Chromosomal rearrangements (CR) initiate leukemogenesis in approximately 50 % of acute myeloid leukemia (AML) patients
however, limited targeted therapies exist due to a lack of accurate molecular and genetic biomarkers of refractory mechanisms during treatment. Here, we investigated the pathological landscape of treatment resistance and relapse in 16 CR-AML patients by monitoring cytogenetic, RNAseq, and genome-wide changes among newly diagnosed, refractory, and relapsed AML. First, in FISH-diagnosed KMT2A (MLL gene, 11q23)/AFDN (AF6, 6q27)-rearrangement, RNA-sequencing identified an unknown CCDC32 (15q15.1)/CBX3 (7p15.2) gene fusion in both newly diagnosed and relapsed samples, which is previously unknown in KMT2A/AFDN-rearranged AML patients. Second, the unreported CCDC32/CBX3 gene fusion significantly affected the expression of wild-type genes of both CCDC32 (essential for embryonic development) and CBX3 (an oncogene for solid tumors) during the relapse, as demonstrated by Quantitative PCR analyses. Third, we further confirmed the existence of triple biomarkers - KMT2A/AFDN (AF6, 6q27) rearrangement, the unknown CCDC32 (15q15.1)/CBX3 (7p15.2) gene fusion and chimeric RNA variants (treatment-resistant leukemic blasts harboring distinct breakpoints) in a 21-year-old male patient of rapid relapsed/refractory AML. Most intriguingly, in this work regarding 16 patients, patients 7 and 20 initially showed the KMT2A/AFDN gene fusion
upon relapse, patient 20 did not show this fusion. On the other hand, patient 7 retained the KMT2A/AFDN fusion at diagnosis and during the relapse, only identified by PCR and Sanger's Sequencing, not by cytogenetics. Interestingly, the chimeric CCDC32/CBX3 gene fusion persisted in the 21-year-old male patient over the diagnostic and relapse phases. Most intriguingly, the overexpression of CCDC32/CBX3 fusion gene in AML patient-specific MV4-11 cells confirms the functional validation, providing experimental evidence of the biological impact of the CCDC32/CBX3 fusion on AML pathogenesis and treatment resistance by promoting cell cycle progression, a mechanism through which AML evolves to become treatment-resistant. All these might exhort differential resistance to treatment. Thus, we found that prognostic and predictive triple biomarkers - KRAS mutated, dual fusions (KMT2A/AFDN, CCDC32/CBX3), and chimeric variants - might evolve with a potential oncogenic role of subclonal evolution for poor clinical outcomes.