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Supplementary Figure from Tyrosine Kinase Inhibition Alters Intratumoral CD8+ T-cell Subtype Composition and Activity

Supplementary Table from Tyrosine Kinase Inhibition Alters Intratumoral CD8+ T-cell Subtype Composition and Activity

Targeted therapy with a tyrosine kinase inhibitor (TKI) such as imatinib is effective in treating gastrointestinal stromal tumor (GIST), but it is rarely curative. Despite the presence of a robust immune CD8+ T-cell infiltrate, combining a TKI with immune-checkpoint blockade (ICB) in advanced GIST has achieved only modest effects. To identify limitations imposed by imatinib on the antitumor immune response, we performed bulk RNA sequencing (RNA-seq), single-cell RNA-seq, and flow cytometry to phenotype CD8+ T-cell subsets in a genetically engineered mouse model of GIST. Imatinib reduced the frequency of effector CD8+ T cells and increased the frequency of naïve CD8+ T cells within mouse GIST, which coincided with altered tumor chemokine production, CD8+ T-cell recruitment, and reduced CD8+ T-cell intracellular PI3K signaling. Imatinib also failed to induce intratumoral T-cell receptor (TCR) clonal expansion. Consistent with these findings, human GISTs sensitive to imatinib harbored fewer effector CD8+ T cells but more naïve CD8+ T cells. Combining an IL15 superagonist (IL15SA) with imatinib restored intratumoral effector CD8+ T-cell function and CD8+ T-cell intracellular PI3K signaling, resulting in greater tumor destruction. Combination therapy with IL15SA and ICB resulted in the greatest tumor killing and maintained an effector CD8+ T-cell population in the presence of imatinib. Our findings highlight the impact of oncogene inhibition on intratumoral CD8+ T cells and support the use of agonistic T-cell therapy during TKI and/or ICB administration.

PDF file - 213K, Gene signatures distinguishing the different MB CSC populations segregate distinct human MB molecular subgroups, with MB1_EXCLUSIVE gene signature distinguishing the WNT molecular subgroup

PDF file - 183K, Long term-cultured hindbrain- and forebrain-derived NSCs are Sonic Hedgehog (Shh) pathway-independent, as cyclopamine treatment negatively affects the survival/proliferation of short-term cultured but not of long term-cultured NSCs

Supplementary Figure Legends

Supplementary Figure 1: Etv1 is required for intramuscular and myenteric ICC development. Supplementary Figure 2: Etv1 ablation with tamoxifen decreases Etv1 and Kit expression in Kit^delta558/+;Etv1^flox/flox;Rosa26^CreERT2/CreERT2 mice. Supplementary Figure 3: Etv1 positively regulates Kit expression in murine stomach. Supplementary Figure 4: ETV1 regulates KIT expression in GIST48 cells. Supplementary Figure 5: MAP kinase pathway inhibition decreases ETV1 target gene expression. Supplementary Figure 6: Dual lineage inhibition inhibits tumor initiation in vivo.

PDF file - 155K, Enforced expression of Ebf3 in CB WM and SVZ NSCs results in premature neuronal differentiation, under proliferative and differentiative conditions

Supplementary Table 2: Gene sets enriched among genes DOWNregulated by tamoxifen treatment (Etv1 ablation) in murine GIST-like tumors.

PDF file - 157K, MB CSC lines are Sonic Hedgehog (Shh) pathway-independent, as their survival/proliferation is not affected by cyclopamine treatment and Smo silencing

Supplementary Table 3: Gene sets enriched among genes UPregulated by tamoxifen treatment (Etv1 ablation) in murine GIST-like tumors.

PDF file - 104K, Details on experimental and bioinformatics procedures.

