Introduction : Fibroblast growth factor receptors (FGFRs) are conserved single transmembrane receptor tyrosine kinases (RTKs) with an extracellular ligand-binding domain and a cytoplasmic tyrosine kinase domain. They play vital roles in cellular proliferation, survival, development, fetal organogenesis, metabolism, and tissue repair. 8p11 myeloproliferative syndrome (EMS) is a hematologic malignancy caused by a translocation of the fibroblast growth factor receptor 1 (FGFR1) gene on chromosome 8p11-12. The most common fusion partner of FGFR1 is the zinc-finger domain ZNF198 (also known as ZMYM2) on chromosome 13q12. Pemigatinib is approved for FGFR fusion positive myeloid/lymphoid neoplasms (MLNs), and other FGFR inhibitors are being tested in clinical trials. However, EMS patients still face a poor prognosis and drug resistance, necessitating further research to overcome these challenges. This study evaluated ex vivo drug screening assay data generated from ZMYM2::FGFR1 positive primary leukemia samples throughout the course of an EMS patient's clinical response and validated these findings on a ZMYM2::FGFR1 positive cell line.
Methods :Ex vivo and drug screening experiments were performed on freshly isolated primary patient samples and a ZMYM2::FGFR1-expressing cell line. The fusion was confirmed using FISH, and additional clinical specimens were collected with informed patient consent. Ex vivo drug sensitivity assays were conducted on patient mononuclear cells isolated from blood and bone marrow. IL-3 dependent BaF3 cells were infected with retrovirus containing a ZMYM2::FGFR1-V5 construct. ZMYM2::FGFR1 transformed BaF3 cells were evaluated with single agent and drug combination panels. Sanger sequencing of the FGFR portion of the fusion was performed on patient samples to confirm the breakpoint and identify potential mutations affecting sensitivity to Ponatinib throughout the course of treatment.
Results : Sample were obtained with informed consent from a 36-year-old man diagnosed with 8p11 myeloproliferative syndrome and a ZMYM2::FGFR1 fusion along with a RUNX1 R201Q mutation. Ex vivo drug screens performed on isolated mononuclear cells demonstrated sensitivity to Ponatinib and Pemigatinib. He underwent treatment with HyperCVAD plus Ponatinib and subsequently HyperCVAD with Ponatinib and Velcade (Bortezomib) to CR1, FISH for FGFR1 rearrangement was undetected. He was scheduled to proceed with hematopoietic stem cell transplant (HSCT), however his pre-transplant bone marrow biopsy showed return of his FGFR1 translocation by FISH. Ex vivo drug screening assays confirmed resistance to Ponatinib, but continued sensitivity to Pemigatinib. He was transitioned to treatment with single agent Pemigatinib and was able to re-clear his FGFR1 fusion by FISH, and has since undergone hematopoietic stem cell transplantation. He remains in remission on post-transplant Pemigatinib maintenance therapy. Sanger sequencing performed at the time of progression on Ponatinib identified a nucleotide alteration resulting in an amino acid substitution (FGFR1 F686L; ZMYM2::FGFR1 F1171L) within the kinase domain that could contribute to decreased Ponatinib sensitivity. Bortezomib and Axitinib exhibited high efficacy on the patient's sample. Ex vivo drug assays performed on ZMYM2::FGFR1 transformed BaF3 cells confirmed the efficacy of Pemigatinib and Ponatinib. Other FGFR inhibitors, including Olverematinib, AZD4547, Axitinib, Cediranib, Dovitinib, and Lenvatinib, also demonstrated exquisite sensitivity. Despite extensive screening, no other single agents or drug combinations exhibited increased effectiveness in the ZMYM2::FGFR1 transformed BaF3 cells except for Trametinib, a MEK inhibitor, and the combination of Belvarafenib (RAF inhibitor) and Gilteritinib (FLT3 inhibitor).
Conclusions :Ex vivo drug sensitivity assays demonstrated the highly selective efficacy of FGFR inhibitors in ZMYM2::FGFR1 fusion-positive leukemia cells and a fusion-expressing BaF3 cell line. Mutations in the FGFR1 kinase domain (ZMYM2::FGFR1 F1171L) could contribute to Ponatinib insensitivity. These ex vivo drug screening results provide further support for ongoing clinical trials which are investigating the use of single agent Pemigatinib and other FGFR1 inhibitors for the treatment of patients with FGFR1-fusion positive leukemias.
Disclosures
Leonard:Takeda: Consultancy; Adaptive Biotechnologies: Consultancy, Membership on an entity's Board of Directors or advisory committees, Other: Travel, accommodations, expenses; Kite/Gilead: Consultancy; Pfizer: Consultancy. Druker:US Patent and Trademark Office: Patents & Royalties: Patents 6958335 (Novartis exclusive license), 4326534, 7416873, 7592142, 10473667, 10664967, 11049247; Aileron Therapeutics: Membership on an entity's Board of Directors or advisory committees; Recludix Pharma, Inc.: Consultancy, Current holder of stock options in a privately-held company; CureOne: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees, Other: PI or Co-investigator on clinical trial(s) funded via contract with OHSU., Research Funding; AstraZeneca: Other: PI or Co-investigator on clinical trial(s) funded via contract with OHSU.; Enliven Therapeutics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees, Research Funding; Oregon Health & Science University: Current Employment, Patents & Royalties: #1518 (exclusive option agreement with CytoImage); #0606/Patent 6958335 (Novartis exclusive license); #2573; #0843; #0996; Gilead: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Incyte: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Tolero: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Syndax: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Therapy Architects, LLC: Membership on an entity's Board of Directors or advisory committees; Bristol Myers Squibb: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Dana-Farber Cancer Institute: Patents & Royalties: #2063 (licensed exclusively to Merck & Co); and #2524, Research Funding; DNA SEQ: Membership on an entity's Board of Directors or advisory committees; Multicancer Early Detection (MCED) Consortium: Membership on an entity's Board of Directors or advisory committees; Beat AML LLC: Membership on an entity's Board of Directors or advisory committees; Vincerx Pharma, Inc.: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; VB Therapeutics: Membership on an entity's Board of Directors or advisory committees; The RUNX1 Research Foundation: Membership on an entity's Board of Directors or advisory committees; Labcorp: Membership on an entity's Board of Directors or advisory committees; Nemucore Medical Innovations, Inc.: Membership on an entity's Board of Directors or advisory committees; Iterion Therapeutics: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Celgene: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; Astellas: Other: Co-investigator on clinical trial(s) funded via contract with OHSU.; GRAIL: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Cepheid: Membership on an entity's Board of Directors or advisory committees; Burroughs Wellcome Fund: Membership on an entity's Board of Directors or advisory committees; Blueprint Medicines: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Aptose Biosciences: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Adela, Inc.: Current holder of stock options in a privately-held company, Membership on an entity's Board of Directors or advisory committees; Amgen: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Tognon:Notable Labs: Research Funding.
