Preclinical Modelling and Therapeutic Development for Other Cancer Indications

Division Of Basic Sciences - Nci

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Abstract

As an illustrative example of CAPR efforts aimed at establishing models for clinically challenging tumor types, we have successfully established several models for pediatric rhabdomyosarcoma and neuroblastoma. In these malignancies, the current paucity of recurrent druggable mutations in pediatric solid tumor cases have significantly restricted effective therapeutic options for patients with high-risk or advanced forms of the disease. Alteration of the MYCN gene is a recurrent feature of pediatric tumors, and high copy amplification of MYCN in neuroblastoma and rhabdomyosarcoma is associated with very high-risk disease. The TOPI inhibitor irinotecan is a standard of care, but treatment is limited by toxic side effects in juvenile patients. These transcription factor-driven tumors may be sensitive to alternative treatment regimens (such as combining TOPI inhibitors with inhibitors of bromodomain and extraterminal (BET) domain containing proteins) that target transcriptionally active sites. The goals of the project were: 1). to assess the single agent activity of BET inhibitor JQ1 and irinotecan in mouse models for pediatric solid tumors using the transplantable MYCN-driven THMYCN GEM-derived allograft neuroblastoma tumor model and a patient-derived xenograft (PDX) PAX3/FOXO1 RMS tumor model; and 2). to assess the toxicity and anti-tumor activity of combinations of the BET inhibitor JQ1 and irinotecan. We have expanded and characterized an alveolar rhabdomyosarcoma PDX tumor provided by the Pediatric Oncology branch. We implanted the PDX orthotopically and evaluated both irinotecan and JQ1 in single agent dose-response tumor growth inhibition studies. Irinotecan as a single agent extended survival, but JQ1 given as a single agent was not effective at the maximum dose. However, JQ1 and irinotecan given together suppressed tumor growth significantly, and better than irinotecan alone. The TH-MYCN model of neuroblastoma was expanded subcutaneously in strain-matched immunocompetent mice and response to JQ1 and irinotecan was evaluated. Irinotecan and JQ1 as single agents at the highest tolerated doses extended survival. In combination, the agents suppressed tumor growth and extended tumor survival better than either agent alone, with three mice remaining tumor-free in the high dose combination after several months. Notably, irinotecan at 2.5mg/kg in combination with JQ1 at 50mg/kg was as effective as irinotecan at 10mg/kg, indicating that treatment with a BET inhibitor may promote greater efficacy of irinotecan at lower doses, potentially reducing toxicity to patients. These studies have led to the development of a pediatric Phase I/II trial currently under review at NCI's Clinical Center. In another large-scale effort, pursuing the development of novel models to support preclinical analyses in rare, "unmet need", malignancies as one of CAPR's critical missions, we have established and characterized several novel mouse lines engineered to express estrogen dependent CreERT2 recombinase variant under the control of endogenous promoter for Dopachrome Tautomerase Type 1 (Dct1) gene. These lines, upon intercrossing with a Cre recombination diagnostic reporter allele, display robust tamoxifen activation of genetic events in several neural crest derived lineages, including Schwann cell precursors and melanoblasts. Combining this novel allele with conditional mutations in Braf oncoprotein (BrafV600E) and recombination-dependent loss-of-function allele for PTEN tumor suppressor resulted in a model producing characteristic skin cancerous lesions when exposed to tamoxifen systemic or topical administration. Histologic evaluation of these neoplastic tissues revealed distinct morphological patterns consistent with Antoni A and Antoni B patterns characteristic for PNST. Additional biomarker analyses further confirmed these tumors as PNST by the abundance of S100 and GFAP positive cells and lack of HMB45 immunoreactivity more typical for melanoma cancers. The uniform infiltration by mast cells, positive by toluidine blue staining, allowed to further qualify these lesions as spontaneous MPNST, a rare malignancy type with prevalence in pediatric cancer patients. We have also established orthotopic engraftment procedure passaging fragments of primary tumors in syngeneic recipient animals and demonstrated pharmacologic vulnerabilities of PNST tumors in vivo by challenging cancer bearing animals with common inhibitors of oncogenic Braf signaling, such as vemurafenib. We are currently completing comparative transcriptome assessment of spontaneous vs. Nf1-dependent PNST with an outlook of publishing these data and making this model available to support preclinical needs of researchers and clinicians working on PNST.