilms tumors.

Recent genomic characterization of PDA reveals that between 20-25 % of PDA harbor recurrent mutations in genes, including BRCA1/2, PALB2, and ATM, which are critical for homologous recombination (HR), an important form of DNA repair. In many patients, these may be germline mutations. This subgroup of PDAs, termed HR-deficient PDA, has emerged as a defined biological entity associated with increased chemoresistance and a more aggressive disease course. The defects in HR observed in these tumors impart cells with a specific vulnerability to PARP inhibitors and platinum-containing therapy. Still, as observed in the case of many other targeted therapies, only a fraction of HR-defective patient tumors respond to PARP inhibition. More so, many patients that initially respond eventually often develop resistance and progress. Therefore, novel therapies which can be effective against HR-defective PDA, alone or in combination with PARP inhibitors or other combinatorial regimens, are urgently needed. We have recently determined that inactivation of the HR pathway is associated with overexpression of polymerase theta (PolO–, also known as POLQ) in PDA. POLQ is a key enzyme that regulates an alternative pathway of DNA repair, known as the alternative non-homologous end-joining (Alt-NHEJ) pathway. In the setting of defective HR, Alt-NHEJ becomes a critical pathway responsible for the repair of DNA breaks and POLQ inhibition in HR-defective tumor cells demonstrates a synthetic lethality phenotype, not observed in cells with intact HR. Furthermore, POLQ knockdown significantly upregulated the cGAS-STING pathway in HR-deficient PDAs linking the DNA damage response to the immune response. Overall, targeting POLQ may represent a novel and in a valuable therapeutic strategy in HR-defective pancreatic cancer, as POLQ inhibitors are currently in development for clinical use. Citation Format: Annie Wang, Andrea Zamperone, Mohammad Sohail, Lidong Wang, Fiyinfolu Balogun, Jiufeng Li, Ende Zhao, Daniel Diolaiti, Agnel Sfeir, Diane M. Simeone. Polymerase theta synthetic lethal interaction in homologous recombination-deficient pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr I14.

NUTM1-rearranged tumors are defined by the presence of a gene fusion between NUTM1 and various gene partners and typically follow a clinically aggressive disease course with poor outcomes despite conventional multimodality therapy. NUTM1-rearranged tumors display histologic features of a poorly differentiated carcinoma with areas of focal squamous differentiation and typically express the BRD4–NUTM1 fusion gene defining a distinct clinicopathologic entity—NUT carcinoma (NC). NCs with mesenchymal differentiation have rarely been described in the literature. In this report, we describe the characterization of two cases of high-grade spindle cell sarcoma harboring a novel MGA–NUTM1 fusion. Whole-genome sequencing identified the presence of complex rearrangements resulting in a MGA–NUTM1 fusion gene in the absence of other significant somatic mutations. Genetic rearrangement was confirmed by fluorescence in situ hybridization, and expression of the fusion gene product was confirmed by transcriptomic analysis. The fusion protein was predicted to retain nearly the entire protein sequence of both MGA (exons 1–22) and NUTM1 (exons 3–8). Histopathologically, both cases were high-grade spindle cell sarcomas without specific differentiation markers. In contrast to typical cases of NC, these cases were successfully treated with aggressive local control measures (surgery and radiation) and both patients remain alive without disease. These cases describe a new subtype of NUTM1-rearranged tumors warranting expansion of diagnostic testing to evaluate for the presence of MGA–NUTM1 or alternative NUTM1 gene fusions in the diagnostic workup of high-grade spindle cell sarcomas or small round blue cell tumors of ambiguous lineage.

We introduce and validate a new precision oncology framework for the systematic prioritization of drugs targeting mechanistic tumor dependencies in individual patients. Compounds are prioritized on the basis of their ability to invert the concerted activity of master regulator proteins that mechanistically regulate tumor cell state, as assessed from systematic drug perturbation assays. We validated the approach on a cohort of 212 gastroenteropancreatic neuroendocrine tumors (GEP-NETs), a rare malignancy originating in the pancreas and gastrointestinal tract. The analysis identified several master regulator proteins, including key regulators of neuroendocrine lineage progenitor state and immunoevasion, whose role as critical tumor dependencies was experimentally confirmed. Transcriptome analysis of GEP-NET-derived cells, perturbed with a library of 107 compounds, identified the HDAC class I inhibitor entinostat as a potent inhibitor of master regulator activity for 42% of metastatic GEP-NET patients, abrogating tumor growth in vivo. This approach may thus complement current efforts in precision oncology.

