Deeksha Vishwamitra

Modeling Ewing sarcoma tumors in vitro with 3D scaffolds

The pronounced biological influence of the tumor microenvironment on cancer progression and metastasis has gained increased recognition over the past decade, yet most preclinical antineoplastic drug testing is still reliant on conventional 2D cell culture systems. Although monolayer cultures recapitulate some of the phenotypic traits observed clinically, they are limited in their ability to model the full range of microenvironmental cues, such as ones elicited by 3D cell–cell and cell–extracellular matrix interactions. To address these shortcomings, we established an ex vivo 3D Ewing sarcoma model that closely mimics the morphology, growth kinetics, and protein expression profile of human tumors. We observed that Ewing sarcoma cells cultured in porous 3D electrospun poly(ε-caprolactone) scaffolds not only were more resistant to traditional cytotoxic drugs than were cells in 2D monolayer culture but also exhibited remarkable differences in the expression pattern of the insulin-like growth factor-1 receptor/mammalian target of rapamycin pathway. This 3D model of the bone microenvironment may have broad applicability for mechanistic studies of bone sarcomas and exhibits the potential to augment preclinical evaluation of antineoplastic drug candidates for these malignancies.

MicroRNA 96 is a post-transcriptional suppressor of anaplastic lymphoma kinase expression.

Anaplastic lymphoma kinase (ALK) constitutes a part of the oncogenic fusion proteins nucleophosmin-ALK and echinoderm microtubule-associated protein like 4-ALK, which are aberrantly expressed in a subset of T-cell anaplastic large-cell lymphoma and non-small-cell lung cancer, respectively. The expression of mutated, constitutively active ALK also occurs in a subset of neuroblastoma tumors. ALK is believed to play an important role in promoting tumor survival. Nevertheless, the mechanisms underlying the expression of ALK in cancer cells are not completely known. MicroRNA (miR) has been implicated in the regulation of the expression of both oncogenes and tumor suppressor genes. We tested the hypothesis that the expression of ALK could be regulated by miR. Three Internet-based algorithms identified miR-96 to potentially bind with the ALK 3-untranslated region. Notably, miR-96 levels were markedly decreased in ALK-expressing cancer cell lines and primary human tumors compared with their normal cellular and tissue counterparts. Transfection of the cell lines with miR-96 decreased levels of the different forms of ALK protein, without significant effects on ALK mRNA. Furthermore, miR-96 decreased the phosphorylation of ALK target proteins, including Akt, STAT3, JNK, and type I insulin-like growth factor receptor, and it down-regulated JunB. These effects were associated with reduced proliferation, colony formation, and migration of ALK-expressing cancer cells. These data provide novel evidence that decreases in miR-96 could represent a mechanism underlying the aberrant expression of ALK in cancer cells.

Expression and effects of inhibition of type I insulin-like growth factor receptor tyrosine kinase in mantle cell lymphoma

Background Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase induces significant oncogenic effects. Strategies to block IGF-IR signaling are being tested in clinical trials that include patients with aggressive solid malignancies. Mantle cell lymphoma is a B-cell neoplasm with poor prognosis and a tendency to develop resistance. The expression and potential significance of IGF-IR in mantle cell lymphoma are not known. Design and Methods We used reverse transcriptase polymerase chain reaction, quantitative real-time polymerase chain reaction, immunoprecipitation, western blotting, flow cytometry, and immunohistochemistry to analyze the expression of IGF-IR mRNA, and IGF-IR and pIGF-IR proteins in mantle cell lymphoma cell lines and patients’ specimens. Selective and specific blockade of IGF-IR was achieved using picropodophyllin and short-interfering RNA, respectively. Cell viability, apoptosis, cell cycle, cellular morphology, cell proliferation, and target proteins were then analyzed. Results We detected the expression of IGF-IR and pIGF-IR in mantle cell lymphoma cell lines. Notably, IGF-IR molecules/cell were markedly increased in mantle cell lymphoma cell lines compared with human B-lymphocytes. IGF-IR and pIGF-IR were also detected in 78% and 74%, respectively, of 23 primary mantle cell lymphoma specimens. Treatment of serum-deprived mantle cell lymphoma cell lines with IGF-I salvaged these cells from apoptosis. Selective inhibition of IGF-IR by picropodophyllin decreased the viability and proliferation of mantle cell lymphoma cell lines, and induced apoptosis and cell cycle arrest. Selective inhibition of IGF-IR was associated with caspase-3, caspase-8, caspase-9, and PARP cleavage, cytochrome c release, up-regulation of cyclin B1, and down-regulation of cyclin D1, pCdc2, pIRS-1, pAkt, and pJnk. Similar results were obtained by using IGF-IR short-interfering RNA. In addition, picropodophyllin decreased the viability and proliferation of primary mantle cell lymphoma cells that expressed IGF-IR. Conclusions IGF-IR is up-regulated and frequently activated in mantle cell lymphoma. Our data suggest that IGF-IR could be a molecular target for the treatment of mantle cell lymphoma.

