Using genome-wide CRISPR library screening with library resistant DCK to find new sources of Ara-C drug resistance in AML.
Acute myeloid leukemia (AML) can display de novo or acquired resistance to cytosine arabinoside (Ara-C), a primary component of induction chemotherapy. To identify genes capable of independently imposing Ara-C resistance, we applied a genome-wide CRISPR library to human U937 cells and exposed to them to Ara-C. Interestingly, all drug resistant clones contained guide RNAs for DCK. To avoid DCK gene modification, gRNA resistant DCK cDNA was created by the introduction of silent mutations. The CRISPR screening was repeated using the gRNA resistant DCK, and loss of SLC29A was identified as also being capable of conveying Ara-C drug resistance. To determine if loss of Dck results in increased sensitivity to other drugs, we conducted a screen of 446 FDA approved drugs using two Dck-defective BXH-2 derived murine AML cell lines and their Ara-C sensitive parental lines. Both cell lines showed an increase in sensitivity to prednisolone. Guide RNA resistant cDNA rescue was a legitimate strategy and multiple DCK or SLC29A deficient human cell clones were established with one clone becoming prednisolone sensitive. Dck-defective leukemic cells may become prednisolone sensitive indicating prednisolone may be an effective adjuvant therapy in some cases of DCK-negative AML.
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.|Department of Comprehensive Pathology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo, Japan.
Morito
M
Kurata
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
Susan K
SK
Rathe
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
Natashay J
NJ
Bailey
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
Natalie K
NK
Aumann
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
Justine M
JM
Jones
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.
G Willemijn
GW
Veldhuijzen
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.|Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.|Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.
Branden S
BS
Moriarity
Masonic Cancer Center, University of Minnesota, Minneapolis, MN, USA.|Department of Pediatrics, University of Minnesota, Minneapolis, MN, USA.|Center for Genome Engineering, University of Minnesota, Minneapolis, MN, USA.|Brain Tumor Program, University of Minnesota, Minneapolis, MN, USA.
David A
DA
Largaespada
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002999:Clone Cells; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003561:Cytarabine / Q000494:pharmacology / Q000627:therapeutic use*; D018076:DNA, Complementary / Q000235:genetics; D003842:Deoxycytidine Kinase / Q000235:genetics; D003907:Dexamethasone / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D033721:Equilibrative Nucleoside Transporter 1 / Q000235:genetics; D015723:Gene Library; D056426:Genetic Loci; D005820:Genetic Testing; D015894:Genome, Human; D005938:Glucocorticoids / Q000494:pharmacology; D006801:Humans; D020128:Inhibitory Concentration 50; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000201:enzymology / Q000235:genetics; D051379:Mice; D009154:Mutation / Q000235:genetics; D011239:Prednisolone / Q000494:pharmacology; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D011965:Receptors, Glucocorticoid / Q000378:metabolism; D015203:Reproducibility of Results; D020298:U937 Cells
D018076:DNA, Complementary; D033721:Equilibrative Nucleoside Transporter 1; D005938:Glucocorticoids; D000094704:RNA, Guide, CRISPR-Cas Systems; D011965:Receptors, Glucocorticoid; D003561:Cytarabine; D003907:Dexamethasone; D011239:Prednisolone; D003842:Deoxycytidine Kinase