PI3K/AKT and NOTCH1 signaling pathways are frequently dysregulated in T-cell acute lymphoblastic leukemias (T-ALL). Although we have shown that the combined activities of the class I PI3K isoforms p110γ and p110δ play a major role in the development and progression of PTEN-null T-ALL, it has yet to be determined whether their contribution to leukemogenic programing is unique from that associated with NOTCH1 activation. Using an Lmo2-driven mouse model of T-ALL in which both the PI3K/AKT and NOTCH1 pathways are aberrantly upregulated, we now demonstrate that the combined activities of PI3Kγ/δ have both overlapping and distinct roles from NOTCH1 in generating T-ALL disease signature and in promoting tumor cell growth. Treatment of diseased animals with either a dual PI3Kγ/δ or a γ-secretase inhibitor reduced tumor burden, prolonged survival, and induced proapoptotic pathways. Consistent with their similar biological effects, both inhibitors downregulated genes involved in cMYC-dependent metabolism in gene set enrichment analyses. Furthermore, overexpression of cMYC in mice or T-ALL cell lines conferred resistance to both inhibitors, suggesting a point of pathway convergence. Of note, interrogation of transcriptional regulators and analysis of mitochondrial function showed that PI3Kγ/δ activity played a greater role in supporting the disease signature and critical bioenergetic pathways. Results provide insight into the interrelationship between T-ALL oncogenic networks and the therapeutic efficacy of dual PI3Kγ/δ inhibition in the context of NOTCH1 and cMYC signaling. Mol Cancer Ther; 16(10); 2069–82. ©2017 AACR.

Pleomorphic xanthoastrocytoma (PXA) is a World Health Organization (WHO) Grade II glioma occurring primarily in children and young adults. Most PXAs harbor the known activating mutation BRAF V600E. We report a case of locally recurrent PXA with anaplastic features in a 10-yr-old female. The PXA was negative by immunohistochemical (IHC) staining for BRAF V600E mutation. Whole-exome and transcriptome sequencing of the tumor confirmed the absence of BRAF V600E, but identified copy-number alterations (including loss of the tumor suppressor CDKN2A) and a novel TMEM106B-BRAF fusion. Based on similar BRAF fusion proteins, this novel fusion is predicted to result in activation of BRAF signaling. Demonstration of positive IHC for phospho-ERK1/2 and phospho-MEK1/2 supported this prediction, and implicated MEK inhibitors as a potential therapeutic strategy.

Background Molecular characterization has the potential to advance the management of pediatric cancer and high-risk hematologic disease. The clinical integration of genome sequencing into standard clinical practice has been limited and the potential utility of genome sequencing to identify clinically impactful information beyond targetable alterations has been underestimated. Methods The Precision in Pediatric Sequencing (PIPseq) Program at Columbia University Medical Center instituted prospective clinical next generation sequencing (NGS) for pediatric cancer and hematologic disorders at risk for treatment failure. We performed cancer whole exome sequencing (WES) of patient-matched tumor-normal samples and RNA sequencing (RNA-seq) of tumor to identify sequence variants, fusion transcripts, relative gene expression, and copy number variation (CNV). A directed cancer gene panel assay was used when sample adequacy was a concern. Constitutional WES of patients and parents was performed when a constitutionally encoded disease was suspected. Results were initially reviewed by a molecular pathologist and subsequently by a multi-disciplinary molecular tumor board. Clinical reports were issued to the ordering physician and posted to the patient’s electronic medical record. Results NGS was performed on tumor and/or normal tissue from 101 high-risk pediatric patients. Potentially actionable alterations were identified in 38% of patients, of which only 16% subsequently received matched therapy. In an additional 38% of patients, the genomic data provided clinically relevant information of diagnostic, prognostic, or pharmacogenomic significance. RNA-seq was clinically impactful in 37/65 patients (57%) providing diagnostic and/or prognostic information for 17 patients (26%) and identified therapeutic targets in 15 patients (23%). Known or likely pathogenic germline alterations were discovered in 18/90 patients (20%) with 14% having germline alternations in cancer predisposition genes. American College of Medical Genetics (ACMG) secondary findings were identified in six patients. Conclusions Our results demonstrate the feasibility of incorporating clinical NGS into pediatric hematology-oncology practice. Beyond the identification of actionable alterations, the ability to avoid ineffective/inappropriate therapies, make a definitive diagnosis, and identify pharmacogenomic modifiers is clinically impactful. Taking a more inclusive view of potential clinical utility, 66% of cases tested through our program had clinically impactful findings and samples interrogated with both WES and RNA-seq resulted in data that impacted clinical decisions in 75% of cases. Electronic supplementary material The online version of this article (doi:10.1186/s13073-016-0389-6) contains supplementary material, which is available to authorized users.

