Tagraxofusp is a CD123-targeted therapy comprised of a recombinant human interleukin-3 (IL-3) fused to a truncated diphtheria toxin payload. It is the first approved treatment specifically for patients with blastic plasmacytoid dendritic cell neoplasm (BPDCN). To identify biomarkers of response, bone marrow samples from 12 BPDCN patients who were treated with tagraxofusp in the pivotal phase II trial (NCT02113982) were profiled longitudinally using a gene panel and single-cell RNA sequencing. Residual tumor cells following tagraxofusp expressed lower levels of TXNRD1 that would reduce the efficacy of tagraxofusp. In support of this, enzymatic inhibition of TXNRD1 resulted in higher viability of CAL-1 BPDCN cells following tagraxofusp. Responders had either wild-type or missense TET2 mutations, while transient and non-responders had at least one truncating TET2 mutation. Examples of these mutations within the catalytic domain of TET2 were constructed and transduced into cells. Missense and truncating mutants displayed reduced sensitivities to hypomethylating agents and prolonged S-phase stasis. These results suggest that the levels of TXNRD1 interact with intrinsic TET2 truncating mutations within the bone marrow to modulate patient response to tagraxofusp.

The upregulation of B-cell lymphoma 2 (BCL2) and B-cell lymphoma-extra large (BCL-XL), 2 proteins in the BCL2 family of proteins, leads to a disproportional expression of prodeath and prosurvival proteins in favor of leukemia survival, tumorigenesis, and chemoresistance. In different subsets of acute lymphoblastic leukemia (ALL), the proportion of these 2 proteins varies, and their potential as therapeutic targets needs detailed characterization. Here, we investigated BCL2 and BCL-XL, the genes that encode BCL2 and BCL-XL, and their expression differences between B-cell acute lymphoblastic leukemia (B-ALL) and T-cell ALL (T-ALL). We also evaluated the therapeutic potential of targeting these proteins with AZD0466, a novel drug-dendrimer conjugate of the BCL2/BCLXL inhibitor AZD4320, and with BCL2 inhibitor venetoclax (ABT-199). Gene expression and activity analyses supported by the protein expression patterns in ALL cell lines and primary samples demonstrated increased levels of BCL2 expression in B-ALL, with high sensitivity to venetoclax or AZD4320. In contrast, strong BCL-XL expression and sensitivity to dual BCL2/ BCL-XL inhibition was observed specifically in T-ALL samples. This observation was confirmed by BH3 profiling, demonstrating BCL2/BCL-XL codependence in T-ALL and BCL2 dependence in B-ALL. In a mouse model of T-ALL, AZD0466 but not venetoclax reduced leukemic burden and prolonged survival without significant toxicities. Our findings therefore suggest that the novel dual BCL2/BCL-XL inhibitor AZD0466 outperforms single BCL2 inhibition by venetoclax in T-ALL. These findings facilitate the translation of dual BCL2/BCL-XL inhibitors into ALL clinical trials, either alone or in combination with standard-of-care chemotherapy and immune therapies.