NPM-ALK up-regulates iNOS expression through a STAT3/microRNA-26a-dependent mechanism

NPM‐ALK chimeric oncogene is aberrantly expressed in an aggressive subset of T‐cell lymphomas that frequently occurs in children and young adults. The mechanisms underlying the oncogenic effects of NPM‐ALK are not completely elucidated. Inducible nitric oxide synthase (iNOS) promotes the survival and maintains the malignant phenotype of cancer cells by generating NO, a highly active free radical. We tested the hypothesis that iNOS is deregulated in NPM‐ALK+ T‐cell lymphoma and promotes the survival of this lymphoma. In line with this possibility, an iNOS inhibitor and NO scavenger decreased the viability, adhesion, and migration of NPM‐ALK+ T‐cell lymphoma cells, and an NO donor reversed these effects. Moreover, the NO donor salvaged the viability of lymphoma cells treated with ALK inhibitors. In further support of an important role of iNOS, we found iNOS protein to be highly expressed in NPM‐ALK+ T‐cell lymphoma cell lines and in 79% of primary tumours but not in human T lymphocytes. Although expression of iNOS mRNA was identified in NPM‐ALK+ T‐cell lymphoma cell lines and tumours, iNOS mRNA was remarkably elevated in T lymphocytes, suggesting post‐transcriptional regulation. Consistently, we found that miR‐26a contains potential binding sites and interacts with the 3‐UTR of iNOS. In addition, miR‐26a was significantly decreased in NPM‐ALK+ T‐cell lymphoma cell lines and tumours compared with T lymphocytes and reactive lymph nodes. Restoration of miR‐26a in lymphoma cells abrogated iNOS protein expression and decreased NO production and cell viability, adhesion, and migration. Importantly, the effects of miR‐26a were substantially attenuated when the NO donor was simultaneously used to treat lymphoma cells. Our investigation of the mechanisms underlying the decrease in miR‐26a in this lymphoma revealed novel evidence that STAT3, a major downstream substrate of NPM‐ALK tyrosine kinase activity, suppresses MIR26A1 gene expression. Copyright

IGF-1R and mTOR Blockade: Novel Resistance Mechanisms and Synergistic Drug Combinations for Ewing Sarcoma

BACKGROUNDnTherapies cotargeting insulin-like growth factor receptor 1 (IGF-1R) and mammalian target of rapamycin (mTOR) have demonstrated remarkable, albeit short-lived, clinical responses in a subset of Ewing sarcoma (ES) patients. However, the mechanisms of resistance and applicable strategies for overcoming drug resistance to the IGF-1R/mTOR blockade are still undefined.nnnMETHODSnTo elucidate predominant mechanism(s) of acquired drug resistance while identifying synergistic drug combinations that improve clinical efficacy, we generated more than 18 ES cell lines resistant to IGF-1R- or mTOR-targeted therapy. Two small-molecule inhibitors of IGF-1R were chosen, NVP-ADW-742 (IGF-1R-selective) and OSI-906 (a dual IGF-1R/insulin receptor alpha [IR-α] inhibitor). Reverse-phase protein lysate arrays (RPPAs) revealed proteomic changes linked to IGF-1R/mTOR resistance, and selected proteins were validated in cell-based assays, xenografts, and within human clinical samples. All statistical tests were two-sided.nnnRESULTSnNovel mechanisms of resistance (MOR) emerged after dalotuzumab-, NVP-ADW-742-, and OSI-906-based targeting of IGF-1R. MOR to dalotuzumab included upregulation of IRS1, PI3K, and STAT3, as well as p38 MAPK, which was also induced by OSI-906. pEIF4E(Ser209), a key regulator of Cap-dependent translation, was induced in ridaforolimus-resistant ES cell lines. Unique drug combinations targeting IGF-1R and PI3K-alpha or Mnk and mTOR were synergistic in vivo and vitro (P < .001) as assessed respectively by Mantel-Cox and isobologram testing.nnnCONCLUSIONSnWe discovered new druggable targets expressed by chemoresistant ES cells, xenografts, and relapsed human tumors. Joint suppression of these newfound targets, in concert with IGF-1R or mTOR blockade, should improve clinical outcomes.

