E3 ubiquitin ligase Atrogin-1 mediates adaptive resistance to KIT-targeted inhibition in gastrointestinal stromal tumor

KIT/PDGFRA oncogenic tyrosine kinase signaling is the central oncogenic event in most gastrointestinal stromal tumors (GIST), which are human malignant mesenchymal neoplasms that often feature myogenic differentiation. Although targeted inhibition of KIT/PDGFRA provides substantial clinical benefit, GIST cells adapt to KIT/PDGFRA driver suppression and eventually develop resistance. The specific molecular events leading to adaptive resistance in GIST remain unclear. By using clinically representative in vitro and in vivo GIST models and GIST patients’ samples, we found that the E3 ubiquitin ligase Atrogin-1 (FBXO32)—the main effector of muscular atrophy in cachexia—resulted in the most critical gene derepressed in response to KIT inhibition, regardless the type of KIT primary or secondary mutation. Atrogin-1 in GISTs is transcriptionally controlled by the KIT-FOXO3a axis, thus indicating overlap with Atrogin-1 regulation mechanisms in nonneoplastic muscle cells. Further, Atrogin-1 overexpression was a GIST-cell-specific pro-survival mechanism that enabled the adaptation to KIT-targeted inhibition by apoptosis evasion through cell quiescence. Buttressed on these findings, we established in vitro and in vivo the preclinical proof-of-concept for co-targeting KIT and the ubiquitin pathway to maximize the therapeutic response to first-line imatinib treatment.This project was funded by the 2014 SARC International Career Development Award (SARC Sarcoma Spore 1U54CA168512–01), Fundación Mari Paz Jiménez Casado, FERO Foundation, Spanish Society of Medical Oncology (SEOM), PERIS SLT006/17/221, ISCIII PI16/01371 and PI19/01271, all to C.S. ISCIII FI20/00275 (to DG-P), and a Ph.D. fellowship from the National Secretary for Higher Education, Science, Technology and Innovation of Ecuador (SENESCYT) (to DFP-J). AE-C is funded by ISCIII PT17/0009/0019 and co-funded by FEDER

The microphthalmia-associated transcription factor is involved in gastrointestinal stromal tumor growth

Gastrointestinal stromal tumors (GISTs) are the most common neoplasms of mesenchymal origin, and most of them emerge due to the oncogenic activation of KIT or PDGFRA receptors. Despite their relevance in GIST oncogenesis, critical intermediates mediating the KIT/PDGFRA transforming program remain mostly unknown. Previously, we found that the adaptor molecule SH3BP2 was involved in GIST cell survival, likely due to the co-regulation of the expression of KIT and Microphthalmia-associated transcription factor (MITF). Remarkably, MITF reconstitution restored KIT expression levels in SH3BP2 silenced cells and restored cell viability. This study aimed to analyze MITF as a novel driver of KIT transforming program in GIST. Firstly, MITF isoforms were characterized in GIST cell lines and GIST patients' samples. MITF silencing decreases cell viability and increases apoptosis in GIST cell lines irrespective of the type of KIT primary or secondary mutation. Additionally, MITF silencing leads to cell cycle arrest and impaired tumor growth in vivo. Interestingly, MITF silencing also affects ETV1 expression, a linage survival factor in GIST that promotes tumorigenesis and is directly regulated by KIT signaling. Altogether, these results point to MITF as a key target of KIT/PDGFRA oncogenic signaling for GIST survival and tumor growth

Multi-omic characterization of gastrointestinal stromal tumor (GIST) in a large real-world patient cohort

