Sustained Oncogenic Signaling in the Cytostatic State Enables Targeting of Nonproliferating Persistent Cancer Cells.

Many advanced therapeutics possess cytostatic properties that suppress cancer cell growth without directly inducing death. Treatment-induced cytostatic cancer cells can persist and constitute a reservoir from which recurrent growth and resistant clones can develop. Current management approaches primarily comprise maintenance and monitoring because strategies for targeting nonproliferating cancer cells have been elusive. Here, we used targeted therapy paradigms and engineered cytostatic states to explore therapeutic opportunities for depleting treatment-mediated cytostatic cancer cells. Sustained oncogenic AKT signaling was common, while nonessential, in treatment-mediated cytostatic cancer cells harboring PI3K-pathway mutations, which are associated with cancer recurrence. Engineering oncogenic signals in quiescent mammary organotypic models showed that sustained, aberrant activation of AKT sensitized cytostatic epithelial cells to proteasome inhibition. Mechanistically, sustained AKT signaling altered cytostatic state homeostasis and promoted an oxidative and proteotoxic environment, which imposed an increased proteasome dependency for maintaining cell viability. Under cytostatic conditions, inhibition of the proteasome selectively induced apoptosis in the population with aberrant AKT activation compared with normal cells. Therapeutically exploiting this AKT-driven proteasome vulnerability was effective in depleting treatment-mediated cytostatic cancer cells independent of breast cancer subtype, epithelial origin, and cytostatic agent. Moreover, transient targeting during cytostatic treatment conditions was sufficient to reduce recurrent tumor growth in spheroid and mouse models. This work identified an AKT-driven proteasome-vulnerability that enables depletion of persistent cytostatic cancer cells harboring PTEN-PI3K pathway mutations, revealing a viable strategy for targeting nonproliferating persistent cancer cell populations before drug resistance emerges. SIGNIFICANCE: This study finds that sustained oncogenic signaling in therapy-induced cytostatic cancer cells confers targetable vulnerabilities to deplete persistent cancer cell populations and reduce cancer recurrence.

Plocabulin, a novel tubulin inhibitor, has potent antitumor activity in patient-derived xenograft models of gastrointestinal stromal tumors.

The majority of patients with gastrointestinal stromal tumors (GIST) eventually become resistant with time due to secondary mutations in the driver receptor tyrosine kinase. Novel treatments that do not target these receptors may therefore be preferable. For the first time, we evaluated a tubulin inhibitor, plocabulin, in patient-derived xenograft (PDX) models of GIST, a disease generally considered to be resistant to cytotoxic agents. Three PDX models of GIST with different KIT genotype were generated by implanting tumor fragments from patients directly into nude mice. We then used these well characterized models with distinct sensitivity to imatinib to evaluate the efficacy of the novel tubulin inhibitor. The efficacy of the drug was assessed by volumetric analysis of the tumors, histopathology, immunohistochemistry and Western blotting. Plocabulin treatment led to extensive necrosis in all three models and significant tumor shrinkage in two models. This histological response can be explained by the drug's vascular-disruptive properties, which resulted in a shutdown of tumor vasculature, reflected by a decreased total vascular area in the tumor tissue. Our results demonstrated the in vivo efficacy of the novel tubulin inhibitor plocabulin in PDX models of GIST and challenge the established view that GIST are resistant to cytotoxic agents in general and to tubulin inhibitors in particular. Our findings provide a convincing rationale for early clinical exploration of plocabulin in GIST and warrant further exploration of this class of drugs in the management of this common sarcoma subtype.

Differential antitumor activity of compounds targeting the ubiquitin-proteasome machinery in gastrointestinal stromal tumor (GIST) cells.

The majority of gastrointestinal stromal tumors (GISTs) are driven by oncogenic KIT signaling and can therefore be effectively treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate. However, most GISTs develop imatinib resistance through secondary KIT mutations. The type of resistance mutation determines sensitivity to approved second-/third-line TKIs but shows high inter- and intratumoral heterogeneity. Therefore, therapeutic strategies that target KIT independently of the mutational status are intriguing. Inhibiting the ubiquitin-proteasome machinery with bortezomib is effective in GIST cells through a dual mechanism of KIT transcriptional downregulation and upregulation of the pro-apoptotic histone H2AX but clinically problematic due to the drug's adverse effects. We therefore tested second-generation inhibitors of the 20S proteasome (delanzomib, carfilzomib and ixazomib) with better pharmacologic profiles as well as compounds targeting regulators of ubiquitination (b-AP15, MLN4924) for their effectiveness and mechanism of action in GIST. All three 20S proteasome inhibitors were highly effective in vitro and in vivo, including in imatinib-resistant models. In contrast, b-AP15 and MLN4924 were only effective at high concentrations or had mostly cytostatic effects, respectively. Our results confirm 20S proteasome inhibitors as promising strategy to overcome TKI resistance in GIST, while highlighting the complexity of the ubiquitin-proteasome machinery as a therapeutic target.

