In vivo activity of the second-generation proteasome inhibitor ixazomib against pediatric T-cell acute lymphoblastic leukemia xenografts.

The overall survival rate of patients with T-cell acute lymphoblastic leukemia (T-ALL) is now 90%, although patients with relapsed T-ALL face poor prognosis. The ubiquitin-proteasome system maintains normal protein homeostasis, and aberrations in this pathway are associated with T-ALL. Here we demonstrate the in vitro and in vivo activity of ixazomib, a second-generation orally available, reversible, and selective proteasome inhibitor against pediatric T-ALL cell lines and patient-derived xenografts (PDXs) grown orthotopically in immunodeficient NOD.Cg-PrkdcscidIL2rgtm1Wjl/SzJAusb (NSG) mice. Ixazomib was highly potent in vitro, with half-maximal inhibitory concentration (IC50) values in the low nanomolar range. As a monotherapy, ixazomib significantly extended mouse event-free survival of five out of eight T-ALL PDXs in vivo.

A proteogenomic surfaceome study identifies DLK1 as an immunotherapeutic target in neuroblastoma.

Cancer immunotherapies have produced remarkable results in B-cell malignancies; however, optimal cell surface targets for many solid cancers remain elusive. Here, we present an integrative proteomic, transcriptomic, and epigenomic analysis of tumor specimens along with normal tissues to identify biologically relevant cell surface proteins that can serve as immunotherapeutic targets for neuroblastoma, an often-fatal childhood cancer of the developing nervous system. We apply this approach to human-derived cell lines (N=9) and cell/patient-derived xenograft (N=12) models of neuroblastoma. Plasma membrane-enriched mass spectrometry identified 1,461 cell surface proteins in cell lines and 1,401 in xenograft models, respectively. Additional proteogenomic analyses revealed 60 high-confidence candidate immunotherapeutic targets and we prioritized Delta-like canonical notch ligand 1 (DLK1) for further study. High expression of DLK1 directly correlated with the presence of a super-enhancer spanning the DLK1 locus. Robust cell surface expression of DLK1 was validated by immunofluorescence, flow cytometry, and immunohistochemistry. Short hairpin RNA mediated silencing of DLK1 in neuroblastoma cells resulted in increased cellular differentiation. ADCT-701, a DLK1-targeting antibody-drug conjugate (ADC), showed potent and specific cytotoxicity in DLK1-expressing neuroblastoma xenograft models. Moreover, DLK1 is highly expressed in several adult cancer types, including adrenocortical carcinoma (ACC), pheochromocytoma/paraganglioma (PCPG), hepatoblastoma, and small cell lung cancer (SCLC), suggesting potential clinical benefit beyond neuroblastoma. Taken together, our study demonstrates the utility of comprehensive cancer surfaceome characterization and credentials DLK1 as an immunotherapeutic target. Highlights: Plasma membrane enriched proteomics defines surfaceome of neuroblastomaMulti-omic data integration prioritizes DLK1 as a candidate immunotherapeutic target in neuroblastoma and other cancersDLK1 expression is driven by a super-enhancer DLK1 silencing in neuroblastoma cells results in cellular differentiation ADCT-701, a DLK1-targeting antibody-drug conjugate, shows potent and specific cytotoxicity in DLK1-expressing neuroblastoma preclinical models.

Targeted Investigational Oncology Agents in the NCI-60: A Phenotypic Systems-based Resource.

The NCI-60 human tumor cell line panel has proved to be a useful tool for the global cancer research community in the search for novel chemotherapeutics. The publicly available cell line characterization and compound screening data from the NCI-60 assay have significantly contributed to the understanding of cellular mechanisms targeted by new oncology agents. Signature sensitivity/resistance patterns generated for a given chemotherapeutic agent against the NCI-60 panel have long served as fingerprint presentations that encompass target information and the mechanism of action associated with the tested agent. We report the establishment of a new public NCI-60 resource based on the cell line screening of a large and growing set of 175 FDA-approved oncology drugs (AOD) plus >825 clinical and investigational oncology agents (IOA), representing a diverse set (>250) of therapeutic targets and mechanisms. This data resource is available to the public (https://ioa.cancer.gov) and includes the raw data from the screening of the IOA and AOD collection along with an extensive set of visualization and analysis tools to allow for comparative study of individual test compounds and multiple compound sets.

