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Secondary fusion proteins as a mechanism of BCR::ABL1 kinase-independent resistance in chronic myeloid leukaemia.

ABSTRACT: 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.

SUBMITTER: Barnes EJ

PROVIDER: S-EPMC9851972 | biostudies-literature | 2023 Feb

REPOSITORIES: biostudies-literature

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Secondary fusion proteins as a mechanism of BCR::ABL1 kinase-independent resistance in chronic myeloid leukaemia.

Barnes Evan J EJ Eide Christopher A CA Kaempf Andy A Bottomly Daniel D Romine Kyle A KA Wilmot Beth B Saunders Dominick D McWeeney Shannon K SK Tognon Cristina E CE Druker Brian J BJ

British journal of haematology 20221020 3

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, differen ...[more]

PMID: 36264026

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Project description:BackgroundDespite improved patient outcome using tyrosine kinase inhibitors (TKIs), chronic myeloid leukaemia (CML) patients require life-long treatment due to leukaemic stem cell (LSC) persistence. LSCs reside in the bone marrow (BM) niche, which they modify to their advantage. The BM provides oncogene-independent signals to aid LSC cell survival and quiescence. The bone-morphogenetic pathway (BMP) is one pathway identified to be highly deregulated in CML, with high levels of BMP ligands detected in the BM, accompanied by CML stem and progenitor cells overexpressing BMP type 1 receptors- activin-like kinases (ALKs), especially in TKI resistant patients. Saracatinib (SC), a SRC/ABL1 dual inhibitor, inhibits the growth of CML cells resistant to the TKI imatinib (IM). Recent studies indicate that SC is also a potent ALK inhibitor and BMP antagonist. Here we investigate the efficacy of SC in overcoming CML BCR::ABL1 dependent and independent signals mediated by the BM niche both in 2D and 3D culture.MethodsCML cells (K562 cell line and CML CD34+ primary cells) were treated with single or combination treatments of: IM, SC and the BMP receptors inhibitor dorsomorphin (DOR), with or without BMP4 stimulation in 2D (suspension) and 3D co-culture on HS5 stroma cell line and mesenchymal stem cells in AggreWell and microfluidic devices. Flow cytometry was performed to investigate apoptosis, cell cycle progression and proliferation, alongside colony assays following treatment. Proteins changes were validated by immunoblotting and transcriptional changes by Fluidigm multiplex qPCR.ResultsBy targeting the BMP pathway, using specific inhibitors against ALKs in combination with SRC and ABL TKIs, we show an increase in apoptosis, altered cell cycle regulation, fewer cell divisions, and reduced numbers of CD34+ cells. Impairment of long-term proliferation and differentiation potential after combinatorial treatment also occurred.ConclusionBMP signalling pathway is important for CML cell survival. Targeting SRC, ABL and ALK kinases is more effective than ABL inhibition alone, the combination efficacy importantly being demonstrated in both 2D and 3D cell cultures highlighting the need for combinatorial therapies in contrast to standard of care single agents. Our study provides justification to target multiple kinases in CML to combat LSC persistence.

