Authors
Evan J. Barnes1, Christopher A. Eide1, Daniel Bottomly2, Beth Wilmot2, Shannon K. McWeeney2, Cristina E. Tognon1, Brian J. Druker1, 1Division of Hematology and Medical Oncology, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA
2Division of Bioinformatics and Computational Biology, Department of Medical Informatics and Clinical Epidemiology, Knight Cancer Institute, Oregon Health & Science University, Portland, Oregon, USA
Disclosures
B.J.D. potential competing interests-- SAB: Aileron Therapeutics, Therapy Architects (ALLCRON), Cepheid, Vivid Biosciences, Celgene, RUNX1 Research Program, Nemucore Medical Innovations, Novartis, Gilead Sciences (inactive), Monojul (inactive); SAB & Stock: Aptose Biosciences, Blueprint Medicines, EnLiven Therapeutics, Iterion Therapeutics, Third Coast Therapeutics, GRAIL (SAB inactive); Scientific Founder: MolecularMD (inactive, acquired by ICON); Board of Directors & Stock: Amgen, Vincera Pharma; Board of Directors: Burroughs Wellcome Fund, CureOne; Joint Steering Committee: Beat AML LLS; Founder: VB Therapeutics; Sponsored Research Agreement: EnLiven Therapeutics; Clinical Trial Funding: Novartis, Bristol-Myers Squibb, Pfizer; Royalties from Patent 6958335 (Novartis exclusive license) and OHSU and Dana-Farber Cancer Institute (one Merck exclusive license and one CytoImage, Inc. exclusive license)
Introduction
Chronic myeloid leukemia (CML) is defined by the presence of the BCR-ABL1 fusion protein, which results in constitutively active ABL1 tyrosine kinase activity. Although most chronic phase CML patients can be successfully treated with ABL1 tyrosine kinase inhibitors (TKIs), such as imatinib, up to one third of CML patients require alternative treatment. While the most common cause of TKI resistance in CML is the acquisition of BCR-ABL1 kinase domain mutations, many patients demonstrate BCR-ABL1 kinase-independent resistance through other secondary molecular changes. Unfortunately, most of these aberrations are poorly understood and may confer cross-resistance to multiple approved BCR-ABL1-targeted therapies. Previous reports have described additional chromosomal rearrangements in CML patients at the time of disease transformation to blast crisis (Nucifora and Rowley, Blood 1995; Branford et al., Blood 2018). We hypothesized that secondary fusion proteins may contribute to BCR-ABL1 kinase-independent drug resistance in CML.
Methods
To explore this, we performed paired-end RNA sequencing on a cohort of 91 unique patients comprising three groups: BCR-ABL1 kinase-independent resistance (n=42), BCR-ABL1 kinase-dependent resistance (n=26), and newly diagnosed disease (n=23). Fusions were called using the STAR and TopHat methods, and in-frame fusion transcripts called by both methods were prioritized for analysis. To further evaluate the potential contribution of these fusions to TKI resistance, we retrovirally co-expressed the confirmed fusion protein constructs with BCR-ABL1 in murine Ba/F3 cells. Cell lines were screened against a panel of approved ABL1 TKIs in vitro for 72 hours and analyzed using methanethiosulfonate (MTS)-based assays.
Results
We identified 11 secondary fusions which were recurrently observed among patients with BCR-ABL1 kinase-independent resistance, including both novel fusions and previously identified fusion proteins such as RUNX1-MECOM and CBFB-MYH11. Fusion breakpoint sequences were amplified via PCR in primary patient specimens at the time of resistance and confirmed by Sanger sequencing for 6 of the identified fusions: RUNX1-MECOM, CBFB-MYH11, KDM7A-MKRN1, PDPK1-ATP6V0C, TRDV2-TRAC, and ZNF292-PNRC1. As a case example, a 49-year-old male with CML in accelerated phase and a history of relapse following interferon-alpha therapy and autologous stem cell transplant was treated with imatinib at 600 mg QD. Despite rapidly achieving a complete cytogenetic response (CCR) and subsequent major molecular response, relapse was observed after 16 months, prompting an increase to 800 mg QD. At this higher dose, slight cytogenetic improvement was observed, though the patient failed to regain CCR. At this time, presence of a secondary KDM7A-MKRN1 fusion was confirmed. Intriguingly, we confirmed that expression of KDM7A-MKRN1 was associated with varying degrees of decreased sensitivity to tested ABL1 TKIs, most pronouncedly for imatinib. Evaluation of drug sensitivity for additional fusions is underway and will be presented.
Conclusion
Our findings suggest that secondary fusions, some of which are cytogenetically cryptic, beyond BCR-ABL1 are present in a subset of patients with BCR-ABL1 kinase-independent resistance and demonstrate decreased sensitivity to TKI treatment in vitro. Further characterization of the molecular mechanisms associated with these fusions in the context of BCR-ABL1 open opportunities for identifying new combination treatment strategies to overcome this type of resistance and improve outcomes for these patients.
References
Nucifora G, Rowley JD. AML1 and the 8;21 and 3;21 translocations in acute and chronic myeloid leukemia. Blood. 1995 Jul 1;86(1):1-14.
Branford S. et al. Integrative genomic analysis reveals cancer-associated mutations at diagnosis of CML in patients with high-risk disease. Blood. 2018 Aug 30;132(9):948-961.
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