Cytarabine-induced differentiation of AML cells depends on Chk1 activation and shares the mechanism with inhibitors of DHODH and pyrimidine synthesis.

Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors.

Tomic B ，Smoljo T ，Lalic H ，Dembitz V ，Batinic J ，Batinic D ，Bedalov A ，Visnjic D ... -  《Scientific Reports》

The ribonucleoside AICAr induces differentiation of myeloid leukemia by activating the ATR/Chk1 via pyrimidine depletion.

Metabolic pathways play important roles in proliferation and differentiation of malignant cells. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr), a precursor in purine biosynthesis and a well-established activator of AMP-activated protein kinase (AMPK), induces widespread metabolic alterations and is commonly used for dissecting the role of metabolism in cancer. We have previously reported that AICAr promotes differentiation and inhibits proliferation of myeloid leukemia cells. Here, using metabolic assays, immunoblotting, flow cytometry analyses, and siRNA-mediated gene silencing in leukemia cell lines, we show that AICAr-mediated differentiation was independent of the known metabolic effects of AMPK, including glucose consumption, but instead depends on the activation of the DNA damage-associated enzyme checkpoint kinase 1 (Chk1) induced by pyrimidine depletion. LC/MS/MS metabolomics analysis revealed that AICAr increases orotate levels and decreases uridine monophosphate (UMP) levels, consistent with inhibition of UMP synthesis at a step downstream of dihydroorotate dehydrogenase (DHODH). AICAr and the DHODH inhibitor brequinar had similar effects on differentiation markers and S-phase arrest, and genetic or pharmacological Chk1 inactivation abrogated both of these effects. Our results delineate an AMPK-independent effect of AICAr on myeloid leukemia differentiation that involves perturbation of pyrimidine biosynthesis and activation of the DNA damage response network.

Dembitz V ，Tomic B ，Kodvanj I ，Simon JA ，Bedalov A ，Visnjic D ... -  《-》

5-aminoimidazole-4-carboxamide ribonucleoside induces differentiation in a subset of primary acute myeloid leukemia blasts.

Dembitz V ，Lalic H ，Kodvanj I ，Tomic B ，Batinic J ，Dubravcic K ，Batinic D ，Bedalov A ，Visnjic D ... -  《BMC CANCER》

Effects of selective checkpoint kinase 1 inhibition on cytarabine cytotoxicity in acute myelogenous leukemia cells in vitro.

Schenk EL ，Koh BD ，Flatten KS ，Peterson KL ，Parry D ，Hess AD ，Smith BD ，Karp JE ，Karnitz LM ，Kaufmann SH ... -  《-》

Bone marrow stromal cells enhance differentiation of acute myeloid leukemia induced by pyrimidine synthesis inhibitors.

Smoljo T ，Lalic H ，Dembitz V ，Tomic B ，Batinic J ，Vrhovac R ，Bedalov A ，Visnjic D ... -  《-》