Project description:BackgroundIt is difficult for chronic myeloid leukemia (CML) patients with BCR::ABL1 independent drug resistance to achieve optimal efficacy. The aim of this study is to investigate the BCR::ABL1 kinase independent mechanism of tyrosine kinase inhibitor (TKI) resistance in CML patients to develop targeted therapeutic strategy.MethodsHerein, we analyzed the long non-coding RNA (lncRNA) and messenger RNA (mRNA) expression profiles of patients who achieved sustained deep molecular response (DMR) after TKI treatment and patients with non-DMR using RNA-seqencing. Furthermore, the differentially expressed lncRNAs and mRNAs were identified. The expression of chosen lncRNA was validated in an expanded cohort, and bioinformatics analysis was performed to analyze the function of selected mRNA.ResultsLncRNA data analysis indicated the diversity lncRNA profiles among healthy individuals, CML patients with non-DMR, and CML patients with DMR. Differential expression analysis and Veen plot of up-regulated lncRNAs in patients with non-DMR (compared with healthy individuals) and down-regulated lncRNAs in patients with DMR (compared to patients with non-DMR) revealed that lncRNA CBR3-AS1 overexpression might be related to BCR::ABL1 independent TKI resistance of CML patients. The expression of CBR3-AS1 was then verified in an expanded cohort, suggesting that, compared with control group, there was no statistical difference of CBR3-AS1 expression in DMR group, whereas, CBR3-AS1 was up-regulated in non-DMR group. Moreover, the mRNA data analysis of RNA-sequencing was performed. We considered genes that up-regulated in non-DMR group (compared with control group), down-regulated in DMR group (compared with non-DMR group), showed no statistical difference between control and DMR group as the potential genes that associated with TKI resistance of CML patients. A total of 55 corresponding mRNAs were obtained including KCNA6, a target gene of CBR3-AS1. Further bioinformatics analysis showed that the major interacted genes of KCNA6 were enriched in several resistance-associated pathways including interleukin -17 signaling pathway and cyclic adenosine monophosphate signaling pathway.ConclusionsIn conclusion, this work indicates that CBR3-AS1 might be involved in BCR::ABL1 independent TKI resistance of CML patients through targeting KCNA6, providing a novel target for intervention treatment of CML patients with BCR::ABL1 independent TKI resistance.

Project description:BackgroundChronic myeloid leukaemia (CML) is one of the most well characterised human malignancies. Most patients have a cytogenetically visible translocation between chromosomes 9 and 22 which generates the pathognomonic BCR::ABL1 fusion gene. The derivative chromosome 22 ('Philadelphia' or Ph chromosome) usually harbours the fusion gene encoding a constitutively active ABL1 kinase domain. A small subset of patients have no visible translocation. Historically, these 'Philadelphia chromosome negative' patients caused diagnostic confusion between CML and other myeloproliferative neoplasms; it is now well established that the BCR::ABL1 fusion gene can be generated via submicroscopic intrachromosomal insertion of ABL1 sequence into BCR, or, more rarely, of BCR into ABL1. The fusion genes arising from cryptic insertions are not detectable via G-banded chromosome analysis [karyotype] but can nevertheless always be detected using fluorescence in situ hybridisation (FISH) and/or qualitative reverse transcriptase PCR.Case presentationA 43-year-old female presented with suspected CML in 2007; however, contemporaneous gold standard laboratory investigations, G-banded chromosome analysis and FISH, were both negative. The reverse transcriptase quantitative PCR (RT-qPCR) assay available at the time, which was capable of detecting the common BCR::ABL1 transcripts (e13a2/e14a2), was also negative. Upon review in 2009, the newly recommended reverse transcriptase multiplex PCR (capable of detecting all BCR::ABL1 transcripts including the atypical ones) subsequently detected an e19a2 fusion. The patient then responded to tyrosine kinase inhibitor therapy. In contrast, FISH studies of both samples with three commercially available probes remained consistently negative. Retrospective whole genome sequencing, undertaken as part of the 100,000 Genomes Project, has now revealed that the patient's BCR::ABL1 fusion gene arose via a uniquely small insertion of 122 kb ABL1 sequences into BCR.ConclusionsWe present a patient with suspected chronic myeloid leukaemia whose genetic investigations were originally negative at the time of diagnosis despite the use of contemporaneous gold standard methods. This is the first report of a FISH-negative, BCR::ABL1 positive CML which demonstrates that, even after sixty years of research into one of the most well understood human malignancies, whole genome sequencing can yield novel diagnostic findings in CML.

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Secondary fusion proteins as a mechanism of BCR::ABL1 kinase-independent resistance in chronic myeloid leukaemia.

Publications

Secondary fusion proteins as a mechanism of BCR::ABL1 kinase-independent resistance in chronic myeloid leukaemia.

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