Abstract Introduction: Osteosarcoma is a primary malignant bone tumor characterized by the production of spindle cells resulting in immature bone formation. The lung is the most frequent site of metastatic disease and relapse occurs in more than 30 % of patients. Recent results show that relapse is due to a subset of cells with different phenotypic and genetic signatures conferring advantage to drive progression and drug resistance within the intra-tumorally heterogeneous population. Thus, developing a defined in vivo model that can identify the rare genetic subpopulation and recapitulate clonal evolution is crucial. In this study, we used whole exome, barcode and RNA sequencing to characterize the landscape of genomic alterations and also track and identify the clonal subpopulations, clonal and genetic drivers of lungs metastasis. Methods: We injected barcoded PDX (OS17) cells intravenously in ten SCID mice to track the clonal subpopulation of cells that metastasize to the lungs. Metastasized lung nodules were collected following death or euthanasia. All samples collected were snap frozen in liquid nitrogen and stored at -80°C. DNA and RNA was extracted and analyzed by PCR, NGS, WES and RNA sequencing to map clonality, mutational and evolutionary profiles. Results: Four (40%) of the mice developed lung metastases with three mice having multiple metastatic lung nodules. Surgery to remove the metastatic nodules was performed 216 ± 55.5 days following tumor cell injection. Nine metastatic lung nodules from three mice were used for sequencing and analysis. A Shannon-Weaver and Jaccard similarity index show a diversity in clonal architecture between the lung nodules. A total of 15,394 somatic variants was identified including 12,547 single nucleotide variants (SNV), 4054 synonymous, 2662 missense, 28 nonsense mutations and 1639 somatic indels. The SNVs accounted for 81% of all variants followed by CNV 11 % and indels 8% respectively. Synonymous and missense mutations were the most frequently observed in all samples. Copy number loss was two-fold compared to copy number gain across all tumors. Furthermore, the deletions span longer genomic distances compared to amplifications. The mutational spectrum showed that the highest substitutions were C>T/G>A followed by T>C/A>G; the least substitutions were T>A/A>T. Also, we identified a large set of genes with genomic aberrations. These include amplifications in VEGF, RB1, RUNX, PARP4, ICAM3, EGFR, BRCA2, COLA6A1, COLA6A2, CCND3, CDKN2D etc. Conclusions: In the current model, we identified multiple tumorigenic seeding clones and the diverse set of genes mutated in osteosarcoma. Further analysis of the mutational signature resulted in the identification of potential genes that act as drivers in the lung metastatic process. Acknowledgments: Swim Across America, the Foster Foundation and the Barbara Epstein Foundation. Citation Format: Sylvester Jusu, Wendong Zhang, Zhongting Zhang, Xu Zhaohui, Sankaranarayanan Kannan, Yifei Wang, Zhou Xin, Yi Yanhua, Michael Roth, Jonathan Gill, Richard Gorlick. Multiomic sequencing reveals diversity in clonal landscape and genomic alterations in a lung metastatic PDX osteosarcoma model [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 148.

(A) Representative histograms of Annexin V abundance in Ph-like B-ALL patient samples cultured with pate-bound IgG-Fc and and increasing concentrations of DLL1-Fc after 3 days. (B) mRNA expression of Notch target gene HES1 in T-ALL and B-ALL cell lines and patient samples (Pt.B1, Pt.B2) was assessed by real-time qRT-PCR (in triplicate).

U133A microarray (Affymetrix) gene expression data for 172 B-ALL patients, 34 T-ALL patients.

Supplementary Figure from Comprehensive Surfaceome Profiling to Identify and Validate Novel Cell-Surface Targets in Osteosarcoma

Primary Ph-like B-ALL (Pt.B1) was treated with PLK1 inhibitor volasertib (100 nm) for 30 min. Cells were harvested and subjected to single cell time of flight mass cytometry (CyTOF) and analyzed using the Spanning Tree Progression of Density Normalized Events (SPADE) algorithm as previously described (ref). SPADE analysis shows an increased median level expression of the p53 protein and the pro-apoptotic Bax protein in cells subjected to PLK1 inhibition.

Acute myeloid leukemia (AML) is a molecularly heterogenous hematological malignancy, with one of the most common mutations being internal tandem duplication (ITD) of the juxtamembrane domain of the fms-like tyrosine kinase receptor-3 (FLT3). Despite the development of FLT3-directed tyrosine kinase inhibitors (TKI), relapse and resistance are problematic, requiring improved strategies. In both patient samples and cell lines, FLT3-ITD raises levels of reactive oxygen species (ROS) and elicits an antioxidant response which is linked to chemoresistance broadly in AML. NF-E2–related factor 2 (NRF2) is a transcription factor regulating the antioxidant response including heme oxygenase -1 (HO-1), a heat shock protein implicated in AML resistance. Here, we demonstrate that HO-1 is elevated in FLT3-ITD-bearing cells compared to FLT3-wild type (WT). Transient knockdown or inhibitor-based suppression of HO-1 enhances vulnerability to the TKI, quizartinib, in both TKI-resistant and sensitive primary AML and cell line models. NRF2 suppression (genetically or pharmacologically using brusatol) results in decreased HO-1, suggesting that TKI-resistance is dependent on an active NRF2-driven pathway. In AML-patient derived xenograft (PDX) models, brusatol, in combination with daunorubicin, reduces leukemia burden and prolongs survival. Cumulatively, these data encourage further development of brusatol and NRF2 inhibition as components of combination therapy for refractory AML.

