Research Project C: Signaling Pathways in Childhood Sarcoma

Program Goals

Approximately 70% of children with sarcoma can be cured using multimodality treatments, but the outcome is still poor for those with advanced or metastatic disease. Specifically, the 5-year event-free survival rates are 30 percent or less in children with advanced or metastatic Ewing sarcoma, osteosarcoma, or rhabdomyosarcoma, and intensive chemo-radiotherapy has not substantially altered this outcome over the past two decades. As additional cytotoxic drugs alone are unlikely to significantly increase cure rates, alternative and complementary approaches need to be explored. Further, given the long-term toxicities associated with standard maximum tolerated dose chemotherapy in the pediatric population, new therapies associated with less genotoxicity are needed. This program centers around three separate but integrated signaling pathways shown to be active in childhood sarcomas.

The overall goals for this Program Project Grant (PO1CA165995) are to comprehensively understand the roles of three signaling pathways (NF-κB, STAT3, and insulin-like growth factors [IGFs]) in the maintenance of malignant phenotypes of childhood sarcomas and to use these findings to develop novel integrated therapies with enhanced efficacy for affected patients. These goals will be achieved through the combined expertise of the principal investigators in childhood sarcoma biology and pediatric cancer drug development, the incorporation of unique small and large animal models of childhood sarcoma, and the broad interactions between Projects 1-3. Together, the projects will characterize the interrelationship of these pathways and identify combinatorial inhibitory approaches most likely to yield biological activity in the clinical setting.

The Program is unique both for its focus on childhood sarcomas and for its focus on combinatorial approaches to suppress glycolysis, tumor cell proliferation, survival, and angiogenesis through modulating NF-κB/TOR/STAT3/IGF signaling pathways.

Redundant Signaling as the Predominant mechanism for Resistance to Antibodies Targeting IGF-1R in Cells Derived from Childhood Sarcoma.
Shackleford TJ, Hariharan S, Vaseva AV, Alagoa K, Espinoza M, Bid HK, Li F, Zhong H, Phelps DA, Roberts RD, Cam H, London CA, Guttridge DC, Chen Y, Rao M, Shiio Y, Houghton PJ.Mol Cancer Ther. 2023 Jan 24:MCT-20-0625. doi: 10.1158/1535-7163.MCT-20-0625. Online ahead of print.PMID: 36696581

Antibodies targeting IGF-1R induce objective responses in only 5-15% of children with sarcoma. Understanding mechanisms of resistance may identify combination therapies that optimize the efficacy of IGF-1R-targeted antibodies. Sensitivity to the IGF1R-targeting antibody TZ-1 was determined in rhabdomyosarcoma (RMS) and Ewing sarcoma (EWS) cell lines. Acquired resistance to TZ-1 was developed and characterized in sensitive Rh41 cells. BRD4 inhibitor, JQ1 was evaluated as an agent to prevent acquired TZ-1 resistance in Rh41 cells. The phosphorylation status of RTKs was assessed. Sensitivity to TZ-1 in vivo was determined in Rh41 parental and TZ-1-resistant xenografts. Of 20 sarcoma cell lines, only Rh41 was sensitive to TZ-1. Cells intrinsically resistant to TZ-1 expressed multiple (>10) activated RTKs or a relatively less complex set of activated RTKs (~5). TZ-1 decreased the phosphorylation of IGF-1R but had little effect on other pRTKs in all resistant lines. TZ-1 rapidly induced activation of RTKs in Rh41 that was partially abrogated by the knockdown of SOX18 and JQ1. Rh41/TZ-1 cells selected for acquired resistance to TZ-1 constitutively expressed multiple activated RTKs. TZ-1 treatment caused complete regressions in Rh41 xenografts and was significantly less effective against the Rh41/TZ-1 xenograft. Intrinsic resistance is a consequence of redundant signaling in pediatric sarcoma cell lines. Acquired resistance in Rh41 cells is associated with rapid induction of multiple RTKs, indicating a dynamic response to IGF-1R blockade and rapid development of resistance. The TZ-1 antibody had greater antitumor activity against Rh41 xenografts compared to other IGF-1R-targeted antibodies tested against this model.

Figure. The sensitivity of RMS and EWS cells to the IGF-1R inhibitor, TZ-1, is dependent on RTK activation status. A panel of RMS (A) and EWS (B) cell lines were treated with TZ-1, and dose-response curves are shown. Cells were treated with TZ-1 for 96 Hr, and cell viability was measured by Alamar Blue assay. Data is representative of three independent experiments in triplicate and presented as the mean ± SD. Phospho-RTK array showing phosphorylation status of 49 RTKs in RMS (C) and EWS (D) cell lines treated with IgG control antibody or TZ-1 (10g/mL) for 48 Hr. Four phospho-tyrosine positive controls are on the corners of each array, and each RTK is represented in duplicate, with each pair of horizontal spots representing one receptor. Upregulated RTKs are indicated.