Assessment of the platelet-derived growth factor receptor alpha antibody olaratumab in a panel of patient-derived soft tissue sarcoma xenografts.

BACKGROUND: Soft tissue sarcoma (STS) comprises a family of rare, heterogeneous tumors of mesenchymal origin. Single-agent doxorubicin remains the first-line standard-of-care treatment for advanced and inoperable STS, but response rates are only around 15%. In 2016, phase Ib/II clinical trial results reported an overall survival benefit of 11.8 months when combining doxorubicin and the platelet-derived growth factor receptor alpha (PDGFRA)-directed antibody olaratumab compared to doxorubicin alone, without providing a scientific rationale for such unprecedented therapeutic effect. We decided to evaluate the efficacy of olaratumab in a panel of STS patient-derived xenografts (PDX). METHODS: NMRI nu/nu mice were bilaterally transplanted with tumor tissue of patient-derived xenograft models expressing PDGFRA, including models of leiomyosarcoma (UZLX-STS22), malignant peripheral nerve sheath tumor (UZLX-STS39), myxofibrosarcoma (UZLX-STS59) and undifferentiated pleomorphic sarcoma (UZLX-STS84). Mice were randomly divided into four different treatment groups: (1) control, (2) doxorubicin (3 mg/kg once weekly), (3) anti-PDGFRA [olaratumab (60 mg/kg twice weekly) + mouse anti-PDGFRA antibody 1E10 (20 mg/kg twice weekly)] and (4) the combination of doxorubicin and anti-PDGFRA (same dose/schedule as in the single treatment arms). Tumor volume, histopathology and Western blotting were used to assess treatment efficacy. RESULTS: Anti-PDGFRA treatment as a single agent did not reduce tumor growth and did not result in significant anti-proliferative or pro-apoptotic activity. Combining doxorubicin and anti-PDGFRA did not reduce tumor burden, though a mild inhibition of proliferation was observed in UZLX-STS39 and -STS59. A pro-apoptotic effect was observed in all models except UZLX-STS22. Antitumor effects on histology were not significantly different comparing doxorubicin and the combination treatment. Moreover, anti-PDGFRA treatment, both as a single agent as well as combined with doxorubicin, did not result in inhibition of the downstream MAPK and PI3K/AKT signaling pathways. CONCLUSIONS: We were not able to demonstrate significant antitumor effects of anti-PDGFRA treatment in selected STS PDX models, neither alone nor in combination with doxorubicin. This is in line with the very recent results of the phase III clinical trial NCT02451943 ANNOUNCE, which did not confirm the clinical benefit of olaratumab in combination with doxorubicin over single agent doxorubicin.

Establishment and Characterization of Histologically and Molecularly Stable Soft-tissue Sarcoma Xenograft Models for Biological Studies and Preclinical Drug Testing.

Soft-tissue sarcomas (STS) represent a heterogeneous group of rare, malignant tumors of mesenchymal origin. Reliable in vivo sarcoma research models are scarce. We aimed to establish and characterize histologically and molecularly stable patient-derived xenograft (PDX) models from a broad variety of STS subtypes. A total of 188 fresh tumor samples from consenting patients with localized or advanced STS were transplanted subcutaneously in NMRI-nu/nu-immunodeficient mice. Once tumor growth was observed, the material was passaged to a next generation of mice. A patient-derived tumor sample was considered "successfully engrafted" whenever the sample was transplanted to passage 1. A PDX model was considered "established" when observing stable morphologic and molecular features for at least two passages. With every passage, histologic and molecular analyses were performed. Specific genomic alterations and copy-number profile were assessed by FISH and low coverage whole-genome sequencing. The tumor engraftment rate was 32% (61/188) and 188 patient samples generated a total of 32 PDX models, including seven models of myxofibrosarcoma, five dedifferentiated liposarcoma, five leiomyosarcoma, three undifferentiated pleomorphic sarcoma, two malignant peripheral nerve sheet tumor models, and single models of synovial sarcoma and some other (ultra)rare subtypes. Seventeen additional models are in early stages of engraftment (passage 1-2). Histopathologic and molecular features were compared with the original donor tumor and were stable throughout passaging. The platform is used for studies on sarcoma biology and suited for in vivo preclinical drug testing as illustrated by a number of completed and ongoing laboratory studies.