11522 Background: Molecular knowledge of GIST is limited due to its rarity, few genes have been identified as relevant determinants of outcomes, tumor evolution and therapeutic targets. Therefore, we aimed to dissect the GIST molecular landscape in the largest series of real-world patients reported to date. Methods: 946 GIST patient samples (536 localized, 369 metastatic, 41 unknown) underwent next-gen sequencing of DNA (592-gene, N = 495; whole exome, N = 451) and RNA (whole transcriptome, N = 592) at Caris Life Sciences (Phoenix, AZ). Gene expression signatures of proliferation (Cristescu, 2021), cell cycle activation (CINSARC; Chibon, 2010), inflammation (T-cell inflamed; Ayers, 2017) and tumor microenvironments (MCP-counter; Becht, 2016) were examined. Statistical significance tested by χ 2 , Fisher’s exact, or Mann-Whitney U as appropriate. Results: GIST samples were comprised of 80% (N = 758) KITmut, 8.1% (N = 77) PDGFRAmut, and 11.7% (N = 111) KIT/PDGFRA wild-type (WT), with 14.8% (N = 140) samples harboring a secondary KIT variant suggestive of TKI resistance. Overall median TMB was 2 mutations/MB (range 0-13). WT GIST were identified with mutations in NF1 (33.7%), DNA repair genes (16.7%), SDHX (8.2%), BRAF (6.3%), and PTEN (1.9%), along with NTRK3 fusions (3.1%). Primary KIT variants occurred in exons 11 (83.5%), 9 (13.9%), and 13 (2.6%), and secondary KIT variants (14.6% of total KIT mutations) were distributed across the ATP binding pocket (36.8%) and activation loop (63.2%). Primary PDGFRA mutations were in exons 18 (62.4%), 12 (11.0%), 14 (4.6%) and other (16.5%). KIT/PDGFRAmut GIST infrequently harbored RB1, TP53, SETD2, ARID1A, PIK3CA, PTEN, TSC1, BRCA1, or CHEK2 co-mutations (1-5% each) . Copy number amplification (≥6 copies) was overall uncommon (≤2% for all genes). Proliferation and cell cycle activation signatures were higher in KIT exon 11 indels v. missense mut (1.2-fold, p < 0.05) and KIT resistant v. KIT primary (1.2-fold, p < 0.05), but not in KIT exon 11 557/558 v. others, nor between KIT v. PDGFRA v. WT subgroups. Deletion of tumor progression genes MAX (40.0%), CDKN2A (32.3%), and DEPDC5 (33.9%) was associated with increased proliferative gene expression (1.2-, 1.3-, and 1.2-fold, p < 0.05 each), while DMD deletion (52.5%) was not (1.1-fold, p = 0.37). Compared to KITmut and WT GIST, PDGFRAmut had increased abundance of several immune cell populations (range 1.2-3.7-fold, p < 0.05), along with enhanced inflammation signatures (1.1- and 1-2-fold, p < 0.05). Conclusions: This series provides unprecedented resolution of KIT/PDGFRAmut GIST with features of clinical aggressiveness associated with KIT exon 11 indels and resistance mutations, illustrating a specific cytogenetic genotype with more aggressive growth and malignant behavior. Identification of less common molecular alterations that drive kinase activation and impaired DNA damage repair warrant further investigation

Circulating tumor DNA (ctDNA) analyses of the phase III VOYAGER trial: KIT mutational landscape and outcomes in patients with advanced gastrointestinal stromal tumor (GIST)

101 Background: The genotype of primary mutations predicts imatinib response in untreated metastatic GIST. However, the sequence of salvage treatments in metastatic GIST is based solely on the chronological order of registration trials. ctDNA sequencing offers a powerful diagnostic tool to detect resistance mutations in GIST but has not been shown to correlate with outcomes in clinical trials of pretreated patients (pts). We analyzed ctDNA samples collected at baseline in the phase III VOYAGER trial (NCT03465722) to describe the landscape of KIT alterations and its association with outcomes of pts treated with avapritinib or regorafenib. Methods: In VOYAGER, 476 pts with advanced KIT-mutant GIST were randomly assigned to avapritinib (240 pts) or regorafenib (236 pts) in 3rd-4th line. Baseline plasma was collected and ctDNA analyzed with the Guardant 360 (G360), 74-gene panel. KIT molecular subgroups were determined and correlated with outcomes. PDGFRA-mutant GISTs were excluded from outcomes analysis. Results: Baseline ctDNA analysis was performed in 386/476 pts (81%). ctDNA was detected in 333 pts (86%), with 250 and 18 pts showing at least one KIT (75%) or PDGFRA (5%) variant, respectively. KIT primary mutations were detected in 71% pts (exon 11, 56%; exon 9, 14%; exon 13, 1%) and KIT secondary mutations in 55% of pts. Activation loop (AL, exons 17 and 18) was more commonly affected (44%) than the ATP-binding pocket (ABP, exons 13 and 14; 23%). Among KIT-mutant tumors, multiple KIT mutations were commonly detected within individual tumors (mean, 2.56; range, 1-14). Notably, 17% of pts had > 3 mutations (mean, 6.07; range, 4 to 14). Median PFS and OS were shorter for patients whose ctDNA was positive for V654A or T670I (ABP hot spots) when treated with avapritinib vs. regorafenib: mPFS, 1.9 mo vs. 7.4 mo; log-rank p <.001; mOS, 8.3 mo vs. 11.7 mo; log rank p =.0651. mPFS was shorter for patients with ctDNA positive for KIT exon 17 mutation if concurrently KIT V654A/T670I was absent when treated with avapritinib, with no difference in OS: mPFS, 4.7 mo vs. 6.7 mo; log-rank p =.03; mOS, 19.2 mo vs. NR; log-rank p =.628. mPFS on avapritinib was longer when ABP mutations were absent when compared to those with ABP present (5.6 vs. 1.9 mo; log-rank p <.001). There were no differences considering AL mutations vs. no AL mutations (3.8 vs. 3.9 mo; log-rank p =.622) when treated with avapritinib. Regorafenib showed similar activity regardless of KIT mutational status and the location of KIT mutation. Conclusions: Hybrid capture-based plasma sequencing detects ctDNA in the majority of patients with advanced TKI-resistant GIST, including heterogeneity of KIT mutations. This study is the first to show that ctDNA sequencing correlates with outcomes in pretreated GIST. Identification of ABP (exon13/14) KIT mutations negatively correlates with avapritinib activity