Yemarshet Kelemework Gebreyohannes

A precision therapy against cancers driven by KIT/PDGFRA mutations

BLU-285 is a selective and potent mutant KIT and PDGFRA inhibitor active in both preclinical and clinical settings. Treatment makes tumors feel BLU Mutations in receptor tyrosine kinases are common in cancer, and a variety of kinase mutations can have oncogenic effects. Not all of these mutations are susceptible to existing kinase inhibitors, and many inhibitors are nonspecific, resulting in undesirable off-target effects. Evans et al. developed BLU-285, an inhibitor that specifically targets the KIT and PDGFRA oncogenic kinases. The authors showed that the compound is very specific for its targets, can inhibit them in the presence of multiple oncogenic mutations, and is selective for the mutant forms of the kinases relative to the wild type. In addition to biochemical and preclinical testing of BLU-285, the authors conducted a phase 1 study, which showed evidence of the compound’s activity in human cancer patients. 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.

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.

New targets and therapies for gastrointestinal stromal tumors

ABSTRACT 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’.

Abstract 1460: Establishment and characterization of a panel of patient-derived soft tissue sarcoma (STS) xenograft models for in vivo testing of novel therapeutic approaches

Background: STS constitutes a rare and very heterogeneous family of tumors of mesenchymal origin. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies. Methods: A panel of patient-derived xenografts was established and is currently expanded in nu/nu NMRI mice by subcutaneous implantation of fresh, surgically resected tumor specimens from consenting patients with STS, treated at the University Hospitals Leuven, Belgium. We mainly focused on STS subtypes which harbor specific genetic alterations (e.g. translocations, gene amplifications). Once tumor growth was observed, pieces of tissue were re-transplanted to the next generation of mice. At each passage tumor fragments were collected for histological and molecular characterization. Fluorescence in situ hybridization (FISH) was performed to confirm specific genomic alterations in selected xenografts. A STS xenograft model was considered established after observing maintained histological and molecular features for at least two passages. Results: Until now a total of 78 STS patient samples have been transplanted. Fifteen well-characterized, stable STS models have already been established, maintaining the features of the original tumor: myxofibrosarcoma (4 models), dedifferentiated liposarcoma (2), synovial sarcoma (2), epithelioid haemangioendothelioma (1), malignant peripheral nerve sheath tumor (2), mesenchymal chondrosarcoma (1), leiomyosarcoma (2) and sarcoma not otherwise specified (1). Some of these models have already been successfully used for in vivo testing of novel agents, including tyrosine kinase inhibitors or cytotoxic (pro-)drugs. Twelve other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of “established model”. Conclusion: This newly established, representative panel of STS xenografts is characterized by stable histological and molecular features reflecting the pheno- and genotype of the original patient samples. This panel is thus well suited for in vivo drug testing of innovative agents. The unique availability of models of some ultra-rare entities will also allow us to study the biology of these diseases. The platform is available for collaboration with academic and commercial partners (contact patrick.schoffski@uzleuven.be). Citation Format: Haifu Li, Jasmien Cornillie, Agnieszka Wozniak, Thomas Van Looy, Yemarshet Gebreyohannes, Jasmien Wellens, Ulla Vanleeuw, Daphne Hompes, Marguerite Stas, Friedl Sinnaeve, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. Establishment and characterization of a panel of patient-derived soft tissue sarcoma (STS) xenograft models for in vivo testing of novel therapeutic approaches. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1460. doi:10.1158/1538-7445.AM2015-1460

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.

Abstract 775: Anti-tumor effects of dovitinib, a multi-target kinase inhibitor, in patient-derived gastrointestinal stromal tumor (GIST) xenograft models

