Abstract GS3-09: Loss of ASXL1 tumor suppressor promotes resistance to CDK4/6 inhibitors in ER+ breast cancer

• Published in: Cancer research (Chicago, Ill.) Ill.), 2022-02, Vol.82 (4_Supplement), p.GS3-09-GS3-09
• Main Authors: Sudhan, Dhivya R., Chatterjee, Sumanta, Kim, Jiwoong, Wang, Yunguan, Kandagatla, Vishal, Ye, Dan, Lin, Chang-Ching, Zanudo, Jorge Gomez Tejeda, Jain, Esha, Marin, Arnaldo, Servetto, Alberto, Lee, Kyung-min, Povedano, Juan Manuel, McFadden, David, Barrett, Alex, Wagle, Nikhil, Hanker, Ariella B., Arteaga, Carlos L.
• Format: Article
• Language: English
• ISSN: 0008-5472; 1538-7445
• DOI: 10.1158/1538-7445.SABCS21-GS3-09

Background: CDK4/6 inhibitors (CDK4/6i) in combination with antiestrogens have prolonged survival of patients with ER+ metastatic breast cancer. However, this combination is not curative mainly due to acquired drug resistance. Knowledge about mechanisms of such resistance remains quite incomplete. We report herein a forward-genetics screen to discover a broad spectrum of novel somatic mutations causal to CDK4/6i resistance. Methods: We used CRISPR/Cas9 to delete the DNA mismatch repair (MMR) gene MSH2 in MCF7 and T47D ER+ breast cancer cells. Deficiency of DNA MMR proteins such as MSH2 results in a high nucleotide substitution rate which, in turn, predisposes cells to acquire drug resistance-associated mutations. MSH2-/- MCF7 and T47D cells were infected with a lentiviral barcode library containing ~1000 unique DNA barcodes. MSH2-/- barcoded cells were expanded for ~25 doublings to allow the accumulation of random mutations. Clones resistant to CDK4/6i were selected in the presence of IC90 of palbociclib (200 nM) or abemaciclib (500 nM) for 4-6 weeks. CDK4/6i resistant clones with unique barcode IDs were subjected to whole exome sequencing (WES). Results: Following drug selection, ~73 uniquely barcoded resistant colonies emerged from MCF7 and T47D MSH2-/- clonal lines. As expected, MCF7 and T47D MSH2-/- clones harbored a high mutation burden compared to parental cells. Candidate variants were distilled based on (a) functionality prediction and (b) mutation frequency in Project GENIE. We observed RB1 (5/73 clones; 6.8%) mutations in CDK4/6i resistant clones, providing proof-of-principle that this approach can identify clinically-relevant drug resistant alterations. Overall, we identified non-synonymous alterations in 2,206 genes in T47D palbociclib-resistant, 2,195 genes in T47D abemaciclib-resistant, and 1,312 genes in MCF7 palbociclib-resistant lines. A secondary screen of the 10 genes recurrently mutated in all three CDK4/6i resistant groups identified loss of ASXL1 as top hit. ASXL1 encodes a polycomb repressive complex protein that regulates chromatin accessibility. Loss of ASXL1 has been implicated in myeloid transformation through epigenetic reprogramming. WES of 76 CDK4/6i resistant tumor biopsies (DFCI/MBCproject cohort) identified ASXL1 alterations in two and four patients with acquired and primary resistance, respectively (6/76=7.9%). One of the tumors that progressed after an initial response to palbociclib had acquired the same ASXL1 R549C muta