Multicellular Complex Tumor Spheroid Response to DNA Repair Inhibitors in Combination with DNA-damaging Drugs.

Multicellular spheroids comprised of malignant cells, endothelial cells, and mesenchymal stem cells served as an in vitro model of human solid tumors to investigate the potentiation of DNA-damaging drugs by pharmacologic modulation of DNA repair pathways. The DNA-damaging drugs, topotecan, trabectedin, and temozolomide were combined with varied inhibitors of DNA damage response enzymes including PARP (olaparib or talazoparib), ATM (ataxia telangiectasia mutated; AZD-1390), ATR (ataxia telangiectasia and Rad3-related protein; berzosertib or elimusertib), and DNA-PK (DNA-dependent protein kinase; nedisertib or VX-984). A range of clinically achievable concentrations were tested up to the clinical Cmax, if known. Mechanistically, the types of DNA damage induced by temozolomide, topotecan, and trabectedin are distinct, which was apparent from the response of spheroids to combinations with various DNA repair inhibitors. Although most combinations resulted in additive cytotoxicity, synergistic activity was observed for temozolomide combined with PARP inhibitors as well as combinations of the ATM inhibitor AZD-1390 with either topotecan or trabectedin. These findings might provide guidance for the selection of anticancer agent combinations worthy of further investigation. Significance: Clinical efficacy of DNA-damaging anticancer drugs can be influenced by the DNA damage response in tumor cells. The potentiation of DNA-damaging drugs by pharmacologic modulation of DNA repair pathways was assessed in multicellular tumor spheroids. Although most combinations demonstrated additive cytotoxicity, synergistic cytotoxicity was observed for several drug combinations.

In vivo activity of the dual SYK/FLT3 inhibitor TAK-659 against pediatric acute lymphoblastic leukemia xenografts.

BACKGROUND: While children with acute lymphoblastic leukemia (ALL) experience close to a 90% likelihood of cure, the outcome for certain high-risk pediatric ALL subtypes remains dismal. Spleen tyrosine kinase (SYK) is a prominent cytosolic nonreceptor tyrosine kinase in pediatric B-lineage ALL (B-ALL). Activating mutations or overexpression of Fms-related receptor tyrosine kinase 3 (FLT3) are associated with poor outcome in hematological malignancies. TAK-659 (mivavotinib) is a dual SYK/FLT3 reversible inhibitor, which has been clinically evaluated in several other hematological malignancies. Here, we investigate the in vivo efficacy of TAK-659 against pediatric ALL patient-derived xenografts (PDXs). METHODS: SYK and FLT3 mRNA expression was quantified by RNA-seq. PDX engraftment and drug responses in NSG mice were evaluated by enumerating the proportion of human CD45+ cells (%huCD45+ ) in the peripheral blood. TAK-659 was administered per oral at 60 mg/kg daily for 21 days. Events were defined as %huCD45+ ≥ 25%. In addition, mice were humanely killed to assess leukemia infiltration in the spleen and bone marrow (BM). Drug efficacy was assessed by event-free survival and stringent objective response measures. RESULTS: FLT3 and SYK mRNA expression was significantly higher in B-lineage compared with T-lineage PDXs. TAK-659 was well tolerated and significantly prolonged the time to event in six out of eight PDXs tested. However, only one PDX achieved an objective response. The minimum mean %huCD45+ was significantly reduced in five out of eight PDXs in TAK-659-treated mice compared with vehicle controls. CONCLUSIONS: TAK-659 exhibited low to moderate single-agent in vivo activity against pediatric ALL PDXs representative of diverse subtypes.

In vivo activity of the dual PI3Kδ and PI3Kγ inhibitor duvelisib against pediatric acute lymphoblastic leukemia xenografts.

