https://openworks.mdanderson.org/sumexp22/1037/thumbnail.jp

Quesada, Andres Ernesto

Yang, Yaling

Ye, Matthew Tianyu

You, M James

You, Yun

English

OpenWorks @ MD Anderson

The Role of HNRNPC::RARG in APL-like AMLMatthew T. Ye, Yun You, Yaling Yang, Andres E. Quesada, M. James  YouDepartment of Hematopathology, MD Anderson Cancer Center❖ Acute promyelocytic leukemia (APL) is a subtype of AML characterized by t(15;17)(q24.1, q21.1)/PML::RARA❖ Standard of care typically involves ATRA and ATO❖ Recent cases have been identified that morphologically resemble APL but are resistant to ATRA and ATO❖ These cases involve RARG instead of RARA, with a myriad of fusion partners, like HNRNPC, a reader protein of m6A involved in pre-mRNA processing❖ Identification and treatment are urgent due to disseminated intravascular coagulopathy (DIC)BackgroundRecombinant cloning❖ HNRNPC and RARG were cloned separately into MSCVI vectors previously ❖ Exon 3 of HNRNPC CDS was then cloned with exons 4-10 of RARG CDS into the retroviral vector MSCVI❖ Fuse, insert, obtainMethodsFigure 1. APL (left) and “APL-like” AML (right). Figure 2. HNRNPC::RARG fusion (exon 3 HNRNPC CDS, exons 4-10 RARG CDS)  HNRNPC::RARG rearrangement plays a significant role in the transformation of “APL-like” leukemia and may serve as a marker for targeted therapyHypothesisa)b)Figure 3. Primers used for a) HNRNPCexon 3 b) RARG exons 4-10; BLUEanneals to RARG, red anneals to HNRNPC5’ ctttatccagccctcac5’ TGCTCTGTGTCTCCACCGctaaaacctggccagcaatc5’ gattgctggccaggttttagCGGTGGAGACACAGAGCAC5’ AGACTGCCTTGGGAAAAGCObtainFuseInsert❖ Primers designed for overlapping PCR were able to allow fusion and digestion by XhoI and BglII❖ Gel images showed correct band sizes for PCR, overlapping PCR, digestion products, and a validation digestion❖ In-silico product was generated using Benchling©❖MAAFT alignment against in-silico product confirmed correct HNRNPC::RARG fusion sequence without evidence of mutationResultsa) b)c) d)Figure 4. Gel images: a) PCR of HNRNPC exon 3, RARG exons 4-10; b) overlapping PCR to make HNRNPC-RARG, c) digestion of HNRNPC-RARG, d) validation digestion Figure 5. MSCVI-HNRNPC-RARG HA; created with Benchling©❖Obtain retrovirus via co-transfectionwith packaging plasmids in 293-T cells❖ Adapt a mouse model, transplanting infected HSCs into mice ❖Other fusions (e.g. HNRNPCL, CPSF6)Future directionsCreated with Biorender©Created with Biorender©Created with Biorender©Figure 6. MAAFT alignment of sequencing results; mismatches were determined to be artifactsZhu HH, Qin YZ, Hu J, Zhang ZL, Huang JY, et al. Acute myeloid leukemia with RARG rearrangement. (Under review)He L, Li H, Wu A, Peng Y, Shu G, et al. Functions of N6-methyladenosine and its role in cancer. Molecular Cancer. 2019; 18:176.References

The Role of HNRNPC-RARG in APL-like AML

https://openworks.mdanderson.org/cgi/viewcontent.cgi?article=1037&context=sumexp22

The Role of HNRNPC-RARG in APL-like AML

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