Fernanda I. Arnaldez

Targeting the Insulin Growth Factor Receptor 1

The IGF axis is a tightly controlled endocrine system that regulates cell growth and development, known to have an important function in cancer biology. IGF1 and IGF2 can promote cancer growth in a GH-independent manner both through paracrine and autocrine secretion and can also confer resistance to chemotherapy and radiation. Many alterations of this system have been found in neoplasias, including increased expression of ligands and receptors, loss of heterozygosity of the IGF2 locus and increased IGF1R gene copy number. The IGF1 network is an attractive candidate for targeted therapy, including receptor blockade with monoclonal antibodies and small molecule inhibitors of receptor downstream signaling. This article reviews the role of the IGF axis in the initiation and progression of cancer, and describes the recent advances in IGF inhibition as a therapeutic tool.

Loss-of-function screen in rhabdomyosarcoma identifies CRKL-YES as a critical signal for tumor growth

To identify novel signaling pathways necessary for rhabdomyosarcoma (RMS) survival, we performed a loss-of-function screen using an inducible small hairpin RNA (shRNA) library in an alveolar and an embryonal RMS cell line. This screen identified CRKL expression as necessary for growth of alveolar RMS and embryonal RMS both in vitro and in vivo. We also found that CRKL was uniformly highly expressed in both RMS cell lines and tumor tissue. As CRKL is a member of the CRK adapter protein family that contains an SH2 and two SH3 domains and is involved in signal transduction from multiple tyrosine kinase receptors, we evaluated CRKL interaction with multiple tyrosine kinase receptor signaling pathways in RMS cells. While we saw no interaction of CRKL with IGFIR, MET or PI3KAKT/mTOR pathways, we determined that CRKL signaling was associated with SRC family kinase (SFK) signaling, specifically with YES kinase. Inhibition of SFK signaling with dasatinib or another SFK inhibitor, sarcatinib, suppressed RMS cell growth in vitro and in vivo. These data identify CRKL as a novel critical component of RMS growth. This study also demonstrates the use of functional screening to identify a potentially novel therapeutic target and treatment approach for these highly aggressive pediatric cancers.

New Strategies in Ewing Sarcoma: Lost in Translation?

Ewing sarcoma is the second most common pediatric malignant bone tumor. Aggressive multimodality therapy has led to an improvement in outcomes, particularly in patients with localized disease. However, therapy-related toxicities are not trivial, and the prognosis for patients with relapsed and/or metastatic disease continues to be poor. In this article, we outline some of the promising therapies that have the potential to change the Ewing sarcoma therapeutic paradigm in the not-too-distant future: insulin-like growth factor receptor inhibitors, targeting of the fusion protein, epigenetic manipulation, PARP inhibitors, and immunotherapy. Clin Cancer Res; 20(12); 3050–6. ©2014 AACR.

Safety and efficacy of catheter directed thrombolysis in children with deep venous thrombosis.