Background: The majority of GISTs are driven by KIT/PDGFRA gain of function mutations. Advanced GIST is routinely treated with tyrosine kinase inhibitors (TKI; e.g imatinib, sunitinib and regorafenib). However, with time, heterogeneous resistance to these agents occurs, mainly due to acquired mutations in KIT. We tested the efficacy of dovitinib, which acts against VEGFR, FGFR, FLT3, PDGFRB and KIT, using patient-derived GIST xenograft models. Methods: NMRI nu/nu mice (n = 47) were bilaterally transplanted with the human GIST xenografts: a dose-dependent-imatinib-sensitive model UZLX-GIST2 (KIT: p.A502_Y503dup) or a model resistant to both imatinib and sunitinib, UZLX-GIST9 (KIT: p.P577del+p.W557LfsX5+p.D820G). Mice were divided to four treatment groups: control (untreated), imatinib (50 mg/kg/bid p.o.), imatinib (100 mg/kg/bid p.o.) and dovitinib (30 mg/kg/qd p.o.). Efficacy was assessed by tumor volume measurement (3x/week), histopathology [hematoxylin & eosin staining (HE p Conclusion: The multi-kinase inhibitor dovitinib showed anti-tumor efficacy in GIST xenograft models, though the effect was more pronounced in KIT exon 9 mutant (UZLX-GIST2). The decrease in MVD in both models suggested that the anti-tumor effect in these models was at least partially mediated by an anti-angiogenic effect of dovitinib. 1 Antonescu et al. Clin Cancer Res. 2005; 11:4182-90 Citation Format: Yemarshet Kelemework Gebreyohannes, Thomas Van Looy, Agnieszka Wozniak, Jasmien Wellens, Haifu Li, Jasmien Cornillie, Ulla Vanleeuw, Lise Vreys, Matthew Squires, Ana-Maria Rodringuez, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. Anti-tumor effects of dovitinib, a multi-target kinase inhibitor, in patient-derived gastrointestinal stromal tumor (GIST) xenograft models. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 775. doi:10.1158/1538-7445.AM2015-775

Abstract 2081: Robust activity of BLU-285, a potent and highly selective inhibitor of mutant KIT and PDGFRα, in patient-derived xenograft (PDX) models of gastrointestinal stromal tumor (GIST)

Objective: Patients with advanced and treatment-resistant GIST are treated with tyrosine kinase inhibitors (TKI) such as imatinib (IMA), sunitinib (SUN) or regorafenib (REG). Resistance to these TKI is mainly caused by the emergence of on-target secondary KIT mutations in exons 13 and 17. We tested the activity of BLU-285 (Blueprint Medicines), a potent and highly selective inhibitor of mutant KIT and PDGFRα, in three PDX models of primary and refractory GIST that respond differently to the approved TKI used in the treatment of GIST. Methods: NMRI nu/nu mice (n=93) were transplanted bilaterally with human xenografts UZLX-GIST3 (KIT: exon 11 p.W557_V559delinsF; IMA-sensitive), GIST2B (KIT: exon 9 p.A502_Y503dup, IMA dose-dependent sensitive) and GIST9 (KIT: exon 11 p.P577del; W557LfsX5; exon 17 D820G; IMA and SUN-resistant). We performed the following experimental comparisons: BLU-285 (10, 30 and 100 mg/kg/qd) vs. control and IMA (50 mg/kg/bid) [GIST3]; BLU-285 (10, 30 and 60 mg/kg/qd) vs. control and IMA (50 and 100 mg/kg/bid or SUN 40 mg/kg/qd) [GIST2B]; and BLU-285 (10 and 30 mg/kg/qd) vs. control, IMA (50 mg/kg/bid) or REG (30 mg/kg/qd) [GIST9]. Animals were dosed orally for 16 days. Activity was assessed by tumor volume measurement, histopathology and Western blotting of the KIT signaling pathway (WB). Mann Whitney U test was used for statistical analysis. A p value Citation Format: Yemarshet K. Gebreyohannes, Agnieszka Wozniak, Madelina-Elena Zhai, Jasmien Wellens, Jasmien Cornillie, Erica Evans, Alexandra K Gardino, Christoph Lengauer, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. Robust activity of BLU-285, a potent and highly selective inhibitor of mutant KIT and PDGFRα, in patient-derived xenograft (PDX) models of gastrointestinal stromal tumor (GIST) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2081. doi:10.1158/1538-7445.AM2017-2081

Abstract 1031: XenoSarc: Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS)—an update on a preclinical platform for early drug testing