An important feature of osteosarcoma is relapse which occurs in more than 30% of patients and metastasis to the lungs. It has been revealed that the metastatic ability and resistance to chemotherapy might be due to the presence of a subset of cells within the intra-tumorally heterogeneous population. However, tracking the rare genetic subpopulations that play a critical role in cancer recurrence, metastasis and resistance and the lack of a defined in vivo model to recapitulate clonal evolution has been a challenge. To further understand clonal dynamics and intra-tumoral heterogeneity a new barcoded PDX (OS17) derived cell line was established by stably transducing the cell line with a lentiviral vector-based system. The highly complex barcode library of ten million enabled labelling each of the cells with a unique molecular identifier and clonally tracking more than one million cells in vitro. Following seventeen passages in vitro and transplant for three generations in vivo, flow cytometry, nested PCR and next generation sequencing showed that the barcoded cells retained the sequence in vitro and in vivo suggesting that it was possible to track cell populations and their clonal lineages both at the genomic DNA and RNA level. The barcoded M17 PDX cells were injected in 25 SCID and NSG mice intratibially to monitor tumor growth and clonally track the cells in both the primary tumors and the lungs. Seventeen (68%) of the mice developed primary tumors. Tumor latency and growth was 61.82 ±34.5 and 51.24 ± 23 days respectively. Seven (35%) of the primary tumor bearing mice had lung metastasis. Limb amputations were performed in 5 mice with 2 surviving the surgery. The results not only show a diversity in clonal subpopulations between the mice injected on different dates but also the primary tumor and matched lung metastatic samples. An in-depth clonal characterization further revealed a high degree of similarity in the subclonal populations between the lung nodules and the primary tumor samples. In vitro, a proportion of the barcodes were very reduced or lost in the early six passages however, these slowly expanded in the late passages suggesting that in a more favorable environment in vivo, these clones can maintain tumorigenic potential. These results suggest a clonal evolutionary dynamic model where pre-existing clones have a fitness for persistence and dissemination. This advantage might include long term tumorigenic and proliferative potential and the ability to grow in different micro-environments. Conclusion: It has been demonstrated that the barcoded PDX models have inter and intra-tumoral heterogeneity of osteosarcoma both in vitro and in vivo and can potentially be used in tracking tumor colonization at the primary and metastatic sites. Using this model will therefore advance our understanding of osteosarcoma lung metastasis which is the critical clinical challenge for these patients. Citation Format: Sylvester Jusu, Sankaranarayanan Kannan, Sahil Seth, Michael D. Peoples, Zhongting Zhang, Wendong Zhang, Zhaohui Xu, Yifei Wang, Xin Zhou, Yizheng Tu, Giuseppe Longo, Michael Roth, Jonathan B. Gill, Richard Gorlick. Comprehensive clonal and molecular profiling of primary tumors and distant lung metastases in an amputation model of osteosarcoma [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 3794.

Abstract Immunoconjugates targeting cell-surface antigens have demonstrated clinical activity to enable regulatory approval in several solid and hematologic malignancies. We hypothesize that a rigorous and comprehensive surfaceome profiling approach to identify osteosarcoma-specific cell-surface antigens can similarly enable development of effective therapeutics in this disease. Herein, we describe an integrated proteomic and transcriptomic surfaceome profiling approach to identify cell-surface proteins that are highly expressed in osteosarcoma but minimally expressed on normal tissues. Using this approach, we identified targets that are highly expressed in osteosarcoma. Three targets, MT1-MMP, CD276, and MRC2, were validated as overexpressed in osteosarcoma. Furthermore, we tested BT1769, an MT1-MMP–targeted Bicycle toxin conjugate, in osteosarcoma patient-derived xenograft models. The results showed that BT1769 had encouraging antitumor activity, high affinity for its target, and a favorable pharmacokinetic profile. This confirms the hypothesis that our approach identifies novel targets with significant therapeutic potential in osteosarcoma.