PLX9486 shows anti-tumor efficacy in patient-derived, tyrosine kinase inhibitor-resistant KIT-mutant xenograft models of gastrointestinal stromal tumors.

The purpose of the present study was to investigate the in vitro and in vivo activity of PLX9486, a tyrosine kinase inhibitor (TKI) targeting both primary KIT exon 9 and 11 and secondary exon 17 and 18 mutations in gastrointestinal stromal tumors (GISTs). Imatinib, a potent inhibitor of mutated KIT, has revolutionized the clinical management of advanced, metastatic GIST. However, secondary resistance develops mainly through acquired mutations in KIT exons 13/14 or exons 17/18. Second-line sunitinib potently inhibits KIT exon 13/14 mutants but is ineffective against exon 17 mutations. In our study, PLX9486 demonstrated in vitro nanomolar potency in inhibiting the growth and KIT phosphorylation of engineered BaF3 cells transformed with KIT exon 17 mutations (p.D816V) and with the double KIT exon 11/17 mutations (p.V560G/D816V). The in vivo efficacy of PLX9486 was evaluated using two imatinib-resistant GIST patient-derived xenograft (PDX) models. In UZLX-GIST9 (KIT: p.P577del;W557LfsX5;D820G), PLX9486 100 mg/kg/day resulted in significant inhibition of proliferation. Pharmacodynamic analysis showed a pronounced reduction in mitogen-activated protein kinase (MAPK) activation and other downstream effects of the KIT signaling pathway but no significant effect on KIT Y703 and Y719 phosphorylation. Similarly, in MRL-GIST1 (KIT: p.W557_K558del;Y823D) PLX9486 treatment led to significant tumor regression and strong inhibition of MAPK activation. Interestingly, the inhibitory effect on MAPK activation was evident even after a single dose of PLX9486. In conclusion, PLX9486 showed anti-tumor efficacy in patient-derived imatinib-resistant GIST xenograft models, mainly through inhibition of KIT signaling. These preclinical efficacy data encourage further testing of PLX9486 in the clinical setting.

Anagrelide for Gastrointestinal Stromal Tumor.

PURPOSE: Gastrointestinal stromal tumor (GIST) is a common type of soft-tissue sarcoma. Imatinib, an inhibitor of KIT, platelet-derived growth factor receptor alpha (PDGFRA), and a few other tyrosine kinases, is highly effective for GIST, but advanced GISTs frequently progress on imatinib and other approved tyrosine kinase inhibitors. We investigated phosphodiesterase 3 (PDE3) as a potential therapeutic target in GIST cell lines and xenograft models. EXPERIMENTAL DESIGN: The GIST gene expression profile was interrogated in the MediSapiens IST Online transcriptome database comprising human tissue and cancer samples, and PDE3A and PDE3B expression was studied using IHC on tissue microarrays (TMA) consisting of 630 formalin-fixed human tissue samples. GIST cell lines were screened for sensitivity to 217 anticancer compounds, and the efficacy of PDE inhibitors on GIST was further studied in GIST cell lines and patient-derived mouse xenograft models. RESULTS: GISTs expressed PDE3A and PDE3B frequently compared with other human normal or cancerous tissues both in the in silico database and the TMAs. Anagrelide was identified as the most potent of the PDE3 modulators evaluated. It reduced cell viability, promoted cell death, and influenced cell signaling in GIST cell lines. Anagrelide inhibited tumor growth in GIST xenograft mouse models. Anagrelide was also effective in a GIST xenograft mouse model with KIT exon 9 mutation that may pose a therapeutic challenge, as these GISTs require a high daily dose of imatinib. CONCLUSIONS: PDE3A and PDE3B are frequently expressed in GIST. Anagrelide had anticancer efficacy in GIST xenograft models and warrants further testing in clinical trials.

Robust Activity of Avapritinib, Potent and Highly Selective Inhibitor of Mutated KIT, in Patient-derived Xenograft Models of Gastrointestinal Stromal Tumors.