Aichberger, K.J., Mayerhofer, M., Krauth, M.T., Vales, A., Kondo, R., Derdak, S., Pickl, W.F., Selzer, E., Deininger, M., Druker, B.J., Sillaber, C., Esterbauer, H. & Valent, P. (2005) Low-level expression of proapoptotic Bcl-2-interacting mediator in leukemic cells in patients with chronic myeloid leukemia: role of BCR/ABL, characterization of underlying signaling pathways, and reexpression by novel pharmacologic compounds. Cancer Research, 65, 9436–9444. Aichberger, K.J., Jagani, Z., Song, K., Kutok, M.R., Melet, A., Santos, T., Grassian, A., Ghaffari, S., Wu, C., Ren, R., Yeckes Rodin, H., Miller, K. & Khosravi-Far, R. (2009) Proteasome inhibition causes regression of leukemia and abrogates BCR-ABL-induced evasion of apoptosis in part through regulation of forkhead tumor suppressors. Cancer Research, 69, 6546–6555. Boultwood, J. & Wainscoat, J.S. (2007) Gene silencing by DNA methylation in haematological malignancies. British Journal of Haematology, 138, 3–11. Jelinek, J., Gharibyan, V., Estecio, M.R.H., Kondo, K., He, R., Chung, W., Lu, Y., Zhang, N., Liang, S., Kantarjian, H.M., Cortes, J.E. & Issa, J.P.J. (2011) Aberrant DNA methylation is associated with disease progression, resistance to imatinib and shortnened survival in chronic myelogenous leukemia. PLoS ONE, 6, e22110. Kuribara, R., Honda, H., Matsui, H., Shinjyo, T., Inukai, T., Sugita, K., Nakazawa, S., Hirai, H., Ozawa, K. & Inaba, T. (2004) Roles of Bim in apoptosis of normal and Bcr-Abl-expressing hematopoietic progenitors. Molecular and Cellular Biology, 24, 6172–6183. Kuroda, J., Puthalakath, H., Cragg, M.S., Kelly, P. N., Bouillet, P., Huang, D.C.S., Kimura, S., Ottmann, O.G., Druker, B.J., Villunger, A., Roberts, A.W. & Strasser, A. (2006) Bim and Bad mediate imatinib-induced killing of Bcr/Abl+ leukemic cells, and resistance due to their loss is overcome by a BH3 mimetic. Proceedings National Academy Science USA, 103, 14907–14912. Mancini, M., Veljkovic, N., Leo, E., Aluigi, M., Borsi, E., Galloni, C., Iacobucci, I., Barbieri, E. & Santucci, M.A. (2012) Cytoplasmatic compartimentalization by Bcr-Abl promotes TET2 lossof-function in chronic myeloid leukemia. Journal of Cellular Biochemistry, 113, 2765–2774. Ng, K.P., Hillmer, A.M., Chuah, C.T., Juan, W.C., Ko, T.K., Teo, A.S., Ariyaratne, P.N., Takahashi, N., Sawada, K., Fei, Y., Soh, S., Lee, W.H., Huang, J.W., Allen, Jr, J.C., Woo, X.Y., Nagarajan, N., Kumar, V., Thalamuthu, A., Poh, W.T., Ang, A.L., Mya, H.T., How, G.F., Yang, L.Y., Koh, L.P., Chowbay, B., Chang, C.T., Nadarajan, V.S., Chng, W.J., Than, H., Lim, L.C., Goh, Y.T., Zhang, S., Poh, D., Tan, P., Seet, J.E., Ang, M.K., Chau, N. M., Ng, Q.S., Tan, D.S., Soda, M., Isobe, K., Nöthen, M.M., Wong, T.Y., Shahab, A., Ruan, X., Cacheux-Rataboul, V., Sung, W.K., Tan, E.H., Yatabe, Y., Mano, H., Soo, R.A., Chin, T.M., Lim, W.T., Ruan, Y. & Ong, S.T. (2012) A common BIM deletion polymorphism mediates intrinsic resistance and inferior response to tyrosine kinase inhibitors in cancer. Nature Medicine, 18, 521– 528. San José-Eneriz, E., Agirre, X., Jiménez-Velasco, A., Cordeu, L., Martin, V., Arqueros, V., Garate, L., Fresquet, V., Cervantes, F., Martinez-Climent, J.A., Heiniger, A., Torres, A., Prosper, F. & Roman-Gomez, J. (2009) Epigenetic downregulation of BIM expression is associated with reduced optimal responses to imatinib treatment in chronic myeloid leukemia. European Journal of Cancer, 45, 1877–1889. Youle, R.J. & Strasser, S. (2008) The BCL-2 protein family: opposing activities that mediate cell death. Nature Reviews. Molecular Cell Biology, 9, 47–59.

Matrix screen identifies synergistic combination of PARP inhibitors and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors in Ewing sarcoma

Purpose: Although many cancers are showing remarkable responses to targeted therapies, pediatric sarcomas, including Ewing sarcoma, remain recalcitrant. To broaden the therapeutic landscape, we explored the in vitro response of Ewing sarcoma cell lines against a large collection of investigational and approved drugs to identify candidate combinations. Experimental Design: Drugs displaying activity as single agents were evaluated in combinatorial (matrix) format to identify highly active, synergistic drug combinations, and combinations were subsequently validated in multiple cell lines using various agents from each class. Comprehensive metabolomic and proteomic profiling was performed to better understand the mechanism underlying the synergy. Xenograft experiments were performed to determine efficacy and in vivo mechanism. Results: Several promising candidates emerged, including the combination of small-molecule PARP and nicotinamide phosphoribosyltransferase (NAMPT) inhibitors, a rational combination as NAMPTis block the rate-limiting enzyme in the production of nicotinamide adenine dinucleotide (NAD+), a necessary substrate of PARP. Mechanistic drivers of the synergistic cell killing phenotype of these combined drugs included depletion of NMN and NAD+, diminished PAR activity, increased DNA damage, and apoptosis. Combination PARPis and NAMPTis in vivo resulted in tumor regression, delayed disease progression, and increased survival. Conclusions: These studies highlight the potential of these drugs as a possible therapeutic option in treating patients with Ewing sarcoma. Clin Cancer Res; 23(23); 7301–11. ©2017 AACR.

