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Purpose: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS-754807, but this combination was not pursued clinically due to intolerability in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor, which would be tolerated in murine models and effective in both cell line and patient-derived xenograft models of RAS-mutant FN RMS. Experimental Design: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a mAb with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of trametinib/ganitumab was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. Results: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in four of six models of RAS-mutant RMS. The combination had little effect on body weight and did not produce thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the phosphorylation of AKT induced by MEK inhibition alone. Therapeutic response to the combination was observed in models without a mutation in the PI3K/PTEN axis. Conclusions: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. Our data also show that the trametinib/ganitumab combination likely has a favorable tolerability profile. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS.

Rhabdomyosarcoma (RMS), a cancer characterized by the defective differentiation of skeletal muscle precursor cells, is the most common pediatric soft tissue sarcoma. Despite significant efforts to characterize the transcriptomic and genetic abnormalities of the disease, the prognosis for patients with advanced or refractory disease has remained unchanged for more than four decades. Preclinically, studies have been hampered by insufficient animal models. For example, the hindlimb orthotopic model, an injection into the gastrocnemius muscle of immunocompromised mice, produces highly encapsulated, non-invasive, non-metastatic tumors. While this model has shown a reasonable recapitulation of tumor proliferation response, investigators using this model are unable to assess the effect of their investigational treatment on local invasion or distant metastasis. Moreover, transgenic mouse models of RMS, while commonly metastatic, diverge significantly from the transcriptomic signature of human tumors. With these considerations, we hypothesized that an alternative orthotopic site, the tongue, may provide a more representative xenograft model of RMS. In a proof of principal experiment, the fusion negative RMS (FN-RMS) cell line RD was injected into the skeletal muscle of the tongue, a lingual intramuscular (LIM) injection. Tongue tumors establish in approximately 60-80% of mice injected within approximately 40 days. By histopathology, the primary tumors show evidence of local invasion. Interestingly, approximately 60% of these tumors also spread to local cervical lymph nodes, a process not observed in hindlimb orthotopic models or subcutaneous models. We expanded our investigation to include additional FN-RMS cell lines: RMS-YM, SMS-CTR, and a MEK inhibitor resistant cell line CTR-557. Mice injected with RMS-YM cells established tumors in at least 60% of mice by 75 days post injection. Additionally, enlarged local and distant lymph nodes are observed in 50% of these mice. Intriguingly, mice injected with SMS-CTR or its MEK inhibitor resistant derivative, CTR-557, frequently have tumors in both local and distant lymph nodes without evidence of a primary tongue tumor. One mouse in each group also had pulmonary metastases. Taken together, this model not only demonstrates the first orthotopic xenograft model of spontaneous metastasis for FN-RMS, it also mimics the spread of disease that is observed clinically in RMS originating in the head and neck. Ongoing and future studies include 1) generating highly metastatic human FN-RMS cell lines; 2) using spatial transcriptomics to compare the tumor microenvironment of non-metastatic hindlimb tumors and invasive and/or metastatic lingual tumors; and 3) identifying drivers of FN-RMS dissemination through transcriptomic, genome accessibility, and phospho-proteomic methods. Citation Format: Katie E. Hebron, Kristine Isanogle, Amy James, Simone Difilippiantonio, Marielle E. Yohe. A novel orthotopic xenograft model of spontaneous metastasis for rhabdomyosarcoma using lingual intramuscular injection. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3537.