BACKGROUND: Acute lymphoblastic leukemia (ALL) remains one of the most common causes of cancer-related mortality in children. Phosphoinositide 3-kinases (PI3Ks) are a family of lipid kinases, and aberrations in the PI3K pathway are associated with several hematological malignancies, including ALL. Duvelisib (Copiktra) is an orally available, small molecule dual inhibitor of PI3Kδ and PI3Kγ, that is Food and Drug Administration (FDA) approved for the treatment of relapsed/refractory chronic lymphocytic leukemia and small lymphocytic lymphoma. Here, we report the efficacy of duvelisib against a panel of pediatric ALL patient-derived xenografts (PDXs). PROCEDURES: Thirty PDXs were selected for a single mouse trial based on PI3Kδ (PIK3CD) and PI3Kγ (PIK3CG) expression and mutational status. PDXs were grown orthotopically in NSG (NOD.Cg-Prkdcscid IL2rgtm1Wjl /SzJAusb) mice, and engraftment was evaluated by enumerating the proportion of human versus mouse CD45+ cells (%huCD45+ ) in the peripheral blood. Treatment commenced when the %huCD45+ reached greater than or equal to 1%, and events were predefined as %huCD45+ greater than or equal to 25% or leukemia-related morbidity. Duvelisib was administered per oral (50 mg/kg, twice daily for 28 days). Drug efficacy was assessed by event-free survival and stringent objective response measures. RESULTS: PI3Kδ and PI3Kγ mRNA expression was significantly higher in B-lineage than T-lineage ALL PDXs (p-values <.0001). Duvelisib was well-tolerated and reduced leukemia cells in the peripheral blood in four PDXs, but with only one objective response. There was no obvious relationship between duvelisib efficacy and PI3Kδ or PI3Kγ expression or mutation status, nor was the in vivo response to duvelisib subtype dependent. CONCLUSIONS: Duvelisib demonstrated limited in vivo activity against ALL PDXs.

Evaluation of the pan-class I phosphoinositide 3-kinase (PI3K) inhibitor copanlisib in the Pediatric Preclinical Testing Consortium in vivo models of osteosarcoma.

Copanlisib is a pan-class I phosphoinositide 3-kinase (PI3K) inhibitor, with activity against all four PI3K class I isoforms (PI3Kα, PI3Kß, PI3Kγ, and PI3Kδ). Whole-genome and RNA sequencing data have revealed several PI3K aberrations in osteosarcoma tumor samples. The in vivo anticancer effects of copanlisib were assessed in a panel of six osteosarcoma models. Copanlisib induced prolonged event-free survival in five of six osteosarcoma models; however, all models demonstrated progressive disease suggesting minimal activity. While copanlisib did not result in tumor regression, more data are needed to fully explore the role of the PI3K pathway in the pathogenesis of osteosarcoma.

Integrative epigenomic analyses of small cell lung cancer cells demonstrates the clinical translational relevance of gene body methylation.

DNA methylation is a key regulator of gene expression and a clinical therapeutic predictor. We examined global DNA methylation beyond the generally used promoter areas in human small cell lung cancer (SCLC) and find that gene body methylation is a robust positive predictor of gene expression. Combining promoter and gene body methylation better predicts gene expression than promoter methylation alone including genes involved in the neuroendocrine classification of SCLC and the expression of therapeutically relevant genes including MGMT, SLFN11, and DLL3. Importantly, for super-enhancer (SE) covered genes such as NEUROD1 or MYC, using H3K27ac and NEUROD1, ASCL1, and POU2F3 ChIP-seq data, we show that genic methylation is inversely proportional to expression, thus providing a new approach to identify potential SE regulated genes involved in SCLC pathogenesis. To advance SCLC transitional research, these data are integrated into our web portal (https://discover.nci.nih.gov/SclcCellMinerCDB/) for open and easy access to basic and clinical investigators.

Evaluation of the DLL3-targeting antibody-drug conjugate rovalpituzumab tesirine in preclinical models of neuroblastoma.