PDF file - 134K, Table S1 A. Lineage-specific differentiation of region-specific clonally derived NSCs is summarized as frequency of neurons, astrocytes and oligodendrocytes. B. Electrophysiological characteristics of neurons derived from the differentiation of region-specific NSCs. C. Analysis of the engraftment of region-specific clonally derived NSCs after transplantation in the early postnatal mouse cerebellum. Table S2 - Statistical analysis of GSEA for gene signatures generated by DEGs up regulated in hindbrain (CB/IVv) or forebrain (SVZ) NSCs. Table S3 - Lineage-specific differentiation of clonally derived MB CSCs is summarized as frequency of neuron-, astrocyte- and oligodendrocyte-like cells. Table S4 - Statistical analysis of GSEA for gene signatures generated by DEGs up regulated in tumorigenic Ptch+/- p53-/- MB CSCs vs. hindbrain NSCs (MB1_EXCLUSIVE), in non-tumorigenic Ptch+/- p53+/+ MB CSCs vs. hindbrain NSCs (MB2_EXCLUSIVE), and commonly expressed in both tumorigenic and non-tumorigenic CSCs vs. hindbrain NSCs (MB1_MB2 COMMON). Table S5 - Expression of EBF proteins in a collection of 33 human MB specimens as determined by immunohistochemistry.

Supplementary Table 1: Differentially expressed genes (p1.5) between oil and tamoxifen treated tumors to conditionally ablate Etv1.

PDF file - 130K, EBF3 expression can be retrieved in silico in human MBs by exploiting publicly available human data sets

PDF file - 132K, The gene signature containing the 297 genes differentially expressed between hindbrain- vs. forebrain-derived NSCs distinguishes the SHH molecular subgroup of human MBs

Introduction: Gastrointestinal stromal tumor (GIST) is the most common human sarcoma and is typically driven by a single mutation in the Kit receptor. While highly effective, tyrosine kinase inhibitors like imatinib are not curative. The natural ligand for the Kit receptor is Kit Ligand (KitL), which exists in both soluble and membrane-bound forms. We sought to determine the role of KitL in the oncogenic Kit signaling of GIST. Methods: KitV558Δ/+ mice, which spontaneously develop an intestinal GIST, were treated with imatinib or vehicle for 1 week, and sorted non-immune (CD45-) cells from tumors were submitted for single-cell RNA sequencing (n=3/group). GIST T1 cells were treated with KitL with or without imatinib and oncogenic Kit signaling was evaluated. KitV558Δ/+ mice were crossed with various KitL mutant and inducible models and tumor weights were measured (n=3-4/group). Human GISTs were stained for KitL expression by IHC, and an existing bulk RNA sequencing human GIST dataset with matched human serum was analyzed. Results: Unsupervised clustering revealed endothelial, smooth muscle, and tumor cells had high expression of KitL RNA in untreated tumors from KitV558Δ/+ mice. Imatinib therapy increased KitL RNA in all 3 cell types, suggesting that extra-tumoral KitL expression is dependent on oncogenic signaling (p Citation Format: Andrew D. Tieniber, Ferdinando Rossi, Andrew Hanna, Mengyuan Liu, Mark Etherington, Kevin Do, Laura Wang, Ronald P. DeMatteo. Multiple intratumoral sources of kit ligand promote oncogenic kit signaling in gastrointestinal stomal tumor [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr A017.

