G3BP2-KIT drives leukemia amenable to kinase inhibition in Ph-like ALL

TO THE EDITOR:

Philadelphia-like (Ph-like) or BCR-ABL1–like B-cell precursor acute lymphoblastic leukemia (B-ALL) is genetically heterogenous,^1,2  with a gene expression profile similar to that of BCR-ABL1⁺ ALL.^3,4  It comprises 15% to 30% of B‐ALLs, with a peak of incidence in young adults.^5,6  The most commonly mutated pathways are the ABL and JAK-STAT pathways with multiple rearrangements and lesions, which are druggable with a variety of tyrosine kinase inhibitors (TKIs).^7,8  Rarer and more heterogeneous alterations, although often targetable with TKIs (eg, ETV6-NTRK3),⁹  may be missed by targeted genomic diagnostic approaches. We describe a case of Ph-like ALL and a novel gene fusion identified by RNA sequencing (RNA-seq) that juxtaposed exon 7 of G3BP2 with exon 11 of KIT and encoded a fusion protein retaining the nuclear transport factor 2 domain of G3BP2 and the entire kinase domain of KIT. In hematological malignancies, activating KIT mutations play a crucial role in systemic mastocytosis and acute myeloid leukemia¹⁰ ; however, fusions have not been described in leukemia or in other tumors.

We report a 52-year-old White man with second relapsed B-ALL after chemotherapy and blinatumomab (registered at www.clinicaltrials.gov as #NCT02013167).¹¹  A bone marrow aspirate specimen obtained at study entry in the INOVATE trial (#NCT01564784)¹²  was analyzed by RNA-seq, given the patient’s therapeutic resistance to date. At relapse, the patient had 51% blast cells in bone marrow by flow cytometric analysis. Of note, the patient was not treated with TKIs, and primary leukemia cells were not available for functional studies.

The fusion gene G3BP2-KIT was synthesized by the BioXp 3200 system (Codex DNA, Inc.) and cloned into a Gateway®-compatible cl20–murine stem cell virus–internal ribosome entry site–green fluorescent protein (cl20-MSCV-G3BP2-KIT-IRES-GFP) lentiviral vector. The truncated (TR) transcript portion of KIT fused to G3BP2 and coding for amino acids 560 to 976 (TR KIT 560-976AA) was amplified and cloned (cl20–MSCV–TR KIT 560-976AA–IRES–GFP). These constructs and the empty vector (MIG) were used for virus production and expression in interleukin-3 (IL-3)–dependent mouse hematopoietic Ba/F3 cells and IL-7–dependent ARF-null pre-B cells.¹³  Cell proliferation was examined in the absence and presence of cytokines. Sublethally irradiated (550 rad) 8-week-old wild-type C57BL/6 female mice underwent transplantation by tail vein injection of ARF-null pre-B cells expressing G3BP2-KIT or empty vector. Leukemia development was monitored by retroorbital bleeding; moribund mice were euthanized, and blood, bone marrow, and spleen samples were analyzed for evidence of leukemia by flow cytometry, histopathology, and genomic sequencing. Full methods are provided in the data supplement.

This B-ALL sample was predicted to be Ph-like B-ALL using prediction analysis of microarrays (Ph score, 1) and clustered with Ph-like B-ALL on t-distributed stochastic neighbor embedding analysis in our B-ALL subtyping pipeline.¹⁴  Moreover, RNA-seq detected a chimeric fusion between exon 7 of G3BP2 (chromosome 4:76580296, hg19) and exon 11 of KIT (chromosome 4:55593611, hg19), juxtaposing the amino acid residues 2227 and 560 of G3BP2 and KIT, respectively, and retaining the nuclear transport factor 2 domain of G3BP2 and part of the catalytic domain and the entire kinase domain of KIT (Figure 1A; supplemental Table 1). This fusion was not previously detected in larger cohorts of B-ALL cases.^2,5,14  Although G3BP2 is ubiquitously expressed in the different hematopoietic lineages (supplemental Figure 1A), KIT is not normally expressed in B-lymphoid cells¹⁴  (supplemental Figure 1B). Thus, the fusion drives ectopic expression of the KIT transcript region fused to G3BP2 in B cells, as demonstrated by RNA-seq (Figure 1B). Analysis of sequence mutations detected few additional alterations, but not in recurrently mutated genes in ALL, except for ETV6, RPL22, and NT5C2¹⁵  (supplemental Table 2), suggesting that the chimeric fusion transcript may have been a sufficient leukemogenic driver lesion in this patient. The fusion protein lacks the transmembrane domain of KIT, and immunofluorescence analysis of transiently transfected HEK293T cells showed a cytoplasmic localization (supplemental Figure 2A-B).

