Abstract A06: Strategies to circumvent resistance to cyclin-dependent kinase-9 inhibition (CDK9i) in non-Hodgkin lymphoma (NHL)
Authors: Thieme Elana , Sun Duanchen , Bruss Nur , Sharma Geeta , Liu Tingting , Coleman Daniel , Nechiporuk Tamilla , Bottomly Daniel , McWeeney Shannon , Pirrotte Patrick , Xia Zheng , Danilov Alexey 
Published: September 2022

Abstract
Introduction Oncogenic programs are facilitated by activators of transcriptional machinery, including certain CDKs. CDK9, a component of the positive transcription elongation factor b (pTEFb) complex, has arisen as an attractive target due to its regulation of MYC and MCL1 transcription (Hashiguchi et al, 2019). Nevertheless, we and others have observed resistance to CDK9i in vitro and in vivo. Here we studied the effects of CDK9 inhibition using the novel selective CDK9 inhibitor AZD4573, currently under evaluation in clinical trials. Methods A panel of NHL cell lines (OCI-LY3/19, SUDHL4/10/16, VAL, U2932) and primary NHL cells were employed. Response to CDK9i was characterized using LC-MS proteomic analysis, RNA-Seq, and CRISPR-Cas9 Screening. Results NHL cells treated with AZD4573 for 6h exhibited a dose dependent reduction in phospho-RNAPIISer2, as well as loss of MYC and Mcl-1. CDK9i potently inhibited proliferation and induced apoptosis in a panel of NHL cell lines (IC50 range 5-30 nM). Two DLBCL cell lines underwent LC-MS proteomic analysis following AZD4573 treatment (30 nM, 3h). Treated cells exhibited rapid loss of MYC, Mcl-1, PIM3 and JUNB protein levels. We observed broad transcriptional repression via RNA-seq, including downregulation of PIM3 and JUNB (30 nM, 3h). However, a subset of genes, including MYC, PIM1 and JUNB underwent early transcriptional recovery, confirmed by immunoblotting, thus identifying candidate genes which may account for resistance to CDK9i. PIM kinases cooperate with the PI3K/ATK signaling pathway, and have been proposed as therapeutic targets in cancer. We next used SGI1776 (PIM1 specific) and AZD1208 (pan-PIM) in combination with AZD4573, and found synergy between them in a panel of 4 cell lines and primary samples. OCI-LY3 xenograft mice treated with a combination of AZD4573 (15 mg/kg; IP; once weekly) and AZD1208 (30 mg/kg; oral gavage, twice weekly) demonstrated restricted tumor growth and increased survival compared to control. To further understand pathways mediating resistance to CDK9i, we carried out a genome-wide loss of function CRISPR-Cas9 library screen. Two Cas9-expressing NHL cell lines were transduced with a CRISPR library comprised of ~5 unique sgRNA per gene. Loss of AKT, RPTOR, or mTOR, among others, sensitized cells to AZD4573. Concurrent treatment with PI3K inhibitors synergistically suppressed proliferation of NHL cell lines and primary cells treated with AZD4573 in vitro. OCI-LY3 xenograft mice were treated with AZD4573 (15 mg/kg; IP; once weekly), Copanlisib (15 mg/kg; IP; twice weekly), or a combination of both. Combo treatment restricted tumor growth and prolonged survival to a greater extent than either drug alone. Conclusions CDK9i with AZD4573 downregulated numerous oncoproteins. However, a subset of genes including MYC and PIM3 recovered transcription. PI3K/AKT pathway was implicated in resistance to CDK9i in CRISPR library screens. Concurrent targeting of pro-survival pathways (e.g., PIM, PI3K) partially reversed resistance to CDK9i.
Citation Format: Elana Thieme, Duanchen Sun, Nur Bruss, Geeta Sharma, Tingting Liu, Daniel Coleman, Tamilla Nechiporuk, Daniel Bottomly, Shannon McWeeney, Patrick Pirrotte, Zheng Xia, Alexey Danilov. Strategies to circumvent resistance to cyclin-dependent kinase-9 inhibition (CDK9i) in non-Hodgkin lymphoma (NHL) [abstract]. In: Proceedings of the Third AACR International Meeting: Advances in Malignant Lymphoma: Maximizing the Basic-Translational Interface for Clinical Application; 2022 Jun 23-26; Boston, MA. Philadelphia (PA): AACR; Blood Cancer Discov 2022;3(5_Suppl):Abstract nr A06.