Introduction: Osteosarcoma (OS) is the most common primary bone tumor in children and adolescents. The cell of origin in OS is still not known, however, OS cells maintain the capacity for tri-lineage differentiation with the ability to differentiate into osteoblasts (OBs), adipocytes and chondroblasts. The impact of surface antigens on OS differentiation capacity has not well studied. ICAM-1 (CD54) is a transmembrane protein that is involved in intercellular adhesion. We previously reported that ICAM-1 is differentially expressed in mesenchymal stem cells (MSCs) compared with mature osteoblasts. The effect of ICAM-1 on MSC and OS differentiation capacity has not been previously assessed. Methods: ICAM-1 RNA expression was measured in MSCs and OS cells lines and protein expression was assessed using western blot and flow cytometry. Human MSCs and OS cells (M1, M17, M31, M33, M36, M39R, M42, M43, M56, and M60) were placed in osteoblastic and adipogenic differentiation media for 21 and 14 days, respectively. Bi-lineage differentiation capacity was assessed by staining the cells with alizarin red and oil red, to determine the extent of OB and adipose differentiation, respectively at 4 times points (day 0, 7, 14, 21) and 3 time points (day 0, 5, 10, 15). Miscrocopic analysis and spectrophotometric assays were utilized to image and quantify the level of differentiation. Results: Flow cytometric analyses identified 3 cell lines with high surface CD54 expression (CD54high =M17, M33, and M39R) and 3 cell lines with low surface CD54 expression (CD54low=M36, M56, and M60). ICAM-1 expression increased significantly throughout osteoblastic differentiation of MSCs. However, when placed in adipogenic media, MSCs expressed decreasing expression of I-CAM-1. CD54high OS cells demonstrated earlier and more robust osteoblastic differentiation compared with CD54low OS cells. CD54low cells demonstrated earlier and more robust adipogenic differentiation. Conclusion and Future Directions: The CD54high expressing OS cells might retain intermittent osteoblast differentiation capacity. OS cells have a higher adipogenic capacity. Current studies are aimed on the validation of inhibitory effects of ICAM-1 towards OS cell proliferation in vitro and local tumor growth and the development of metastatic disease in preclinical patient derived xenograft in vivo models. We believe that further exploration of the role of surface markers in normal and aberrant osteogenic differentiation may lead to the identification of common tumor progenitor cells and new cell surface based therapeutic targets for the treatment of osteosarcoma. Citation Format: Sankaranarayanan Kannan, Yidan Zhang, Yifei Wang, Sylvester Jusu, Zhongting Zhang, Zhang Wendong, Zhaohui Xu, Michaeal Roth, Gill Jonathan Benjamin, Richard Gorlick. ICAM-1(CD54) mediated bi-lineage differentiation in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3110.

Upregulation of BCL-2 family proteins, such as BCL-2 and BCL-XL, leads to imbalanced ratio between pro-death and pro-survival proteins that favors leukemic survival, tumorigenesis, and is an important driver of chemoresistance. B- and T-ALL JAK-STAT signaling pathway is positively correlated with increased BCL-2 family function. Importantly, this pathway plays an important role in the switch between BCL-2 and BCL-XL dependencies in normal developing T-cells. However, recent studies have shown that co-expression of BCL-2 and BCL-XL is linked with higher therapeutic resistance in different types of ALL. In this study, we tested BCL-2 and BCL-XL dependencies among different subtypes of ALL and the potential to revert these using AZD0466, a novel drug-dendrimer conjugate, of a BCL-2/ BCL-XL inhibitor AZD4320 and an FDA-approved BCL-2 inhibitor, ABT-199 (Venetoclax). The analysis of protein expression patterns in ALL cell lines revealed increased levels of BCL-2 in B-ALL cell lines, and of BCL-XL in Ph-like B-ALL and T-ALL cell lines. The analysis of cell viability assay revealed most B-ALL cell lines show sensitivity to both ABT-199 and AZD4320, except NALM6 which lacks the expression of the executioner Bax protein. Ph-like B-ALL and T-ALL cell lines on the other hand are most sensitive to the BCL-2/BCL-XL dual inhibitor, AZD4320. Additionally, Ph-like B-ALL and T-ALL PDX cells responded greater to dual inhibitor. The analysis of BH3 profiling of ALL cell lines demonstrated BCL-2 and BCL-XL codependence in Ph-like B-ALL and T-ALL cell lines while major BCL-2 dependence was seen in B-ALL cell lines. Our pre-clinical studies in T-ALL PDX models showed moderate response to ABT-199 and significantly prolonged survival and decreased leukemic burden mice treated with dual BCL-2/BCL-XL inhibitor AZD0466 (30mg/kg/weekly/IV). Importantly, AZD0466 treated cohorts did not show any significant change in the body weight and platelet counts when compared to control animals. Comparison of pharmacological response to dual inhibitor AZD0466 treatments in B-ALL PDX models demonstrated lower therapeutic efficacy than observed in T-ALL PDX, while in the same T-ALL PDX ABT-199 (100mg/kg/daily/oral) showed reduced efficacy. Further studies including BH3 profiling to measure the dynamic response of mitochondria and determine prior BH3 domain dependencies are in progress and will be reported. Our findings suggest that the novel dual BCL-2/BCL-XL inhibitor AZD0466 outperforms single BCL-2 inhibition by ABT-199 in T-ALL and Ph-like B-ALL. These findings will facilitate translation of AZD0466 dual BCL-2/BCL-XL inhibitor into ALL clinical trials, alone or in combination with standard chemotherapy and monoclonal antibodies. Citation Format: Sankaranarayanan Kannan, Sanaz Ghotbaldini, Julia E. Wells, Qi Zhang, Srividya Balachander, Justin Cidado, Marina Konopleva. Anti-leukemic activity of BCL-2/BCL-XL dual inhibitor - AZD0466 in T-acute lymphoblastic leukemia preclinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3075.