The transcription factors Ik-1 and MZF1 downregulate IGF-IR expression in NPM-ALK+ T-cell lymphoma

BackgroundThe type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase promotes the survival of an aggressive subtype of T-cell lymphoma by interacting with nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) oncogenic protein. NPM-ALK+ T-cell lymphoma exhibits much higher levels of IGF-IR than normal human T lymphocytes. The mechanisms underlying increased expression of IGF-IR in this lymphoma are not known. We hypothesized that upregulation of IGF-IR could be attributed to previously unrecognized defects that inherently exist in the transcriptional machinery in NPM-ALK+ T-cell lymphoma.Methods and resultsScreening studies showed substantially lower levels of the transcription factors Ikaros isoform 1 (Ik-1) and myeloid zinc finger 1 (MZF1) in NPM-ALK+ T-cell lymphoma cell lines and primary tumor tissues from patients than in human T lymphocytes. A luciferase assay supported that Ik-1 and MZF1 suppress IGF-IR gene promoter. Furthermore, ChIP assay showed that these transcription factors bind specific sites located within the IGF-IR gene promoter. Forced expression of Ik-1 or MZF1 in the lymphoma cells decreased IGF-IR mRNA and protein. This decrease was associated with downregulation of pIGF-IR, and the phosphorylation of its interacting proteins IRS-1, AKT, and NPM-ALK. In addition, overexpression of Ik-1 and MZF1 decreased the viability, proliferation, migration, and anchorage-independent colony formation of the lymphoma cells.ConclusionsOur results provide novel evidence that the aberrant decreases in Ik-1 and MZF1 contribute significantly to the pathogenesis of NPM-ALK+ T-cell lymphoma through the upregulation of IGF-IR expression. These findings could be exploited to devise new strategies to eradicate this lymphoma.

Type I insulin-like growth factor receptor signaling in hematological malignancies

The insulin-like growth factor (IGF) signaling system plays key roles in the establishment and progression of different types of cancer. In agreement with this idea, substantial evidence has shown that the type I IGF receptor (IGF-IR) and its primary ligand IGF-I are important for maintaining the survival of malignant cells of hematopoietic origin. In this review, we discuss current understanding of the role of IGF-IR signaling in cancer with a focus on the hematological neoplasms. We also address the emergence of IGF-IR as a potential therapeutic target for the treatment of different types of cancer including plasma cell myeloma, leukemia, and lymphoma.

SUMOylation Confers Posttranslational Stability on NPM-ALK Oncogenic Protein.

Nucleophosmin-anaplastic lymphoma kinase–expressing (NPM-ALK+) T-cell lymphoma is an aggressive form of cancer that commonly affects children and adolescents. The expression of NPM-ALK chimeric oncogene results from the chromosomal translocation t(2;5)(p23;q35) that causes the fusion of the ALK and NPM genes. This translocation generates the NPM-ALK protein tyrosine kinase that forms the constitutively activated NPM-ALK/NPM-ALK homodimers. In addition, NPM-ALK is structurally associated with wild-type NPM to form NPM/NPM-ALK heterodimers, which can translocate to the nucleus. The mechanisms that sustain the stability of NPM-ALK are not fully understood. SUMOylation is a posttranslational modification that is characterized by the reversible conjugation of small ubiquitin-like modifiers (SUMOs) with target proteins. SUMO competes with ubiquitin for substrate binding and therefore, SUMOylation is believed to protect target proteins from proteasomal degradation. Moreover, SUMOylation contributes to the subcellular distribution of target proteins. Herein, we found that the SUMOylation pathway is deregulated in NPM-ALK+ T-cell lymphoma cell lines and primary lymphoma tumors from patients. We also identified Lys24 and Lys32 within the NPM domain as the sites where NPM-ALK conjugates with SUMO-1 and SUMO-3. Importantly, antagonizing SUMOylation by the SENP1 protease decreased the accumulation of NPM-ALK and suppressed lymphoma cell viability, proliferation, and anchorage-independent colony formation. One possible mechanism for the SENP1-mediated decrease in NPM-ALK levels was the increase in NPM-ALK association with ubiquitin, which facilitates its degradation. Our findings propose a model in which aberrancies in SUMOylation contribute to the pathogenesis of NPM-ALK+ T-cell lymphoma. Unraveling such pathogenic mechanisms may lead to devising novel strategies to eliminate this aggressive neoplasm.