NUTM1-rearranged tumors are defined by the presence of a gene fusion between NUTM1 and various gene partners and typically follow a clinically aggressive disease course with poor outcomes despite conventional multimodality therapy. NUTM1-rearranged tumors display histologic features of a poorly differentiated carcinoma with areas of focal squamous differentiation and typically express the BRD4–NUTM1 fusion gene defining a distinct clinicopathologic entity—NUT carcinoma (NC). NCs with mesenchymal differentiation have rarely been described in the literature. In this report, we describe the characterization of two cases of high-grade spindle cell sarcoma harboring a novel MGA–NUTM1 fusion. Whole-genome sequencing identified the presence of complex rearrangements resulting in a MGA–NUTM1 fusion gene in the absence of other significant somatic mutations. Genetic rearrangement was confirmed by fluorescence in situ hybridization, and expression of the fusion gene product was confirmed by transcriptomic analysis. The fusion protein was predicted to retain nearly the entire protein sequence of both MGA (exons 1–22) and NUTM1 (exons 3–8). Histopathologically, both cases were high-grade spindle cell sarcomas without specific differentiation markers. In contrast to typical cases of NC, these cases were successfully treated with aggressive local control measures (surgery and radiation) and both patients remain alive without disease. These cases describe a new subtype of NUTM1-rearranged tumors warranting expansion of diagnostic testing to evaluate for the presence of MGA–NUTM1 or alternative NUTM1 gene fusions in the diagnostic workup of high-grade spindle cell sarcomas or small round blue cell tumors of ambiguous lineage.

The amplification of the gene MYCN (V-myc myelocytomatosis viral-valeted oncogene, neuroblastoma derived) has been a well-documented indicator for poor prognosis in neuroblastoma, a childhood cancer. Unfortunately, there has been limited success in understanding MYCN functionality in the landscape of neuroblastoma and more importantly, given that MYCN has been deemed "undruggable," the need to potentially illuminate key opportunities that indirectly target MYCN is of great interest. To this end, this work employs an emerging quantitative technique from network science, namely network curvature, to quantify the biological robustness of MYCN and its surrounding neighborhood. In particular, when amplified in Stage IV cancer, MYCN exhibits higher curvature (more robust) than those samples with under expressed MYCN levels. When examining the surrounding neighborhood, the above argument still holds for network curvature, but is lost when only analyzing differential expression - a common technique amongst oncologists and computational/molecular biologists. This finding points to the problem (and possible solution) of drug targeting in the context of complexity and indirect cell signaling affects that have often been obfuscated through traditional techniques.

10552Background: Malignant rhabdoid tumors (MRTs) are rare, aggressive pediatric solid tumors characterized by a 22q11 chromosome rearrangement that inactivates the SMARCB1 gene. Outcomes remain po...

Cancer cells exhibit altered metabolic requirements compared to their normal counterparts. This metabolic reprogramming is mediated by activation of oncogenes such as MYC. Here, we summarize our recent findings demonstrating a metabolic dependency of deregulated MYC on MLXIP-MLX, critical components of the nutrient-sensing arm of the extended MYC transcriptional network.

Advances in risk-adapted cytotoxic chemotherapy, hematopoietic stem cell transplantation and supportive care have contributed to significant improvements in the survival of patients with acute lymphoid leukemia (ALL) and acute myeloid leukemia (AML) over the past few decades. However, despite such progress, a significant percentage of both adult and pediatric leukemia patients become refractory to therapy or relapse and eventually die of disease. Hence, there remains an urgent need for the development of effective and targeted therapies for acute leukemia. Recent genetic profiling of solid and hematologic malignancies has identified epigenetic factors as a critical group of genes recurrently mutated in cancer. Additionally, epigenetic dysregulation has been shown to play an important role in the development, progression and maintenance of leukemia. Therefore, pharmacological inhibition of epigenetic factors represents a potential avenue for the development of novel epigenetic-targeted therapies. In order to identify epigenetic vulnerabilities in leukemia, we developed an epigenetic-focused shRNA screen to search for novel therapeutic targets in human leukemia cell lines both in vitro and in vivo. Specifically, T- and B-ALL cell lines were transduced with a library of shRNAs targeting 449 genes including epigenetic readers, writers and erasers and other chromatin-related factors. Selected cells were subsequently cultured in vitro and concurrently injected into mice. Engraftment of inoculated cells and disease progression were monitored through bioluminescence imaging. Amongst the universe of epigenetic regulatory proteins, the arginine methyl transferase, PRMT5, emerged as the most significantly depleted factor in both in vitro and in vivo screenings. Chemical inhibition of PRMT5 enzymatic activity effectively reduced protein symmetric dimethyl arginine methylation, altered splicing, inhibited cell growth and promoted apoptosis of both ALL and AML cell lines in vitro. In addition, inhibition of PRMT5 in vivo using patient-derived xenograft (PDX) T-ALL mouse models demonstrated diminished tumor growth and prolonged survival. Notably, quantification of peripheral blood cell numbers showed that pharmacologic PRMT5 inhibition was well tolerated and did not affect normal hematopoiesis in mice suggesting that a therapeutic window exists for anticancer drugs targeting PRMT5 in acute leukemia. Overall, our data indicates that pre-mRNA processing and in particular RNA splicing modulation may represent novel therapeutic targets in leukemia. Note: This abstract was not presented at the meeting. Citation Format: Yunyue Wang, Hui Huang, Daniel Diolaiti, Marta Sanchez Martin, Beata Modzelewski, Lianna J. Marks, Allison R. Rainey, Ervin S. Gaviria, Maria L. Sulis, Filemon S. Dela Cruz, Adolfo A. Ferrando, Andrew L. Kung. Identification of arginine methyltransferase PRMT5 as a novel therapeutic target in T-cell acute lymphoblastic leukemia [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 1128. doi:10.1158/1538-7445.AM2017-1128