Abstract IA33: Combination targeted therapy in pediatric sarcomas

Our group, and many others have documented a crucial role for IGFIR signaling in several pediatric sarcomas. These studies, along with the development of fully human IGFIR antibodies led to clinical testing of these blocking antibodies in rhabdomyosarcoma, Ewing9s sarcoma, and osteosarcoma, along with a variety of other sarcomas. In general these single agent Phase 2 studies showed objective responses rates below 20%. Furthermore, even in responding patients, the duration of response is short-lived, typically less than 18 weeks. Thus the majority of patients do not have long term benefit from IGFIR blockade, indicating the presence of innate or acquired resistance to this therapeutic approach. We have been using xenografts of human rhabdomyosarcoma cell lines RD(embryonal) and RH30 (alveolar) to model both innate and acquired resistance to IGFIR blockade. Our xenograft models of rhabdomyosarcoma predicted short duration responses that mimicked the clinical observations, and we have been attempting to use these models to better understand how various signaling pathways in addition to IGFIR may interact and indeed be used as “by-pass” resistance pathways. Our hope is that this knowledge will lead to rational combination targeted therapy that we assume will be necessary for maximal clinical benefit. I will discuss one example of an additional signaling pathway that was activated upon IGFIR blockade, and how this information led to combination targeted therapy that holds therapeutic promise. We recently demonstrated that Src-Family Kinase (SFK) YES is highly expressed and functional in rhabdomyosarcomas. Since SFK signaling has been previously linked to IGF signaling in other tumor types, we evaluated the effect of IGFIR on YES activation. YES is rapidly activated upon IGFIR blockade using either a monoclonal antibody directed against the IGFIR or an IGFIR kinase inhibitor, suggesting that YES activation could play a role in resistance to IGFIR blockade. We next tested dual SFK and IGFIR inhibition and found that combined SFK and IGFIR inhibition led to more potent inhibition of both RD and RH30 rhabdomyosarcoma cell lines and perhaps most importantly led to increased apoptosis. Based on these promising in vitro effects, we evaluated the combination of IGFIR Ab, R1507 plus the SFK inhibitor, dasatinib, in our RD and RH30 xenografts. While both R1507 and dasatinib led to tumor growth inhibition as single agents, mice developed resistance to both single agents within 70-90 days of treatment. In contrast, the mice treated with the combination of R1507 and dasatinib did not develop resistance after 90 days and also showed enhanced tumor growth inhibition. This example highlights the need to better understand how signaling pathways interact so that one may predict consequences of therapeutic intervention using targeted agents and develop combinations that will undoubtedly be necessary to achieve the maximal clinical benefit of agents designed to inhibit specific signaling pathways. Citation Format: Lee J. Helman, Fernanda Arnaldez, Christine Heske, Xiaolin Wan, Choh Yeung. Combination targeted therapy in pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr IA33.

Abstract A46: HSP-90 inhibition in pediatric sarcomas

Hsp90 is a molecular chaperone that regulates post-translational folding, stability, and function of many client proteins, which play critical roles in key signal transduction pathways implicated in cell growth, differentiation, and survival. Several relevant oncogenic proteins are described as clients, such as insulin growth factor receptor 1, AKT/PKB, and src-family kinases. Signaling through these molecules is critical in the biology of rhabdomyosarcoma and Ewing9s sarcoma, which are two of the most common pediatric sarcomas. Novel therapies for these tumors are clearly needed since patients with relapsed and metastatic disease continue to have poor outcomes and multimodality therapy, even when successful, is often accompanied by long-term morbidity. Ganetespib (Synta Pharmaceuticals) is a small molecule Hsp90 inhibitor that binds to the ATP pocket in the N-terminus of Hsp90, leading to down-regulation of Hsp90 client protein levels. We sought to evaluate the preclinical activity of ganetespib in a panel of Ewing9s Sarcoma and rhabdomyosarcoma cell lines. We evaluated in vitro activity of ganetespib in Ewing Sarcoma cell lines (TC71, TC32, EW8, RDES) and in rhabdomyosarcoma (RMS) cell lines (RD, RH30) using kinetic proliferation assays and MTS. In all cases, marked inhibition of cell growth was achieved in a dose-dependent manner with drug IC50s in the low nanomolar range (3-6 nM). We found that ganetespib exposure is associated with both induction of apoptosis and alterations in cell cycle progression. In addition to upregulation of Hsp70, which is considered a biomarker of Hsp90 inhibition, ganetespib treatment was associated with loss of expression of IGF1R-beta as well as downregulation of key signaling pathways including mammalian target of rapamycin (mTOR), pAKT, and Src-family kinases, particularly YES1, a kinase we previously identified as critical in rhabdomyosarcoma biology. Moreover, we observed a cooperative effect between Hsp90 inhibition and IGF1R blockade with R1507, a monoclonal antibody targeting this receptor (Hoffman-La Roche). Furthermore, synergy was seen when ganetespib was combined with the SFK inhibitor dasatinib. While in-vivo validation of these results is ongoing, we conclude that Hsp90 inhibition with ganetespib is an attractive strategy to explore for the therapy of pediatric sarcomas, possibly in combination with other targeted therapies. Citation Format: Fernanda I. Arnaldez, Toby Terwilliger, Choh Yeung, Len Neckers, Lee Helman. HSP-90 inhibition in pediatric sarcomas. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A46.