Osteosarcoma (OS) is the most common primary malignant bone tumor in pediatric patients. Current developments in targeted therapeutic strategies show potential clinical implications in cancer treatments. However, immunotherapies such as antibody-drug conjugates (ADCs) or CAR-T cell therapy have not been sufficiently studied in OS because of the lack of tumor-specific target. In this study, we developed a multi-step RNA-seq based pipeline. First, we did RNA sequencing for our OS PDX models and 17 patient-derived OS cell lines. The results are then pooled with the RNQ-seq data from 111 OS patient via the Therapeutically Applicable Research to Generate Effective Treatments project (TARGET). All these tumor data are subsequently compared with normal tissue RNA-sequencing data from NIH Genotype-Tissue Expression (GTEx) database. The significantly differentially expressed genes (log fold change tumor versus normal >1 for each tissue, p < 0.01) are selected. We further filtered this gene list by cell surface protein prediction based on Gene Ontology, the TransMembrane prediction using hidden Markov models (TMHMM), and glycosylphosphatidylinositol (GPI)-anchored protein annotations. Based on the transcriptomic result, ranks of differentially expressed genes were generated. CD276, LCLAT1, and OR8B12 are the surface markers with high ranks. To validate the transcriptomic results, we prepared surface protein extraction of the PDX models and patient-derived OS cell lines. We are profiling these surface proteins by proteomic mass spectrometry. Further validation for the expression level and location of these markers are underway. Our current finding is not previously reported in OS. The results warrant further development of ADC and car-T cell therapy.Note: This abstract was not presented at the meeting.Citation Format: Yifei Wang, Zhongting Zhang, Sankaranarayanan Kannan, Wendong Zhang, Michael Roth, Jonathan B. Gill, Zhaohui Xu, Xiangjun Tian, Jing Wang, Richard Gorlick. Surfaceome profiling in osteosarcoma: Identification of the candidate immunotherapeutic target [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2866.

Notch signaling pathway is a mediator of cell differentiation and is critical for normal bone development. Four functional Notch ligands DLL1 and DLL4, and JAG1 and JAG2 showed various levels of affinity for Notch1-4 receptors. Dose dependent Notch signaling activation or blocking is attributed to anchored or soluble form of Notch ligands binding. Interestingly, Notch pathway is shown to play a dual role, either oncogenic or tumor suppressive, depending on signal dose and tissue-context. To understand Notch signaling activities in osteosarcoma (OS), we first studied the cell surface levels of Notch receptor expression in OS cell lines and patient derived xenograft (pdx) models. Cytometry based screening of NOTCH1-4 receptors showed NOTCH2 as a predominant surface expressed protein in most of the samples. Interestingly, the canonical Notch target genes HES1 and DTX1 were higher (~5-fold) in OS cells compared to mesenchymal cells (MSC). Subsequently, we evaluated selected Notch pathway gene expression in 48 patients with recurrent/metastatic OS by RNAseq analysis. High expression of Notch pathway-related genes was seen in a subset of patients. NOTCH2 expression was comparable between primary and metastatic OS specimens, highest as compared to other Notch receptors and was significantly higher than normal tissue. Thus we aimed to elucidate soluble Notch ligands mediated blocking of Notch signaling using immunoglobulin-G bound DLL-1, -3, and -4 and JAG-1, and -2 in in vitro studies using OS cells. Soluble DLL1-Fc treatment increased OS cell death as compared to the other Notch ligands as well as control-Fc cells. Addition of soluble DLL1 resulted in the decreased expression of Notch downstream genes such as HES1, DTX1 and HEY1. Taken together, these findings identify soluble DLL1 as a potent Notch pathway inhibitor in OS in vitro and support further exploration of NOTCH2 as a potential therapeutic target in OS. Citation Format: Sankaranarayanan Kannan, John A. Livingston, Michael Roth, Jonathan Benjamin, Yifei Wang, Zhongting Zhang, Wendong Zhang, Chia-Chin Wu, Hannah Beird, Andrew Futreal, Richard Gorlick. Notch2 inhibition as a therapeutic intervention in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1291.