Background: STS constitutes a rare and very heterogeneous family of mesenchymal tumors. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies. Methods: A panel of patient-derived xenografts (PDX) was established by subcutaneous implantation of fresh, surgically resected or biopsied tumor specimens in immunodeficient, athymic nude NMRI mice. Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of mice. At each passage tumor fragments were collected for histopathological and molecular characterization. A model was considered established after observing stable histological and molecular features for at least two passages. Results: Until now 171 STS samples from consenting patients treated at the University Hospitals, Leuven, Belgium, have been transplanted. Twenty-eight well-characterized, stable PDX models of STS have been established, maintaining the histopathological and molecular features of the original tumor. The detailed clinical information about a donor patient, including sensitivity to standard and experimental drugs, is linked to every model. At this point the XenoSarc platform includes models of gastrointestinal stromal tumor (6 models), myxofibrosarcoma (6), dedifferentiated liposarcoma (3), malignant peripheral nerve sheath tumor (3), synovial sarcoma (1), leiomyosarcoma (4), epithelioid haemangioendothelioma (1), mesenchymal chondrosarcoma (1), pleomorphic rhabdomyosarcoma (1) and high-grade undifferentiated pleomorphic sarcoma (2). From these models we have also available tissue microarray (TMA) as well as data on genomic and/or expression profile including mutations (by RNA-Seq). Some of these models have already been successfully used for in vivo testing of novel agents, including both targeted and cytotoxic (pro-)drugs, and results served as a rationale for several prospective clinical trials. In addition, 24 other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of an “established model”. Conclusion: Our XenoSarc platform contains a number of well-annotated models, characterized by stable histological and molecular features. This platform is a reliable tool for the evaluation of new anticancer treatments for STS and for studying the biology of these rare diseases. The platform is made available to collaborators from academia and industry. Citation Format: Agnieszka Wozniak, Jasmien Cornillie, Yemarshet K. Gebreyohannes, Jasmien Wellens, Lise Vreys, Daphne Hompes, Marguerite Stas, Friedl Sinnaeve, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. XenoSarc: Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS), an update on a preclinical platform for early drug testing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4811. doi:10.1158/1538-7445.AM2017-4811

Abstract 5197: Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS): a preclinical platform for early drug testing

Background: STS constitutes a rare and very heterogeneous family of mesenchymal tumors. The limited treatment options available for advanced STS underline the need for reliable preclinical models to test novel therapeutic strategies. Methods: A panel of patient-derived xenografts (PDX) was established by subcutaneous implantation of fresh, surgically resected tumor specimens in immunodeficient athymic nude NMRI mice. Once tumor growth was observed, pieces of tumor were re-transplanted to next generations of mice. At each passage tumor fragments were collected for histolopathological and molecular characterization. A model was considered established after observing stable histological and molecular features for at least two passages. To evaluate the stability of the genomic profile we performed low-coverage whole-genome sequencing on DNA isolated from tumors obtained from at least two passages. In addition RNA-seq was used to better characterize the mutational profile of the ex-mouse tumors. Results: Until now 139 STS samples from consenting patients treated at the University Hospitals, Leuven, Belgium, have been transplanted. Twenty six well-characterized, stable STS PDX models have been established, maintaining the histopathological and molecular features of the original tumor. Seventeen models, analyzed with low-coverage DNA sequencing, showed a stable genomic profile in at least two different tumor passages. At this point the panel includes models of gastrointestinal stromal tumor (6 models), myxofibrosarcoma (5), dedifferentiated liposarcoma (3), malignant peripheral nerve sheath tumor (3), synovial sarcoma (2), leiomyosarcoma (3) epithelioid haemangioendothelioma (1), mesenchymal chondrosarcoma (1) and undifferentiated high grade sarcoma (2). Some of these models have already been successfully used for in vivo testing of novel agents, including both targeted and cytotoxic (pro-)drugs, and results served as a rationale for at least four prospective clinical trials. In addition 22 other xenografts are still in early stages of engraftment, not yet fulfilling our criteria of an “established model”. Conclusion: Our panel of mesenchymal PDX models is characterized by stable histological and molecular features. These clinically well-annotated models can contribute to reliable preclinical studies for new anticancer treatments for STS. The availability of unique, rare STS xenografts also allows to study the biology of these diseases. The platform is made available to collaborators from academia and industry. Citation Format: Agnieszka Wozniak, Jasmien Cornillie, Yemarshet K. Gebreyohannes, Bram Boeckx, Haifu Li, Thomas Van Looy, Giuseppe Floris, Jasmien Wellens, Lise Vreys, Daphne Hompes, Marguerite Stas, Friedl Sinnaeve, Diether Lambrechts, Maria Debiec-Rychter, Raf Sciot, Patrick Schoffski. Patient-derived xenograft (PDX) models of soft tissue sarcoma (STS): a preclinical platform for early drug testing. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5197.