Neuroblastomas have neuroendocrine features and often show similar gene expression patterns to small cell lung cancer including high expression of delta-like ligand 3 (DLL3). Here we determine the efficacy of rovalpituzumab tesirine (Rova-T), an antibody drug conjugated (ADC) with a pyrrolobenzodiazepine (PBD) dimer toxin targeting DLL3, in preclinical models of human neuroblastoma. We evaluated DLL3 expression in RNA sequencing data sets and performed immunohistochemistry (IHC) on neuroblastoma patient derived xenograft (PDX), human neuroblastoma primary tumor and normal childhood tissue microarrays (TMAs). We then evaluated the activity of Rova-T against 11 neuroblastoma PDX models using varying doses and schedules and compared anti-tumor activity to expression levels. DLL3 mRNA was differentially overexpressed in neuroblastoma at comparable levels to small cell lung cancer, as well as Wilms and rhabdoid tumors. DLL3 protein was robustly expressed across the neuroblastoma PDX array, but membranous staining was variable. The human neuroblastoma array, however, showed staining in only 44% of cases, whereas no significant staining was observed in the normal childhood tissue array. Rova-T showed a clear dose response effect across the 11 models tested, with a single dose inducing a complete or partial response in 3/11 and stable disease in another 3/11 models. No overt signs of toxicity were observed, and there was no treatment-related mortality. Strong membranous staining was necessary, but not sufficient, for anti-tumor activity. Rova-T has activity in a subset of neuroblastoma preclinical models, but heterogeneous expression in these models and the near absence of expression seen in human tumors suggests that any DLL3-targeting clinical trial should be only performed with a robust companion diagnostic to evaluate DLL3 expression for patient selection.

The State of Preclinical Modeling for Early Phase Cancer Trials Using Molecularly Targeted Agents with Radiation.

Preclinical studies inform and guide the development of novel treatment combination strategies that bridge the laboratory with the clinic. We aimed to evaluate approaches cancer researchers used to justify advancing new combinations of molecularly targeted agents and radiation treatment into early-phase human clinical trials. Unsolicited early phase clinical trial proposals submitted to the National Cancer Institute's Cancer Therapy Evaluation Program between January 2016 and July 2020 were curated to quantify key characteristics and proportion of preclinical data provided by trialists seeking to conduct molecularly targeted agent-radiation combination studies in cancer patients. These data elucidate the current landscape for how the rationale for a molecularly targeted agent-radiation combination therapy is supported by preclinical research and illustrate unique challenges faced in translation at the intersection of precision medicine and radiation oncology.

Trastuzumab Deruxtecan, Antibody-Drug Conjugate Targeting HER2, Is Effective in Pediatric Malignancies: A Report by the Pediatric Preclinical Testing Consortium.

HER2 is expressed in many pediatric solid tumors and is a target for innovative immune therapies including CAR-T cells and antibody-drug conjugates (ADC). We evaluated the preclinical efficacy of trastuzumab deruxtecan (T-DXd, DS-8201a), a humanized monoclonal HER2-targeting antibody conjugated to a topoisomerase 1 inhibitor, DXd, in patient- and cell line-derived xenograft (PDX/CDX) models. HER2 mRNA expression was determined using RNA-seq and protein expression via IHC across multiple pediatric tumor PDX models. Osteosarcoma (OS), malignant rhabdoid tumor (MRT), and Wilms tumor (WT) models with varying HER2 expression were tested using 10 mice per group. Additional histologies such as Ewing sarcoma (EWS), rhabdomyosarcoma (RMS), neuroblastoma (NB), and brain tumors were evaluated using single mouse testing (SMT) experiments. T-DXd or vehicle control was administered intravenously to mice harboring established flank tumors at a dose of 5 mg/kg on day 1. Event-free survival (EFS) and objective response were compared between treatment and control groups. HER2 mRNA expression was observed across histologies, with the highest expression in WT (median = 22 FPKM), followed by MRT, OS, and EWS. The relationship between HER2 protein and mRNA expression was inconsistent. T-DXd significantly prolonged EFS in 6/7 OS, 2/2 MRT, and 3/3 WT PDX models. Complete response (CR) or maintained CR (MCR) were observed for 4/5 WT and MRT models, whereas stable disease was the best response among OS models. SMT experiments also demonstrated activity across multiple solid tumors. Clinical trials assessing the efficacy of a HER2-directed ADC in pediatric patients with HER2-expressing tumors should be considered.