Summary Drug resistance in chronic myeloid leukaemia (CML) may occur via mutations in the causative BCR::ABL1 fusion or BCR::ABL1‐independent mechanisms. We analysed 48 patients with BCR::ABL1‐independent resistance for the presence of secondary fusion genes by RNA sequencing. We identified 10 of the most frequently detected secondary fusions in 21 patients. Validation studies, cell line models, gene expression analysis and drug screening revealed differences with respect to proliferation rate, differentiation and drug sensitivity. Notably, expression of RUNX1::MECOM led to resistance to ABL1 tyrosine kinase inhibitors in vitro. These results suggest secondary fusions contribute to BCR::ABL1‐independent resistance and may be amenable to combined therapies.
27 28 8p11 myeloproliferative syndrome (EMS) represents a unique WHO-classified hematologic malignancy 29 defined by translocations of the FGFR1 receptor. The syndrome is a myeloproliferative neoplasm 30 characterized by eosinophilia and lymphadenopathy, with risk of progression to either AML (acute myeloid 31 leukemia) or T or B-lymphoblastic lymphoma/leukemia. Within the EMS subtype, translocations between 32Breakpoint Cluster Region (BCR) and Fibroblast Growth Factor Receptor 1 (FGFR1) have been shown to 33 produce a dominant fusion protein that is notoriously resistant to tyrosine kinase inhibitors (TKIs). Here, 34 we report two cases of BCR-FGFR1 + EMS identified via RNA-seq and confirmed by FISH. Sanger 35 sequencing revealed that both cases harbored the exact same breakpoint. In the first case, the patient 36 presented with AML-like disease, and in the second, the patient progressed to B-ALL. Additionally, we 37 observed that that primary leukemia cells from Case 1 demonstrated sensitivity to the tyrosine kinase 38 inhibitors Ponatinib and Dovitinib that can target FGFR1 kinase activity, while primary cells from Case 2 39 were resistant to both drugs. Taken together these results suggest that some but not all BCR-FGFR1 fusion 40 positive leukemias may respond to TKIs that target FGFR1 kinase activity.
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 causes of TKI resistance in CML are BCR-ABL1 kinase domain mutations, many patients demonstrate BCR-ABL1 kinase-independent resistance through other poorly understood secondary molecular changes that mediate cell survival despite effective BCR-ABL1 kinase inhibition. 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), and we hypothesized that secondary fusion proteins may contribute to BCR-ABL1 kinase-independent resistance in CML. 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 analyzed. 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, TPM4-ACTB, TRDV2-TRAC, and ZNF292-PNRC1. To further evaluate the contribution of these fusion constructs to TKI resistance, we retrovirally co-expressed them with BCR-ABL1 in murine Ba/F3 cells and screened the cells against a panel of approved ABL1 TKIs in vitro using methanethiosulfonate (MTS)-based assays. 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. 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. Citation Format: Evan J. Barnes, Christopher A. Eide, Daniel Bottomly, Beth Wilmot, Shannon K. McWeeney, Cristina E. Tognon, Brian J. Druker. Secondary fusions as a mechanism of BCR-ABL1 kinase-independent resistance in chronic myeloid leukemia [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 38.
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