Abstract In B-cell acute lymphoblastic leukemia (B-ALL), activation of Notch signaling leads to cell-cycle arrest and apoptosis. We aimed to harness knowledge acquired by understanding a mechanism of Notch-induced cell death to elucidate a therapeutically viable target in B-ALL. To this end, we identified that Notch activation suppresses Polo-like kinase 1 (PLK1) in a B-ALL–specific manner. We identified that PLK1 is expressed in all subsets of B-ALL and is highest in Philadelphia-like (Ph-like) ALL, a high-risk subtype of disease. We biochemically delineated a mechanism of Notch-induced PLK1 downregulation that elucidated stark regulation of p53 in this setting. Our findings identified a novel posttranslational cascade initiated by Notch in which CHFR was activated via PARP1-mediated PARylation, resulting in ubiquitination and degradation of PLK1. This led to hypophosphorylation of MDM2Ser260, culminating in p53 stabilization and upregulation of BAX. shRNA knockdown or pharmacologic inhibition of PLK1 using BI2536 or BI6727 (volasertib) in B-ALL cell lines and patient samples led to p53 stabilization and cell death. These effects were seen in primary human B-ALL samples in vitro and in patient-derived xenograft models in vivo. These results highlight PLK1 as a viable therapeutic target in B-ALL. Efficacy of clinically relevant PLK1 inhibitors in B-ALL patient-derived xenograft mouse models suggests that use of these agents may be tailored as an additional therapeutic strategy in future clinical studies.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and young adults. Neo-adjuvant chemotherapy combined with wide resection increased the 5-year survival rate to 60%-70%. However, treatment and outcome of OS have not changed in several decades. OS is histologically defined by the presence of malignant osteoid, suggesting it could be derived from anywhere in the osteogenic lineage between mesenchymal stem cells (MSC) and mature osteoblasts, but the cell of origin of OS is still undefined. Tracing the cells from which most cancers formed are characterized by its multipotent and/or unipotent stem cell nature. Studies on the cellular origin of tumors from phenotypic lineage-tracking and its derived progeny can reveal tissue-specific tumor-initiating cells. Investigating cell of origin of OS and osteogenic differentiation using developmentally relevant model using MSC to OB differentiation is promising. Here we developed a high-dimensional analytic pipeline using single-cell mass cytometry (CyTOF) and RNA sequencing to discover the unrecognized progenitor-osteoblast populations in the osteogenic lineage. For the data analysis, the population clustering using Flowsom and ConsensusClusterPlus on arcsinh transformed FCS data combined with t-SNE plot visualization shows that combination of CD49f, CD95, CD56, CD117 expression enables the identification and future isolation of the progenitor populations. Also, when comparing OS with the populations from MSC to OB, there are progenitor -osteoblast / mature osteoblast-like populations in tumor cells, but no MSC-like component is found. The expression level of CD56 and CD117 are significantly higher in OS compare to MSC and OB, which warrants future investigation of new therapeutic strategy. Isolation of the progenitor-osteoblast cells, gene expression comparison between OS and these progenitors, validation of their differentiation potential and tumor-initiating capacity is still underway. In this study, we developed a CyTOF based pipeline to discover the progenitor populations in the osteogenic lineage. By comparison of the OS and these progenitor cells, we may be able to unveil the cell of origin and new targeted treatment strategy of OS. Citation Format: Yifei Wang, Sankaranarayanan Kannan, Zhongting Zhang, Wendong Zhang, Michael Roth, Jonathan B. Gill, Zhaohui Xu, Xiangjun Tian, Jing Wang, Richard Gorlick. Mapping the osteogenic lineage and identification of the cell of origin of osteosarcoma through high-dimensional analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3655.

