A selective BCL-X PROTAC degrader achieves safe and potent antitumor activity
Authors: Janet S Wiegand
Daohong Zhou
Adolfo A Ferrando
Guangcun Huang
Natalia Baran
Yaxia Yuan
Anna Rogojina
Guangrong Zheng
Dinesh Thummuri
Peiyi Zhang
Vinitha M Kuruvilla
Peter J Houghton
Xingui Liu
Marina Konopleva
Yonghan He
Xuan Zhang
Sajid Khan
Yong-Mi Kim
Qi Zhang
Robert Hromas
Jing Pei
Christopher R McCurdy
Dongwen Lv
Abhisheak Sharma
Weizhou Zhang
Journal: Nature Medicine (2019)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: BCL-XL; PROTAC; ABT263; DT2216; Von Hippel-Lindau
Affiliations: Department of Pharmacodynamics, University of Florida, Gainesville, FL, USA., Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL, USA., Department of Leukemia, University of Texas M.D. Anderson Cancer Center, Houston, TX, USA., Department of Pathology, Immunology and Laboratory Medicine, College of Medicine, University of Florida, Gainesville, FL, USA., Department of Pharmaceutics, College of Pharmacy, University of Florida, Gainesville, FL, USA., Department of Pediatrics, Pathology, Cell Biology and Systems of Biology and Institute for Cancer Genetics, Columbia University, New York, NY, USA., Department of Pediatrics, Children's Hospital Los Angeles, Los Angeles, CA, USA., Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA., Department of Medicine, the Long School of Medicine, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA., Department of Medicinal Chemistry, College of Pharmacy, University of Florida, Gainesville, FL, USA. ., Department of Pharmacodynamics, University of Florida, Gainesville, FL, USA. .

Abstract:  B-cell lymphoma extra large (BCL-XL) is a well-validated cancer target. However, the on-target and dose-limiting thrombocytopenia limits the use of BCL-XL inhibitors, such as ABT263, as safe and effective anticancer agents. To reduce the toxicity of ABT263, we converted it into DT2216, a BCL-XL proteolysis-targeting chimera (PROTAC), that targets BCL-XL to the Von Hippel-Lindau (VHL) E3 ligase for degradation. We found that DT2216 was more potent against various BCL-XL-dependent leukemia and cancer cells but considerably less toxic to platelets than ABT263 in vitro because VHL is poorly expressed in platelets. In vivo, DT2216 effectively inhibits the growth of several xenograft tumors as a single agent or in combination with other chemotherapeutic agents, without causing appreciable thrombocytopenia. These findings demonstrate the potential to use PROTAC technology to reduce on-target drug toxicities and rescue the therapeutic potential of previously undruggable targets. Furthermore, DT2216 may be developed as a safe first-in-class anticancer agent targeting BCL-XL. 

Retracted: No

PubPeer comments:
 According to its description:  The target guide sequences are as follows: BAK-sg1, forward (5u2032-CACCGTCATCGGGGACGACATCAAC-3u2032) and reverse (5u2032-AAACGTTGATGTCGTCCCCGATGAC-3u2032); BAK-sg2, forward (5u2032-CACCGCTGCAACCTAGCAGGTGAGC-3u2032) and reverse (5u2032- AAACGCTCACCTGCTAGGTTGCAGC-3u2032); , it seems that none of the two guide RNAs would target the BAK. The first gRNA (TCATCGGGGACGACATCAAC) doesn t have the PAM following it, the second gRNA (CTGCAACCTAGCAGGTGAGC) has only 70% overlapping with the BAK gene. , 
 It looks like neither of the guides have a PAM following them, but they do have a PAM on the opposite side, so either the authors swapped the forward and reverse oligo sequences, or they cloned guides that cannot target the BAK gene. , 
 #1 Thank you very much for your comment. We checked the primers and noticed that the forward and reverse oligo sequences were inadvertently switched when preparing the manuscript. The correct primers are as follows: Bak-sg1: forward (5u2019-CACCGGTTGATGTCGTCCCCGATGA-3u2019) and reverse (5u2019-AAACTCATCGGGGACGACATCAACC-3u2019); The forward Oligo sequence was selected from Feng Zhangu2019s human_geckov2_library_a_09mar2015.csv with ID (HGLibA_04134). Bak-sg2: forward (5u2019-CACCGGCTCACCTGCTAGGTTGCAG-3u2019) and reverse (5u2019-AACCTGCAACCTAGCAGGTGAGCC-3u2019); The forward Oligo sequence was selected from Feng Zhangu2019s human_geckov2_library_a_09mar2015.csv with ID (HGLibA_04135). The sg2 gRNA targets an exon-intron junction. Thanks for pointing out this error. We will contact the journal and make corrections as soon as possible. , 
 #2 Thank you very much for your comment. We checked the primers and noticed that the forward and reverse oligo sequences were inadvertently switched when preparing the manuscript. The correct primers are as follows: Bak-sg1: forward (5u2019-CACCGGTTGATGTCGTCCCCGATGA-3u2019) and reverse (5u2019-AAACTCATCGGGGACGACATCAACC-3u2019); The forward Oligo sequence was selected from Feng Zhangu2019s human_geckov2_library_a_09mar2015.csv with ID (HGLibA_04134). Bak-sg2: forward (5u2019-CACCGGCTCACCTGCTAGGTTGCAG-3u2019) and reverse (5u2019-AACCTGCAACCTAGCAGGTGAGCC-3u2019); The forward Oligo sequence was selected from Feng Zhangu2019s human_geckov2_library_a_09mar2015.csv with ID (HGLibA_04135). The sg2 gRNA targets an exon-intron junction. Thanks for pointing out this error. We will contact the journal and make corrections as soon as possible. , 
 To Whom It May Concern: I am sorry for my oversight on the response submitted by one of the coauthors on the manuscript on my behalf. It was my fault that I did not carefully review the original experimental record before allowing him to submit the response. After carefully reviewing the record, it appeared that we made a mistake in the design of the gRNAs as indicated by you. The design of the gRNAs was based on the sequence information from Feng Zhangu2019s group as indicated in our previous response. The sg2 gRNA targets an exon-intron junction. However, we had the PAM on the opposite side of the gRNAs as pointed out by you. We were surprised that these gRNAs appeared reduced the expression of Bak in H146 cells. However, we did not perform DNA sequencing to confirm this finding as we did for other knockout experiments. We will make the following efforts to validate the results: 1. We will extract DNA from our Bak knockout H146 cells and perform Sanger sequencing to determine whether these gRNAs can induce frameshift on the corresponding region of Bak. 2. We will repeat the knockout experiment with these original gRNAs to determine if the knockout of Bak in the cells is an off-target effect or something unusual for the gene. 3. We will design correct gRNAs to repeat the experiment to confirm the findings reported in our manuscript. After completion of these experiments, I will send you an update and contact the editor at Nature Medicine to make necessary corrections. Meantime, I greatly appreciate your careful review of our manuscript and your concerns of our mistake. Thank you. Daohong Zhou ,