PURPOSE: Gastrointestinal stromal tumors (GIST) are commonly treated with tyrosine kinase inhibitors (TKI). The majority of patients with advanced GIST ultimately become resistant to TKI due to acquisition of secondary KIT mutations, whereas primary resistance is mainly caused by PDGFRA p.D842V mutation. We tested the activity of avapritinib, a potent and highly selective inhibitor of mutated KIT and PDGFRA, in three patient-derived xenograft (PDX) GIST models carrying different KIT mutations, with differential sensitivity to standard TKI.Experimental Design: NMRI nu/nu mice (n = 93) were transplanted with human GIST xenografts with KIT exon 11+17 (UZLX-GIST9 KIT 11+17 ), exon 11 (UZLX-GIST3 KIT 11 ), or exon 9 (UZLX-GIST2B KIT9 ) mutations, respectively. We compared avapritinib (10 and 30 mg/kg/once daily) versus vehicle, imatinib (50 mg/kg/bid) or regorafenib (30 mg/kg/once daily; UZLX-GIST9 KIT11+17 ); avapritinib (10, 30, 100 mg/kg/once daily) versus vehicle or imatinib [UZLX-GIST3 KIT11 ]; and avapritinib (10, 30, 60 mg/kg/once daily) versus vehicle, imatinib (50, 100 mg/kg/twice daily), or sunitinib (40 mg/kg/once daily; UZLX-GIST2B KIT9 ). RESULTS: In all models, avapritinib resulted in reduction of tumor volume, significant inhibition of proliferation, and reduced KIT signaling. In two models, avapritinib led to remarkable histologic responses, increase in apoptosis, and inhibition of MAPK-phosphorylation. Avapritinib showed superior (UZLX-GIST9 KIT 11+17 and -GIST2B KIT 9 ) or equal (UZLX-GIST3 KIT 11 ) antitumor activity to the standard dose of imatinib. In UZLX-GIST9 KIT 11+17 , the antitumor effects of avapritinib were significantly better than with imatinib or regorafenib. CONCLUSIONS: Avapritinib has significant antitumor activity in GIST PDX models characterized by different KIT mutations and sensitivity to established TKI. These data provide strong support for the ongoing clinical trials with avapritinib in patients with GIST (NCT02508532, NCT03465722).

A precision therapy against cancers driven by KIT/PDGFRA mutations.

Targeting oncogenic kinase drivers with small-molecule inhibitors can have marked therapeutic benefit, especially when administered to an appropriate genomically defined patient population. Cancer genomics and mechanistic studies have revealed that heterogeneous mutations within a single kinase can result in various mechanisms of kinase activation. Therapeutic benefit to patients can best be optimized through an in-depth understanding of the disease-driving mutations combined with the ability to match these insights to tailored highly selective drugs. This rationale is presented for BLU-285, a clinical stage inhibitor of oncogenic KIT and PDGFRA alterations, including activation loop mutants that are ineffectively treated by current therapies. BLU-285, designed to preferentially interact with the active conformation of KIT and PDGFRA, potently inhibits activation loop mutants KIT D816V and PDGFRA D842V with subnanomolar potency and also inhibits other well-characterized disease-driving KIT mutants both in vitro and in vivo in preclinical models. Early clinical evaluation of BLU-285 in a phase 1 study has demonstrated marked activity in patients with diseases associated with KIT (aggressive systemic mastocytosis and gastrointestinal stromal tumor) and PDGFRA (gastrointestinal stromal tumor) activation loop mutations.

New targets and therapies for gastrointestinal stromal tumors.

INTRODUCTION: The majority of gastrointestinal stromal tumors (GIST) are driven by an abnormal receptor tyrosine kinase (RTK) signaling, occurring mainly due to somatic mutations in KIT or platelet derived growth factor receptor alpha (PDGFRA). Although the introduction of tyrosine kinase inhibitors (TKIs) has revolutionized therapy for GIST patients, with time the vast majority of them develop TKI resistance. Advances in understanding the molecular background of GIST resistance allows for the identification of new targets and the development of novel strategies to overcome or delay its occurrence. Areas covered: The focus of this review is on novel, promising therapeutic approaches to overcome heterogeneous resistance to registered TKIs. These approaches involve new TKIs, including drugs specific for a mutated form of KIT/PDGFRA, drugs with inhibitory effect against multiple RTKs, compounds targeting dysregulated downstream signaling pathways, drugs affecting KIT expression and degradation, inhibitors of cell cycle, and immunotherapeutics. Expert commentary: As the resistance to standard TKI treatment can be heterogeneous, a combinational approach for refractory GIST could be beneficial. Moreover, the understanding of the molecular background of resistant disease would allow development of a more personalized approach for these patients and their response to targeted therapy could be monitored closely using 'liquid biopsy'.

In Vivo Antitumoral Efficacy of PhAc-ALGP-Doxorubicin, an Enzyme-Activated Doxorubicin Prodrug, in Patient-Derived Soft Tissue Sarcoma Xenograft Models.