Introduction: MHC class I (MHC I) expression is necessary for the antitumoral lymphocyte response but its relationship to oncogene signaling is not well characterized. Gastrointestinal stromal tumor (GIST) is a common sarcoma caused by an activating Kit mutation and is sensitive to the specific Kit inhibitor imatinib. Using KitV558Δ/+ mice, a murine model of GIST, we found imatinib reduced MHC I expression on tumor cells, which we hypothesized was due to attenuation of type I interferon (IFN) signaling in the setting of oncogene inhibition. Methods: Using flow cytometry and Western blot, we studied tumoral MHC I expression in human GIST cell lines T1 and HG129 under conditions of IFN stimulation and imatinib inhibition. We applied the same methods in vivo in KitV558Δ/+ mice. To show that IFN signaling was responsible for tumoral MHC I expression, we used a blocking anti-IFNAR antibody to abrogate IFN signaling in KitV558Δ/+ mice. Finally, we validated our findings with RNA seq of 35 Kit exon 11 human GIST. Results: In human GIST cell lines, IFNα (1000U/mL) stimulated MHC I expression at 24h as measured on cell surface by mean florescence intensity (MFI) and by Western blot. This was accompanied by increased Stat1 and pStat1 protein. Imatinib (100nM) inhibited Stat1 activity and reduced MHC I expression by 12-34% on MFI, with and without IFNα stimulation. When Stat1 was knocked down with SiRNA, imatinib did not further decrease MHC I. In vivo, KitV558Δ/+ mice treated with imatinib had 50% reduction in tumoral MHC I expression MFI by flow (n=5/group). Concurrently, imatinib diminished mRNA expression of IFNb1 in bulk KitV558Δ/+ tumors by 8-fold. To demonstrate that IFN signaling contributed to GIST MHC I expression, we administered a blocking anti-IFNAR antibody in KitV558Δ/+ mice and found MHC I decreased by 34% on MFI, along with decreased pStat1 on western blot. In human GIST, imatinib significantly decreased MHC I gene expression on RNA seq (p = 0.03) and reduced IFNb1 mRNA by 77% (p = 0.04). Furthermore, there was significant correlation between MHC I genes and Stat1 expression (p< 0.0001). Taken together, this suggests that oncogene-dependent IFN signaling is responsible for MHC I expression in GIST. Conclusion: Kit oncogene inhibition reduced MHC I expression in GIST through decreased type I interferon production and signaling via Stat1. Thus, targeted therapies may reduce the antitumor immune response. Citation Format: Mengyuan Liu, Benjamin D Medina, Geraldo A Vitiello, Mark S Etherington, Nesteene J. Param, Mia E DiLolle, Timothy G. Bowler, Ferdinando Rossi, Ronald P. DeMatteo. Kit inhibition decreases tumoral MHC class I expression in gastrointestinal stromal tumors through reduction of type I interferon signaling [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2018 Nov 27-30; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(4 Suppl):Abstract nr B27.

Gastrointestinal stromal tumour (GIST) is one of the most common types of human sarcoma and is primarily defined by activating mutations in the KIT or PDGFRA receptor tyrosine kinases. Despite the initial clinical success of imatinib that specifically target mutant KIT and PDGFRA, imatinib resistance has become the biggest challenge in the management of advanced GIST patient. Novel therapeutics that can improve the efficacy of first line imatinib therapy and/or prevent and overcome imatinib resistance is imperative. The ETS family member, ETV1, has been previously identified as a lineage-specific survival factor that cooperates with mutant KIT by ETV1 protein stabilization by active MAP kinase signaling downstream of KIT signaling in GIST oncogenesis. Here we demonstrate that ETV1 is required for GIST initiation and maintenance in vivo using compound genetically engineered mouse models (GEMM). We further identified that ETV1 forms a feed forward circuit in GIST oncogenesis where the ETV1 protein is stabilized by active MAP kinase signaling downstream of KIT and stabilized ETV1enhances KIT expression through direct binding to the KIT enhancer regions in both GIST mouse models and human GIST cell lines. The dual lineage targeting of KIT by imatinib and ETV1 by MEK162 (an MEK inhibitor) disrupts the ETV1-KIT feed forward circuit and induces more apoptosis than single agent imatinib or MEK162 in human GIST cells. The combination therapy resulted in complete tumor regression whereas single agent imatinib or MEK162 resulted in stabilization of disease in human GIST xenograft studies. Moreover, the combination therapy also induced more tumor fibrosis than single agent imatinib or MEK162 in GIST GEMM. These observations demonstrate the in vivo role of ETV1 in GIST oncogenesis and the feasibility of targeting ETV1 protein stability by inhibiting MAP kinase signaling. They also suggest that the dual lineage targeting of ETV1 and KIT by the combination therapy may provide a more effective therapeutic strategy than imatinib alone in GIST management. Citation Format: Leili Ran, Inna Sirota, Zhen Cao, Devan Murphy, Shipra Shukla, Ferdinando Rossi, John Wongvipat, William D. Tap, Peter Besmer, Cristina R. Antonescu, Yu Chen, Ping Chi. Dual lineage inhibition of ETV1 and KIT disrupts the ETV1-KIT feed forward circuit and potentiates imatinib antitumor effect in GIST oncogenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3396. doi:10.1158/1538-7445.AM2014-3396