To investigate its oncogenic role, we cloned and expressed G3BP2-KIT fusion transcript, TR KIT (TR KIT 560-976AA), or empty viral vector in IL-3–dependent Ba/F3 (Figure 1C) and IL-7–dependent ARF-null pre-B cells (Figure 1D). Only the cells expressing G3BP2-KIT sustained cytokine-independent proliferation, suggesting that 5′ G3BP1 is necessary to confer oncogenic properties to TR KIT, likely by promoting KIT dimerization. Both Ba/F3 and ARF-null pre-B cells expressing G3BP2-KIT, but not those transduced with empty vector, showed phosphorylation of KIT, activation of JAK-STAT signaling with phosphorylated STAT3 and STAT5, MAPK pathway activation with phosphorylation of p44/p42 MAPK, and phosphorylation of phospholipase C (Figure 1E; supplemental Figure 3). Notably, transplantation of ARF-null pre-B cells expressing G3BP2-KIT in C57BL6 sublethally irradiated mice resulted in the development of serially transplantable B-ALL (Figure 2A), positive for B-cell lymphoid markers B220, CD19, and PAX5 (supplemental Figure 4A-B). Tumors were characterized by splenomegaly (Figure 2B) and multiorgan infiltration (supplemental Figure 4C). Whole-exome sequencing did not detect pathogenetic cooperating driver sequence mutations or copy-number changes (supplemental Table 3), confirming that G3BP2-KIT is a necessary and sufficient oncogenic driver alteration. Mouse leukemic cells showed enrichment of genes involved in JAK/STAT signaling (eg, CSF3R, IL2RA, CXCL3, IL10RA, and PIK3R5; supplemental Figure 4D-E), concordant with phosphorylation of STAT5 and STAT3 in Ba/F3 and ARF-null pre-B cells expressing G3BP2-KIT (Figure 1E).

Activating mutations in KIT have been described in acute myeloid leukemia, gastrointestinal stromal tumor, most cases of systemic mastocytosis, and other tumors.¹⁰  They result in activation of the receptor in the absence of ligand, playing a critical role in cell proliferation and differentiation. In these tumors where KIT deregulation is the driver oncogenic event, treatment with TKIs has been a successful approach.¹⁶  Therefore, we next examined the effect of kinase inhibition in Ba/F3 and ARF-null cell lines expressing G3BP2-KIT. These cells showed sensitivity to a wide range of TKIs with different specificities against KIT and the highest sensitivity to the dual SRC/ABL inhibitor dasatinib (Figure 2C; supplemental Figure 5A). In vitro treatment resulted in cell death and loss of KIT phosphorylation, indicating that constitutive activation of KIT is an important oncogenic signaling pathway for this fusion (supplemental Figure 5B). To confirm the efficacy of tyrosine kinase inhibition on leukemia cells in vivo, the established genetically engineered models of G3BP2-KIT–driven leukemia were randomized to receive dasatinib (20 mg/kg per day by oral gavage; n = 10) or vehicle (n = 10). Dasatinib prolonged survival in the engineered fusion mouse cell models, but it was not curative (Figure 2D).

G3BP2 is a member of the family of Ras-GTPase–activating SH3 domain–binding proteins, which includes 2 additional family members, G3BP1 and G3BP3. These are RNA-binding proteins that are ubiquitously expressed and, under extracellular stresses, bind to untranslated regions of target transcripts and downregulate translation by either degrading the transcript or blocking ribosomal recruitment.¹⁷  Recently, a chimeric G3BP1-PDGFRB fusion sensitive to ABL1/PDGFRB kinase inhibition¹⁸  was described in a patient with a myeloid neoplasm with eosinophilia.

In conclusion, these results identify KIT rearrangement as a driver oncogenic alteration in leukemia and provide the rationale for therapeutic targeting with TKIs in KIT-rearranged Ph-like B-ALL.

Acknowledgments: The authors thank the Animal Resources Center and the Small Animal Imaging Center, the Compound Management Center, the Department of Chemical Biology & Therapeutics, the Department of Structural Biology for the use of BioXp 3200 system, the Genome Sequencing Facility of the Hartwell Center for Bioinformatics and Biotechnology, and the Flow Cytometry and Cell Sorting core facility of St. Jude Children’s Research Hospital.

This work was supported in part by the American Lebanese Syrian Associated Charities of St. Jude Children’s Research Hospital, the Leukemia and Lymphoma Society Translational Research Program (C.G.M.), a St. Baldrick’s Foundation Robert J. Arceci Innovation Award (C.G.M.), the Henry Schueler 41 & 9 Foundation (C.G.M.), National Cancer Institute, National Institute of Health, Outstanding Investigator Award R35 CA197695 (C.G.M.), and a Research Agreement with Pfizer, Inc. (C.G.M).

Contribution: I.I. designed, directed, and performed research, analyzed data, and wrote the manuscript; R.F., J.D.F., P.B., and E.B. performed research and analyzed data; C.Q. performed genomic analyses; L.J.J. performed histopathology analyses; Y.Z. performed sample processing; P.C. contributed to phosphoproteomic analysis; A.S. contributed to drug screening analysis; A.D.L. and A.S.A contributed to conception and design of clinical sample analyses; C.G.M. edited the manuscript and gave final approval; and all authors critically reviewed the manuscript before submission.

Conflict-of-interest disclosure: I.I. reports honoraria from Amgen and Mission Bio. A.D.L. reports employment and stock and/or other ownership interests in Pfizer. A.S.A. reports consulting for Jazz Pharmaceuticals, Taiho Pharmaceutical, Team Pharmaceuticals, Amgen, Pfizer, Nkarta, Kite Pharmaceuticals, and Glycomimetics and research funding from Servier, Incyte, Amgen, Pfizer, Glycomimetics, Kite Pharmaceuticals, Seattle Genetics, Immunogen, and Macrogenics. C.G.M. reports research funding from AbbVie, Loxo Oncology, and Pfizer; speaking and travel fees from Illumina and Amgen; and stock ownership in Amgen. The remaining authors declare no competing financial interests.

Correspondence: Ilaria Iacobucci, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 343, Memphis TN 38105; e-mail: ilaria.iacobucci@stjude.org; and Charles G. Mullighan, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, Mail Stop 342, Memphis TN 38105; e-mail: charles.mullighan@stjude.org.