Background: PAX-fusion negative rhabdomyosarcoma (FN RMS) is driven by alterations in the RAS/MAP kinase pathway and is partially responsive to MEK inhibition. Overexpression of IGF1R and its ligands is also observed in FN RMS. Preclinical and clinical studies have suggested that IGF1R is itself an important target in FN RMS. Our previous studies revealed preclinical efficacy of the MEK1/2 inhibitor, trametinib, and an IGF1R inhibitor, BMS75807, but this combination was not pursued clinically due to excessive toxicity in preclinical murine models. Here, we sought to identify a combination of an MEK1/2 inhibitor and IGF1R inhibitor that would be better tolerated in murine models and effective in both cell line and patient derived xenograft models of RAS-mutant FN RMS. Methods: Using proliferation and apoptosis assays, we studied the factorial effects of trametinib and ganitumab (AMG 479), a monoclonal antibody with specificity for human and murine IGF1R, in a panel of RAS-mutant FN RMS cell lines. The molecular mechanism of the observed synergy was determined using conventional and capillary immunoassays. The efficacy and tolerability of the combination was assessed using a panel of RAS-mutated cell-line and patient-derived RMS xenograft models. Results: Treatment with trametinib and ganitumab resulted in synergistic cellular growth inhibition in all cell lines tested and inhibition of tumor growth in five out of six models of RAS-mutant RMS. Evidence suggests that the combination had little effect on body weight loss, thrombocytopenia, neutropenia, or hyperinsulinemia in tumor-bearing SCID beige mice. Mechanistically, ganitumab treatment prevented the AKT phosphorylation that is induced by MEK inhibition alone. Therapeutic response to the combination was observed in models with an intact PI3K/PTEN axis. Conclusions: We demonstrate that combined trametinib and ganitumab is effective in a genomically diverse panel of RAS-mutated FN RMS preclinical models. The trametinib/ganitumab combination also likely has an improved tolerability profile compared to other IGF1R/MEK inhibitor combinations. These data support testing this combination in a phase I/II clinical trial for pediatric patients with relapsed or refractory RAS-mutated FN RMS. Citation Format: Marielle E. Yohe, Katie E. Hebron, Xiaolin Wan, Jacob S. Roth, David J. Liewehr, Nancy E. Sealover, Stacey Stauffer, Olivia Feehan-Nelson, Wenyue Sun, Kristine A. Isanogle, Christina M. Robinson, Amy James, Parirokh Awasthi, Priya Shankarappa, Xiaoling Liu, Haiyan Lei, Donna Butcher, Roberta Smith, Elijah F. Edmonson, Jin-Qui Chen, Noemi Kedei, Cody S. Peer, Jack F. Shern, W. Douglas Figg, Lu Chen, Matthew D. Hall, Simone Difillipantonio, Frederic G. Barr, Robert L. Kortum, Angelina V. Vaseva, Javed Khan. Therapeutic efficacy of trametinib and ganitumab in RAS-mutated rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr IA023.

Despite aggressive, multimodal therapies, the prognosis of patients with refractory or recurrent rhabdomyosarcoma (RMS) has not improved in four decades. RMS is thought to arise due to defective differentiation in skeletal muscle precursor cells. Differentiation-inducing therapy may improve outcomes for RMS patients with advanced disease. In RAS-mutant PAX fusion-negative RMS (FN-FMS), targeting ERK1/2 activation through MEK1/2 inhibition (MEKi) induces differentiation, slows tumor growth, and extends survival in preclinical studies. However, the duration of therapeutic response is short lived. Identifying additional targets for differentiation therapy is necessary. We propose that ASAP1, an Arf GTPase-activating protein (Arf GAP) highly expressed in FN-RMS and implicated in breast and colorectal cancer progression as well as osteogenic and retinal endothelium differentiation, contributes to FN-RMS differentiation. We find that knockdown (KD) of ASAP1 inhibits differentiation in myoblasts and FN-RMS cells, and its overexpression enhances differentiation. Moreover, myogenic differentiation-associated genes are not enriched upon loss of ASAP1. We discover that KD of ASAP1 homologs, ASAP2 and ASAP3, also blocks differentiation. However, loss of a paralogous Arf GAP, ARAP1, does not, indicating that ASAP regulates differentiation through a mechanism not explained by GAP activity alone. Interestingly, KD of Arf1 or Arf5, small GTPases inactivated by ASAP, also blocks differentiation of FN-RMS, suggesting a novel relationship between Arf and ASAP. Using RNAseq, qPCR, and immunoblotting techniques, we show that loss of ASAP blocks myogenic transcription factor expression. To determine the mechanism by which ASAP1 regulates myogenic transcription factor expression, we investigate the PI3K/AKT, MAPK, and Hippo pathways, which are known to be regulated by ASAP1. While the PI3K/AKT and MAPK pathways are unaffected, downstream components of the Hippo pathway are modulated by ASAP1 KD in FN-RMS cells treated with MEKi. YAP and TAZ are transcriptional coactivators that promote proliferation. Upon Hippo pathway activation, YAP/TAZ activity is blocked by phosphorylation at a nuclear exclusion site. Using cell fractionation and immunoblotting techniques, we find that induction of differentiation by MEKi increases TAZ phosphorylation, excluding it from the nucleus and blocking pro-proliferative transcription. Further, upon ASAP1 KD, TAZ phosphorylation is blocked, restoring nuclear localization, and inhibiting MEKi-induced differentiation. In conclusion, we discover that ASAP1 regulates MEKi-induced differentiation of FN-RMS cells by modulating TAZ localization and supports targeting the YAP pathway as a strategy for FN-RMS differentiation therapy. Our work also identifies ASAP1 as a potential effector of Arf1 activity, a novel interaction of these two proteins. The results described herein provide a deeper understanding of differentiation in FN-RMS and establish the groundwork for advancing differentiation therapy in FN-RMS. Citation Format: Katie E. Hebron, Olivia Feehan-Nelson, Angela Kim, Xiaoying Jian, Sofia A Girald, Paul Randazzo, Marielle E. Yohe. ASAP1 regulates myogenic differentiation in rhabdomyosarcoma by modulating YAP localization [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A003.

