Surmounting Cytarabine-resistance in acute myeloblastic leukemia cells and specimens with a synergistic combination of hydroxyurea and azidothymidine

Abstract

Acute myeloid leukemia (AML) patients display dismal prognosis due to high prevalence of refractory and relapsed disease resulting from chemoresistance. Treatment protocols, primarily based on the anchor drug Cytarabine, remained chiefly unchanged in the past 50 years with no standardized salvage regimens. Herein we aimed at exploring potential pre-clinical treatment strategies to surmount Cytarabine resistance in human AML cells. We established Cytarabine-resistant sublines derived from human leukemia K562 and Kasumi cells, and characterized the expression of Cytarabine-related genes using real-time PCR and Western blot analyses to uncover the mechanisms underlying their Cytarabine resistance. This was followed by growth inhibition assays and isobologram analyses testing the sublines’ sensitivity to the clinically approved drugs hydroxyurea (HU) and azidothymidine (AZT), compared to their parental cells. All Cytarabine-resistant sublines lost deoxycytidine kinase (dCK) expression, rendering them refractory to Cytarabine. Loss of dCK function involved dCK gene deletions and/or a novel frameshift mutation leading to dCK transcript degradation via nonsense-mediated decay. Cytarabine-resistant sublines displayed hypersensitivity to HU and AZT compared to parental cells; HU and AZT combinations exhibited a marked synergistic growth inhibition effect on leukemic cells, which was intensified upon acquisition of Cytarabine-resistance. In contrast, HU and AZT combination showed an antagonistic effect in non-malignant cells. Finally, HU and AZT synergism was demonstrated on peripheral blood specimens from AML patients. These findings identify a promising HU and AZT combination for the possible future treatment of relapsed and refractory AML, while sparing normal tissues from untoward toxicity.

Venetoclax and alvocidib are both cytotoxic to acute myeloid leukemia cells resistant to cytarabine and clofarabine

“…Thus, more reduced kinases produced less CAFdATP in HL-60/CAFdA30 cells than in HL-60/CAFdA4 cells. The importance of the kinases was demonstrated previously in studies of cellular resistance to nucleoside analogs [7,17,39,43,44] and found to be associated with therapeutic outcomes [43,45,46]. Previously established nucleoside analog-resistant cell lines showed reduced cell surface hENT1 expression [16,17].…”

Deciphering molecular mechanisms underlying chemoresistance in relapsed AML patients: towards precision medicine overcoming drug resistance

“…Performing an analysis, such as the one presented here, prior to the treatment decision might have revealed a superior personalized treatment modality, for example by targeting the DNSP using a combination of azidothymidine (AZT) and hydroxyurea (HU) as we have recently demonstrated [42]. This plausible treatment modality could be specifically viable in light of the upregulation of TK1 (Fig.…”

“…Similarly, leukemic cells that survived Ara-C cytotoxicity might be cross-resistant to other nucleoside analog prodrugs that share the same cellular uptake and activation routes. Consideration of drug action mechanisms and metabolism could further suggest potential combinations that may readily overcome chemoresistance modalities [42,[140][141][142][143].…”

“…Thereafter, Ara-C is sequentially phosphorylated to Ara-CTP by deoxycytidine kinase (dCK), deoxycytidylate kinase (dCMPK), and finally by nucleotide diphosphate kinase (NDK) [30]. Accordingly, commonly reported Ara-C resistance mechanisms include downregulation of ENT1 [11,[33][34][35] or CNT3 [36], loss of function of dCK [21,34,35,[37][38][39][40][41][42], or upregulation of the catabolic enzymes cytidine deaminase (CDA) and deoxycytidine monophosphate deaminase (dCMPD) [17,24,43]. Unlike other chemotherapeutic agents, enhanced Ara-C efflux is not an established mechanism of drug resistance.…”

Deciphering the Therapeutic Resistance in Acute Myeloid Leukemia

“…In particular, the authors studied the impact of 10 different polymorphisms in Ara-C metabolic pathway genes in a cohort of 225 adult patients with de novo AML showing the association of activating (deoxycytidine kinase, DCK) and deactivating (cytidine deaminase, CDA) genes variants with PIF [ 47 ]. As a matter of fact, increased levels of CDA are associated with Ara-C resistance in AML [ 48 , 49 ]. Moreover, in vitro studies showed a higher level of CDA activity in Ara-C resistant AML cell lines and demonstrated also the negative impact on the CDA A79C polymorphism [ 50 ].…”