Given the very limited efficacy of doxorubicin (doxo) in soft tissue sarcoma, there is a clear need for more active and less toxic treatments for this family of diseases. However, due to the rarity of these malignancies and lack of reliable preclinical models, development of new therapies has lagged behind. We evaluated the efficacy of PhAc-ALGP-doxorubicin (ALGP-doxo), a prodrug metabolized to doxo by peptidases present in tumor cells and/or tumor microenvironment, in a synovial sarcoma (SynSa) and two dedifferentiated liposarcoma (DDLPS) patient-derived xenograft models. Sixty-eight mice were engrafted bilaterally with human DDLPS or SynSa and randomized to control, doxo, or ALGP-doxo treatment, which were administered using an intraperitoneal minipump. Tumor volume measurement, histopathology, and Western blotting were used to assess treatment efficacy. Tumor regrowth was evaluated in a subset of mice over a period of 2 weeks after treatment cessation. Although tumor volume in the control and doxo groups increased steadily, ALGP-doxo caused tumor volume stabilization in the DDLPS xenografts and significant tumor shrinkage in the SynSa model, continuing after treatment cessation. A significant decrease in proliferation and increase in apoptosis compared with control and doxo was observed during and after treatment with ALGP-doxo in all models. In conclusion, ALGP-doxo shows considerably higher antitumoral efficacy compared with doxo in all patient-derived xenograft models tested. Administration of a 30- to 40-fold higher dose of ALGP-doxo than doxo is tolerated without significant adverse events. These results warrant further testing of this prodrug in anthracycline-sensitive and -resistant models of soft tissue sarcoma. Mol Cancer Ther; 16(8); 1566-75. ©2017 AACR.

Volatile organic compounds in gastrointestinal stromal tumour tissue originating from patient-derived xenografts.

Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and localize mainly in the stomach or small intestine. The metabolomic signatures of GISTs driven by different KIT gene mutations remain undiscovered and unexplored. The main aim of this pilot study was to determine and compare metabolomic profiles in GIST xenograft models with different genetic backgrounds. Metabolomic profiling using gas chromatography coupled with mass spectrometry followed by univariate and multivariate statistical analyses was applied to select metabolites that differentiated the GIST models studied. The significant differences observed in the metabolites were mainly derived from glycolysis, the citric acid cycle and glutamine and lipid metabolism. The obtained results may suggest variable metabolomic signatures of tumours, possibly related to the different underlying, specific KIT gene mutations and with potential implications for the biological behaviour and natural course of this rare disease. This study constitutes a proof of concept in GISTs and reveals the potential of the metabolomic approach in orphan malignancies.

Cabozantinib Is Active against Human Gastrointestinal Stromal Tumor Xenografts Carrying Different KIT Mutations.

In the majority of gastrointestinal stromal tumors (GIST), oncogenic signaling is driven by KIT mutations. Advanced GIST is treated with tyrosine kinase inhibitors (TKI) such as imatinib. Acquired resistance to TKI is mainly caused by secondary KIT mutations, but can also be attributed to a switch of KIT dependency to another receptor tyrosine kinase (RTK). We tested the efficacy of cabozantinib, a novel TKI targeting KIT, MET, AXL, and vascular endothelial growth factor receptors (VEGFR), in patient-derived xenograft (PDX) models of GIST, carrying different KIT mutations. NMRI nu/nu mice (n = 52) were bilaterally transplanted with human GIST: UZLX-GIST4 (KIT exon 11 mutation, imatinib sensitive), UZLX-GIST2 (KIT exon 9, imatinib dose-dependent resistance), or UZLX-GIST9 (KIT exon 11 and 17 mutations, imatinib resistant). Mice were grouped as control (untreated), imatinib (50 mg/kg/bid), and cabozantinib (30 mg/kg/qd) and treated orally for 15 days. Cabozantinib resulted in significant tumor regression in UZLX-GIST4 and -GIST2 and delayed tumor growth in -GIST9. In all three models, cabozantinib inhibited the proliferative activity, which was completely absent in UZLX-GIST4 and significantly reduced in -GIST2 and -GIST9. Increased apoptotic activity was observed only in UZLX-GIST4. Cabozantinib inhibited the KIT signaling pathway in UZLX-GIST4 and -GIST2. In addition, compared with both control and imatinib, cabozantinib significantly reduced microvessel density in all models. In conclusion, cabozantinib showed antitumor activity in GIST PDX models through inhibition of tumor growth, proliferation, and angiogenesis, in both imatinib-sensitive and imatinib-resistant models. Mol Cancer Ther; 15(12); 2845-52. ©2016 AACR.