Abstract CT100: First-in-human phase 1 trial of pyrimidine anti-metabolite FF-10502-01 in patients with advanced cancer

Background: FF10502-01 is a synthetic pyrimidine nucleoside analogue that is structurally similar to gemcitabine with a substitution of sulfur for oxygen in the pentose ring. FF-10502-01 showed potent anti-tumor activity in preclinical studies. In Capan-1 and SUIT-2 pancreatic ca xenograft models, FF-502-01 achieved superior tumor growth suppression and survival, respectively, compared to gemcitabine (gem), with less toxicity at clinically relevant doses. In gem-resistant pancreatic PDX models, FF-10502-01, alone and in combination with nab-paclitaxel (nab-pac), had higher efficacy and tolerability than gem/gem+nab-pac. Methods: We conducted a standard 3+3, dose-escalation phase 1 trial with FF-10502-01 to determine safety, maximum tolerated dose (MTD), pharmacokinetics (PK), pharmacodynamics (PD) i.e. FF-10502 incorporation into peripheral blood cellular DNA, and preliminary antitumor activity. Eligibility criteria included age >18 years, solid tumors refractory to standard treatment and adequate organ function. FF-10502-01 was administered IV over 60 minutes on days 1, 8, and 15 every 4 weeks. Planned dosing cohorts included 8, 12, 18, 27, 40, 60, 90, 135 and 200 mg/m2. Supportive medications such as anti-nausea prophylaxis were allowed. Results: 22 patients (pts) have been treated in 6 dose cohorts of 8-60 mg/m2. The median number of cycles received was 2 (range 1 to >12). Pts with the following cancers were enrolled: pancreatic (5 pts); ovarian and cholangiocarcinoma (3 each); parotid gland, prostate, and sarcoma (2 each); and endometrial, squamous cell carcinoma of the head and neck, anal, colon-neuroendocrine, and unknown primary (1 pt each). 11 pts were male and 11 female, median age 63 years (range 21-80), average number of prior cytotoxic therapies 3 (range 1-7) and 9 pts with prior gemcitabine therapy. Common related adverse events were Gr 1/2 nausea (43%), rash (38%), fever (29%), fatigue (19%), and vomiting despite prophylactic ondansetron and dexamethasone (14%). One pt had Gr 3 nausea (a DLT). Cytopenias have been minimal: 2 pts with Gr 1 and 2 anemia; with no neutropenia or thrombocytopenia. No pts have required dose reduction for toxicity. To date, an MTD has not been identified and enrollment continues. A pt with chondroblastic osteogenic sarcoma (18 mg/m2) achieved an unconfirmed partial response (73% decrease) in a maxillary mass but developed progressive disease at another site. 3 currently active patients (1 each of acinar pancreatic, prostate, parotid gland), demonstrated durable stable disease for > 48, 40 and 28 weeks, respectively, at doses ranging from 8 to 27 mg/m2. FF-10502 plasma concentrations increased with dose. Additional PK and PD data will be presented. Conclusions: The pyrimidine nucleoside antimetabolite FF-10502-01 is well tolerated with prophylactic anti-emetics and demonstrated preliminary antitumor activity in heavily pretreated patients. Citation Format: Gerald Steven Falchook, Lindsay Bramwell, Lori Hannan, Deeksha Vishwamitra, Takayuki Yamada, Michele Rosner, David Wages, Thomas Myers, Linda Paradiso, Filip Janku. First-in-human phase 1 trial of pyrimidine anti-metabolite FF-10502-01 in patients with advanced cancer [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 CT100. doi:10.1158/1538-7445.AM2017-CT100

Abstract A36: Decreased levels of the transcription factors Ik-1 and MZF1 contribute to upregulation of IGF-IR expression in NPM-ALK+ T-cell anaplastic large-cell lymphoma.