Normal physiology relies on the organization of transmembrane proteins by molecular scaffolds, such as tetraspanins. Oncogenesis frequently involves changes in their organization or expression. The tetraspanin CD151 is thought to contribute to cancer progression through direct interaction with the laminin-binding integrins α3β1 and α6β1. However, this interaction cannot explain the ability of CD151 to control migration in the absence of these integrins or on non-laminin substrates. We demonstrate that CD151 can regulate tumor cell migration without direct integrin binding and that integrin-free CD151 (CD151free) correlates clinically with tumor progression and metastasis. Clustering CD151free through its integrin-binding domain promotes accumulation in areas of cell–cell contact, leading to enhanced adhesion and inhibition of tumor cell motility in vitro and in vivo. CD151free clustering is a strong regulator of motility even in the absence of α3 expression but requires PKCα, suggesting that CD151 can control migration independent of its integrin associations. The histologic detection of CD151free in prostate cancer correlates with poor patient outcome. When CD151free is present, patients are more likely to recur after radical prostatectomy and progression to metastatic disease is accelerated. Multivariable analysis identifies CD151free as an independent predictor of survival. Moreover, the detection of CD151free can stratify survival among patients with elevated prostate-specific antigen levels. Cumulatively, these studies demonstrate that a subpopulation of CD151 exists on the surface of tumor cells that can regulate migration independent of its integrin partner. The clinical correlation of CD151free with prostate cancer progression suggests that it may contribute to the disease and predict cancer progression. Cancer Res; 74(1); 173–87. ©2013 AACR.

Heterogeneity in the tumor microenvironment is dictated in part by context-dependent changes in cell-cell interactions. Subpopulations of tumor cells can responding to local micro-environmental changes in a manner that distinguishes them biochemically as well as behaviorally from the rest of the tumor. We identified such a phenomenon in the proteolytic shedding of the cell adhesion molecule ALCAM (Activated Leukocyte Cell Adhesion Molecule). ALCAM controls cell adhesion in a variety of cell types including T-cells, epithelial cells, neurons and endothelial cell. Although ALCAM mRNA is detected in all epithelial tissues and the tumors they give rise to, the detection of cell-surface ALCAM is highly variable, being strong in some regions of the tumor while completely absent in adjacent tumor cells. Through molecular analysis of cell-surface ALCAM we determined that this adhesion molecule is shed from the surface by ADAM17 in response to cytokines from the local milieu. Although ALCAM shedding is common in cancer, the loss of ALCAM greatly diminishes metastasis, thereby suggesting that ALCAM remains necessary for tumor progression. Indeed, ALCAM shedding supports a dynamic regulation of cell adhesion which, in turn, supports tumor cell dissemination. Considering that the proteolytic processing of ALCAM is functionally important in tumor progression, the detection of ALCAM shedding should be a molecular indicator of disease progression. Indeed, through analysis of retrospective patient cohorts, we demonstrated ALCAM shedding to be a prognostic indicator of disease progression in cancers of the colon, kidney and bladder. Our observations demonstrate that spatial and context-dependent heterogeneity can be accomplished through post-translational proteolysis of cell adhesion molecules. This process enables tumor cells to respond dynamically to the local milieu and disseminate to a more hospitable environment. Identifying this proteolysis in cancer patients through the detection of ALCAM shedding can predict metastatic progression and long-term patient outcome. Citation Format: Amanda G. Hansen, Katie Hebron, Trenis D. Palmer, Shanna Arnold, Andries Zijlstra. Proteolytic shedding of the cell adhesion molecule ALCAM generates a spatial and context-dependent heterogeneity important for metastasis.. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr B65. doi:10.1158/1538-7445.CHTME14-B65