Background: The proto-oncogene MYC encodes a bHLHLZ transcription factor that binds to its obligate DNA-binding partner, MAX, leading to heterodimer occupancy of E-Box motifs within the genome. MYC is normally mitogen-dependent and tightly regulated where it plays a role in transcriptionally coordinating metabolic sufficiency for cell cycle progression, regulating both proliferation and viability. MYC is deregulated in the majority of human cancers. Importantly, MYC exists within an extended family of related bHLHLZ transcription factors including the MYC antagonizing MAX dimerization (MXD) proteins, MYC paralogs, such as MYCN, as well as MAX-like protein X (MLX), and its nutrient-sensing heterodimerization partners MondoA and carbohydrate response element binding protein (ChREBP). We recently defined a role for the extended MAX/MLX network in the regulation of metabolism and survival of cancer cells specifically with deregulated MYC or MYCN, in which MYC-MAX or MYCN-MAX heterodimers coordinately regulate transcription of important metabolic targets with MondoA-MLX to facilitate tumor cell survival. Targeting a number of these downstream effectors is sufficient to kill cancer cells with deregulated MYC. Methods: Expanding on our initial findings we have used molecular and biochemical approaches to demonstrate non-redundant roles for both MondoA-MLX and ChREBP-MLX in the transcriptional regulation of metabolism and survival of human cancer cells with deregulated MYC, including liver and colon cancer cells. We have also characterized the metabolic and biological phenotypes associated with targeted deletion of Mlx in the mouse, combining in vivo and ex vivo analysis with both whole-body deletion and tissue-specific ablation to unmask novel functions of Mlx and its binding partners. Results: Our results demonstrate that MLX and its binding partners regulate pathways associated with mitochondrial function as well as glucose and lipid metabolism in a non-redundant, cell-type-dependent manner. The presence of MLX stabilizes MondoA and ChREBP, and deletion of Mlx in the mouse leads to decreased levels of MondoA and Chrebp proteins in vivo. Whole-body deletion of Mlx in the mouse is well tolerated but leads to male infertility and teratozoospermia. This sterility phenotype is recapitulated by specific deletion of Mlx in the Sertoli cells of the testis, a major metabolic cell type within the tissue and the central regulator of multiple steps associated with germ cell fate, including production and/or transport of critical metabolites required by the germ cells. This indicates both cell-autonomous (germ cell) and non-cell-autonomous (Sertoli cell) regulation of survival and metabolism in spermatogenesis mediated by Mlx. Importantly, human patients with teratozoospermia exhibit diminished expression of MondoA, MLX and their metabolic targets. Moreover, overexpression of MYCN, MondoA, MLX and their metabolic targets occurs in patients with germ cell-derived tumors, such as seminomas. Conclusions: Transcriptional coordination between the mitogen- and nutrient-responsive arms of the MAX/MLX network exhibits context-dependent lethality. MondoA and Chrebp exhibit non-redundant functions in both cancer cells and normal mouse tissue, and inactivation of Mlx phenocopies loss of both factors, suggesting that targeting Mlx can inhibit both MondoA and Chrebp transcriptional activity and subsequent metabolic output. Metabolic regulation by Mlx impacts normal testicular tissue homeostasis and differentiation in the mouse, and its deregulation is associated with human diseases such as loss of function associated with infertility and gain of function associated with germ cell malignancy. While deletion of Myc, Mycn or Max is embryonic lethal in the mouse, deletion of MondoA, Chrebp or Mlx is well tolerated, indicating that targeting this arm of the extended MYC network, or their critical downstream targets, could be of therapeutic value in metabolic syndrome, fertility and cancer. Citation Format: Patrick A. Carroll, Daniel Diolaiti, Pei-Feng Cheng, Haiwei Gu, Danijel Djukovic, Daniel Raftery, Donald E. Ayer, Charles H. Muller, Robert N. Eisenman. Transcriptional regulation of metabolism by MLX and its binding partners is essential for tumor cell survival and spermatogenesis. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr PR12.