Abstract 5348: In vivo and in vitro alveolar rhabdomyosarcoma cell growth is affected by TNK2 expression

Rhabdomyosarcoma is the most common pediatric soft tissue tumor. The two major histologic subtypes are embryonal (ERMS) and alveolar rhabdomyosarcoma (ARMS); which have different genetic alterations: 11p15 LOH in ERMS and PAX3 (or PAX7)-FOXO1 translocation in ARMS. Despite utilization of aggressive multimodal therapy, patients with recurrent or metastatic disease remain essentially incurable. This is particularly true in patients with alveolar rhabdomyosarcoma. New therapies are needed to improve outcomes in this disease. To detect genes that are critical for rhabdomyosarcoma cell growth and survival, we performed a loss-of-function shRNA screen in ARMS and ERMS cell lines using a regulated shRNA library that was delivered to RMS cells using retrovirus. We hypothesized that genes whose loss of function significantly influence the growth and survival of ARMS cells with relatively no impact on ERMS cells represent a critical vulnerability or “Achilles’ heel” for ARMS. We sorted our gene list using this approach. In this study, we identified TNK2 (tyrosine kinase, non-receptor 2; also known as ACK1) as a critical gene is ARMS cell survival and as a potential therapeutic target. TNK2 is a non-receptor tyrosine kinase that integrates signals from different membrane receptors, including integrins, EGFR and IGFR. Reported downstream targets include CDC42 and AKT. The TNK2 gene is located at 3q29. TNK2 amplification has been described in prostate, lung and breast cancer and its role in invasion and metastasis has recently been suggested. However, its role in rhabdomyosarcoma has not been previously explored. We initially confirmed that TNK2 is uniformly expressed RMS. In addition, analysis of TNK2 expression in primary tumor samples of RMS using a publicly available database indicated that rhabdomyosarcomas with higher TNK2 expression show lower overall survival. We used a tetracycline-inducible shRNA construct to knock down TNK2 expression in rhabdomyosarcoma cells. TNK2 knock down resulted in significant decrease in cell growth in ARMS. We characterized the specificity of our findings using 5 different shRNA sequences targeting this gene delivered via lentivirus. In addition, we have been able to rescue the effect of an shRNA targeting the 3’UTR of this gene using TNK2 cDNA. We validated our in vitro data in an in vivo model using tumor xenografts. Downstream pathways under investigation include but are not limited to AKT, MAPK and CDC42. These data suggest that TNK2 could represent a therapeutic target in ARMS and highlight the role of loss of function shRNA screens to improve the understanding of tumor biology. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5348. doi:10.1158/1538-7445.AM2011-5348

Use of loss of function RNA interference screen to evaluate TNK-2 as a factor involved in rhabdomyosarcoma cell growth.

9525 Background: Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue tumor. Embryonal (ERMS) and alveolar rhabdomyosarcoma (ARMS) represent different genetic alterations: 11p15 LOH in ERMS and PAX3 (or PAX7)-FOXO1 translocation in ARMS. Patients with metastatic or recurrent disease are essentially non-curable. To find novel therapeutic approaches for these patients, we sought to identify genes necessary for survival of both subtypes of RMS cells using a loss of function shRNA screen. We identified TNK-2 (ACK-1), a non-receptor protein tyrosine kinase known to interact with Cdc-42, a rho family GTPase critical for growth and differentiation, and possibly p130Cas, a scaffold protein involved in regulating growth and development. TNK-2 has not previously been linked to RMS. Methods: RD and RH30 cell lines (ERMS and ARMS respectively) engineered to tightly regulate gene expression via the bacterial tetracycline repressor were infected by a doxycycline regulated retroviral shRNA library. Each shRNA...