Fluorescent or luminescent imaging of tumors in animal models is a critical element in dynamic and longitudinal monitoring of tumor burden, tumor growth, and metastatic dissemination. Currently, most imaging strategies are limited to one or two different colors per animal. To improve utility of current imaging technologies, we have developed a library of fluorescent and luminescent tracking vectors designed to expand the color dimensions possible within a single model through a multi-label approach to fluorescent barcoding. The library was built using Multisite Gateway® Cloning technology, which offers a fast and simple recombination-based cloning approach that allows for efficient expression of several genes driven by one promoter on a single vector backbone. Our lentiviral-based vectors express firefly luciferase and one of seven spectrally unique fluorescent proteins covering the entire fluorescent protein color palette from blue to far-red. We have stably labeled bone-derived MDA-MB-231 cells with the vectors by viral transduction. Using these individually colored cell lines, we have demonstrated that the seven FPs are uniquely identifiable by spectral un-mixing with the Maestro Q Imaging System and by flow cytometry. The combination of luciferase and fluorescent labeling allows us to monitor tumor growth by luciferase activity and distinguish individual cell populations by their fluorescent label. We are currently verifying that the cells retain stable expression of the vectors in mouse models; preliminary results are promising. In the future, this system will be used to develop an elegant multi-label-based fluorescent barcoding strategy that will allow us to identify individual cell populations from a heterogeneous environment in vivo. By exploiting combinations of the seven fluorescent proteins, we could potentially create over 200 uniquely identifiable cells populations. This ability will not only reduce the number of animals necessary per experiment (as control and several experimental populations can be individually analyzed in a single mouse); but will also allow us to mimic the heterogeneous environment common to all human tumors. Citation Format: Katie E. Hebron, Tatiana Ketova, Shanna Arnold, H. Charles Manning, Julie Sterling, Florent Elefteriou, Andries Zijsltra. Fluorescent barcoding offers increased dimensionality in tracking tumor cells in vitro and in vivo. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4958. doi:10.1158/1538-7445.AM2014-4958

The dysregulation of cell migration enables tumor cells to escape their tissue of origin and disseminate. Since cancer-related deaths are primarily caused by the dissemination of tumor cells, mechanisms of migration are both a target for therapy and an indicator of disease progression. The regulation of cell adhesion is widely recognized as a rate-limiting step in metastasis but how tumor cells achieve dynamic control over their adhesion receptors is poorly understood. During an analysis of prostate cancer progression we discovered that α3β1 expression is reduced and that its tetraspanin partner, CD151, is not “integrin-free”. We were able to detect integrin-free CD151 using antibodies specific to the integrin-binding domain of CD151. Dual staining of tumor tissue and normal tissue from prostate cancer patients for total CD151 and integrin-free CD151 revealed that the appearance of integrin-free CD151 corresponds with poor-patient outcome. In fact, the detection of integrin-free CD151 is an independent predictor of prostate cancer progression. Surprisingly, the clustering of integrin-free CD151 immobilizes tumor cells in vivo and prevents metastasis suggesting that the ability of CD151 to control migration does not depend on its α3β1 integrin partner. Indeed, integrin-free CD151 is now associated with non-integrin partners through which it can regulate tumor cell motility. These observations demonstrate that the appearance of integrin-free CD151 reflects the disruption of the CD151/ α3β1/laminin axis and thereby reveals an altered migratory ability in tumor cells. This has clinical as well molecular implications. Integrin-free CD151 can be used as a molecular indicator of disease progression and assist in the distinction between indolent (benign) and advanced disease (Palmer et al. 2013). In addition, the identification of new CD151 partners can provide new therapeutic targets to inhibit the motility of tumor cells that have undergone this change in migratory status. A preliminary evaluation identified a similar appearance of integrin-free CD151 in cancers derived from other tissues, suggesting that this change in molecular status is broadly applicable to most solid tumors. Palmer, et al. (2013). Integrin-free tetraspanin CD151 can inhibit tumor cell motility upon clustering and is a clinical indicator of prostate cancer progression. Cancer Research. Citation Format: Trenis Palmer, Carlos Martinez, Catalina Vasquez, Katie Hebron, Shanna Arnold, Celestial Jones-Paris, Susanne Chan, Venu Chalasani, Jose Gomez-Lemus, Andrew Williams, Joseph Chin, Giovanna Giannico, Tatiana Ketova, John Lewis, Andries Zijlstra. Separation of tetraspanin CD151 from its integrin partner α3β1 reflects an alter migratory state and predicts prostate cancer progression. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 55. doi:10.1158/1538-7445.AM2014-55