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Thus, it is plausible that GCA inhibits CUL3-KLHL20 or another K48-linked ubiquitin ligase to stabilize ULK1. in imatinib resistance. Our findings represent the basis for novel therapeutic strategies against CML. Abbreviation: ACTB/-actin: actin beta; ADM: adrenomedullin; AMBRA1: autophagy and beclin 1 regulator 1; AMPK: AMP-activated protein kinase; ANXA5: annexin A5; CP: cytogenetic response; CML: chronic myeloid leukemia; CUL3: cullin 3; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GCA: grancalcin; Dx: at diagnosis; E-64-d: (2S,3S)-trans-Epoxysuccinyl-L-leucylamido-3-methylbutane ethyl ester; IMres: Imatinib resistance; KLHL20: Kelch-like protein 20; LRMP: lymphoid-restricted membrane protein; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MMR: major molecular response; NH4Cl: ammonium chloride; PBMCs: peripheral blood mononuclear cells; PTPRC: protein tyrosine phosphatase, receptor type, C; SQSTM1/p62: sequestosome 1; SYK: spleen associated tyrosine kinase; TAP1: transporter 1, ATP binding cassette subfamily B member; TKIs: ABL-specific tyrosine kinase inhibitors; TLR9: toll- like receptor 9; TRAF6: TNF receptor associated factor 6; ULK1: unc-51 like autophagy activating kinase 1 [5] and [6] are widely used to treat CML [7]. Imatinib, the first designed TKI, represents effective therapy for CML [8]. Despite these COG 133 impressive results, a few patients are COG 133 resistant to imatinib. Approximately 10% of CML patients show primary imatinib resistance within 3?months of imatinib treatment [9,10]. Furthermore, nearly 20% of CML patients in chronic phase (CML-CP), who were previously sensitive to imatinib, develop resistance [11,12]. The most frequently identified mechanism of acquired secondary imatinib resistance involves point mutations associated with BCR-ABL kinase domain name that inhibit imatinib binding [13]. However, mutation or amplification is not observed in 50% or more of IM-resistant CML patients [14C16] and the molecular basis of such BCR-ABL-independent imatinib resistance is poorly comprehended. Autophagy (self [auto]-eating [phagy]) is usually a lysosomal recycling mechanism that leads to sequestration of intracellular components into autophagosomes and lysosomal degradation by autophagosome-lysosome fusion [17]. Autophagy initiation starts with the activation of ULK1 and AMBRA1 complexes following AMPK activation and MTOR inhibition [18,19]. Interestingly, autophagy induction leads to dephosphorylation of AMBRA1 and COG 133 conversation with the E3-ligase TRAF6 to support Lys63-linked ubiquitination of ULK1, and its subsequent activation [19]. Notably, the inhibition of BCR-ABL by TKIs induces autophagy [20] that is a cell-survival response in CML stem cells [21]. Several recent studies have shown that the combination of TKIs and autophagy inhibitors represent an effective treatment for CML [22C25]. However, the molecular bridge linking autophagy with imatinib resistance remains in vague. GCA (grancalcin) is usually a cytosolic protein that is translocated to the granule membrane upon neutrophil activation [26,27]. It belongs to a group of EF-hand Ca2+?binding proteins including CAPN (calpain), ALG2, and SRI (sorcin), which show structural rather than functional similarity [28]. Although it has been reported that GCA modulates Toll-like receptor 9 (TLR9)-mediated signaling via direct conversation with TLR9 [29], a genetic knockout of in mice failed to show relevant phenotypes and the physiological role of GCA remains to be elucidated [30]. In this report, we identified GCA as a potential novel molecule driving imatinib resistance in CML-CP through activating autophagy. Our study showed that GCA facilitates K63-linked ubiquitination of ULK1 by activating TRAF6, resulting in autophagy induction, which is COG 133 critical for imatinib resistance in CML patients. These findings open new perspectives for the treatment of CML. Results GCA was highly expressed in imatinib-resistant CML-CP patients To identify candidate genes for imatinib resistance in CML-CP patients, we performed microarray-based transcriptome profiling of peripheral blood mononuclear cells (PBMCs) obtained from two groups: imatinib-sensitive (achieving major molecular response within 6?months of imatinib treatment, with no mutations detected; n =?5) and resistant (CP but, no cytogenetic response within 6?months of imatinib treatment, and mutations were not detected; n =?6) patients (Physique 1a). We also confirmed mRNA expression in the same samples indicating that imatinib treatment was effective in sensitive but not in the resistant group (Physique S1a). We selected 970 candidate genes based on fold change greater than 1.5 and DDR1 a p-value less than 0.01 (Supplementary Material 1) and performed unsupervised clustering. Two major clusters emerged: one group of 5 imatinib-sensitive patients clustered; another group of 6 imatinib-resistant COG 133 samples clustered, of which most genes are upregulated while some portion of genes are downregulated in imatinib-resistant CML-CP patients (Physique S1b). Accordingly, principal component analysis (PCA) showed successful transcriptomic reprogramming in imatinib-resistant patient group away from imatinib sensitive patient group (Physique S1c). For further analysis, microarray data were submitted.