Type I insulin-like growth factor receptor (IGF-IR) tyrosine kinase is a homodimeric protein that is composed of 2 extracellular α; and 2 transmembranous β; subunits connected by disulfide bonds. IGF-IR belongs to the insulin receptor family whose members exhibit a common structural characteristic in the form of an amino acid motif (YXXXYY) within the activation loop of their respective kinase domains. Ligand stimulation of IGF-IR causes its dimerization, phosphorylation, and the subsequent activation of downstream signaling systems. It has been shown that IGF-IR is aberrantly overexpressed in and contributes to the survival of a variety of human solid tumors and hematological neoplasms and, therefore, it may represent an important therapeutic target. Pathological activation of IGF-IR induces cellular transformation and protection from apoptosis-- prerequisites for the establishment and growth of malignant tumors. Of important note is that the mechanisms underlying increased expression of IGF-IR in cancer cells are not completely understood. For instance, only a few transcription factors are known to bind the IGF-IR gene promoter (15q26.3) and modulate its activity through stimulation or inhibition. These transcription factors include Sp1, WT1, E2F1, STAT1, and EGR-1. Recently, we identified IGF-IR as a major survival molecule that interacts reciprocally with nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) in a subtype of T-cell anaplastic large-cell lymphoma. NPM-ALK+ T-cell lymphoma is an aggressive type of cancer that is frequently encountered in children and young adults. Compared with its expression in normal human T lymphocytes and reactive lymphoid tissues, the expression of IGF-IR mRNA and protein is remarkably upregulated in NPM-ALK+ T-cell lymphoma cell lines and primary human tumors. Similar to other types of cancer, the mechanisms leading to increased expression of IGF-IR in NPM-ALK+ T-cell lymphoma have not been identified. In this study, we hypothesized that increased IGF-IR expression can be explained by transcriptional aberrancies that exist inherently in NPM-ALK+ T-cell lymphoma. To identify transcription factors that may potentially bind to the human IGF-IR gene promoter, 3 web-based transcription factor search algorithms were used: TFSearch (www.cbrc.jp/research/db/TFSEARCH.html), Genomatix (www.genomatix.de), and MATCH (www.gene-regulation.com/pub/programs.html). These algorithms identified the transcription factors Ikaros (Ik) and myeloid zinc finger 1 (MZF1) to potentially bind to IGF-IR gene promoter. Western blotting (WB) studies demonstrated lower levels of Ik-1 and MZF1 in 5 NPM-ALK+ T-cell lymphoma cell lines than in T lymphocytes. WB also showed that Ik-1 and MZF1 are decreased in 85% and 100%, respectively, of 15 ALK+ T-cell lymphoma patient specimens. A luciferase assay supported that Ik-1 and MZF1 regulate the IGF-IR gene promoter. Furthermore, ChIP assay showed that these transcription factors bind specific sites located within the IGF-IR gene promoter. Forced expression of Ik-1 and MZF1 in NPM-ALK+ T-cell lymphoma cells significantly decreased IGF-IR mRNA and protein expression levels. This decrease in IGF-IR was associated with downregulation of pIGF-IR and the phosphorylation of its interacting molecules, including IRS-1, AKT, and NPM-ALK. In addition, overexpression of Ik-1 and MZF1 decreased the viability, proliferation, migration, and anchorage-independent colony formation of NPM-ALK+ T-cell lymphoma cells. Collectively, our findings provide novel evidence that aberrant decreases in the transcription factors Ik-1 and MZF1 play important roles in the pathogenesis of NPM-ALK+ T-cell lymphoma through upregulation of IGF-IR expression. These findings could be exploited to devise more effective strategies to treat this lymphoma. Citation Format: Deeksha Vishwamitra, Choladda V. Curry, Serhan Alkan, Ping Shi, Hesham M. Amin. Decreased levels of the transcription factors Ik-1 and MZF1 contribute to upregulation of IGF-IR expression in NPM-ALK+ T-cell anaplastic large-cell lymphoma. [abstract]. In: Proceedings of the AACR Special Conference on Hematologic Malignancies: Translating Discoveries to Novel Therapies; Sep 20-23, 2014; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(17 Suppl):Abstract nr A36.