Transcription factor NF-E2-related factor 2 (Nrf2) regulates antioxidant response element (ARE)-mediated expression and coordinated induction of chemoprotective proteins in response to chemical stress. In this report, we investigated Nrf2 response to low and high dose UVB irradiation. Low dose (7.5 J/m(2)) UVB exposure of mouse hepatoma, mouse keratinocyte, and human skin fibroblast cells led to the nuclear accumulation of Nrf2 and up-regulation of ARE-mediated gene expression. On the contrary, and intriguingly, high dose (20 J/m(2)) UVB exposure of cells led to the nuclear exclusion of Nrf2 and down-regulation of chemoprotective gene expression with possible implications in UVB carcinogenesis. We investigated the mechanism by which high dose UVB induced the nuclear exclusion of Nrf2. Prior treatment with nuclear export inhibitor, leptomycin B, abrogated the UVB-induced nuclear exclusion of Nrf2, indicating that the decrease of Nrf2 in the nucleus was due to the nuclear export of Nrf2. High dose UVB increased the phosphorylation of Nrf2Y568 which stimulated the nuclear export of Nrf2. Mutation of Nrf2Y568 to phenylalanine and src kinase inhibitor PP2 abrogated/reduced the UVB-induced phosphorylation of Nrf2Y568 and nuclear exclusion of Nrf2. Transfection with src family member Fyn small interfering RNA resulted in the nuclear accumulation of Nrf2 and an increase in the expression and UVB induction of ARE-mediated gene expression. UVB exposure also induced the nuclear localization of Fyn. These results suggest that high dose UVB induced the activation/nuclear localization of Fyn which led to increased phosphorylation of Nrf2Y568 and enhanced nuclear export of Nrf2. This resulted in nuclear exclusion of Nrf2 and down-regulation of ARE-mediated chemoprotective gene expression.

Tropomyosin-related kinase A (TRKA) translocations have oncogenic potential and have been found in rare cases of solid tumors.Accumulating evidence indicates that TRKA and its ligand, nerve growth factor (NGF), may play a role in normal hematopoiesis and may be deregulated in leukemogenesis.Here, we report a comprehensive evaluation of TRKA signaling in normal and leukemic cells.TRKA expression is highest in common myeloid progenitors and is overexpressed in core binding factor and megakaryocytic leukemias, especially Down syndrome-related AML.Importantly, NGF can rescue GM-CSF dependent TF-1 AML cells, but does not drive proliferation in other TRKA-expressing lines.Although TRKA expression is heterogeneous between and within AML samples, NGF stimulation broadly induces ERK signaling, demonstrating the functional ability of AML cells to respond to NGF/ TRKA signaling.However, neither shRNA knockdown nor pharmacologic inhibition have significant anti-proliferative effects on human AML cells in vitro and in vivo.Thus, despite functional NGF/TRKA signaling, the importance of TRKA in AML remains unclear.

Selectins and their ligands have been implicated in tumor growth and progression in carcinomas, but their role in neuroblastoma has not been systematically examined. In the current study we evaluated L-, P- and E-selectin binding to neuroblastoma cells and the expression of some of their known ligands, namely CD44, CD24 and P-selectin glycoprotein ligand-1 (PSGL-1). Genetic loss of PSGL-1 or CD24 and pharmacological inhibition of P-selectin reduced P-selectin binding to neuroblastoma cells in vitro. Targeting P-selectin using specific antibodies promoted a significant reduction in the growth of neuroblastoma tumors in vivo. In mechanistic studies binding of P-selectin to neuroblastoma cells activated Src and several other pro-survival kinases such as ERK1, AKT, FAK and p38. Interestingly, comparative mass single cell cytometry (CyTOF) analyses revealed considerable intra- and inter-cell line heterogeneity with respect to response to P-selectin binding. Additionally, the downstream response to all selectins showed general similarity. Our findings reported here not only provide pre-clinical evidence in support of therapeutic targeting of P-selectin, but also highlight the heterogeneity in response of tumor cells to P-selectin binding. These observations provide the basis for combining P-selectin inhibition with other targeted therapies for neuroblastoma.

Background: Chronic oxidative stress (COS) is the consequence of prolonged elevation of reactive oxygen species (ROS). In the context of AML, COS leads to sustained induction of antioxidant pathways to compensate for COS. The presence of abundant antioxidants may contribute to chemoresistance in AML. NRF2 is a master regulator of the antioxidant response, is induced by oxidative stress and functions as a cyto-protective mechanism in acute oxidative stress. In AML, sustained NRF2-activation can be viewed as a pathological maladaptation as it leads to elevated reductive metabolite formation, termed reductive stress (RS). In this study we describe a novel link between NRF2-induced reductive stress and the Notch pathway in AML.