Abstract C083: SMNDC1 alters the splicing of ERK to potentiate its activity in pancreatic cancer
Authors: Siraj Md Afjalus , Zhang Yushan , Chakraborty Prabir , Tseng Robert , Ku Li-Ting , Goda Grant , Giri Gilbert , Yugawa Deanne , Wang Mathew , Das Shamik , Dey Anindya , Dwivedi Shailendra Dhar , Rao Geeta , Zhang Min , Yang Da , Hossen Md Nazir , Ding Wei-Qun , Fung Kar-Ming , Dominguez Daniel , Bhattacharya Resham , Escobar-Hoyos Luisa , Mukherjee Priyabrata 
Published: January 2024

Abstract
While oncogenic KRAS is believed to drive pancreatic cancer by maintaining active ERK signaling, our studies suggest that ERK signaling can be also regulated in a KRAS-independent manner. We found that survival motor neuron domain containing 1 (SMNDC1), a poorly studied splicing factor is amplified or upregulated in 30% of pancreatic ductal adenocarcinomas (PDACs) and associated with lower survival rates in patients. To determine the role of SMNDC1 in PDACs, we conducted loss- and gain-of-function studies and found that this splicing factor drives tumor growth by promoting the retention of A/C-rich cassette exons, which otherwise are excluded or retained at a lower rate after RNA splicing in normal cells. Importantly, across tumors, SMNDC1 promoted the inclusion of the cassette exon 4 (E4) of MAPK3 (ERK1), which encodes the kinase-activating phosphorylation sites (Thr202/Tyr204). Forced exclusion of MAPK3-E4 using antisense oligonucleotide morpholino (ASO), inhibited tumor establishment, ERK1 phosphorylation, and expression of ERK-targets. We then tested if retention of E4 in MAPK3 by SMNDC1 is a mechanism to resist chemical inhibition of the KRAS-MEK-ERK pathway. We found that PDAC cells acutely treated with selective MEK inhibitors (MEKi), trametinib, or selumetinib increased SMNDC1 and MAPK3 E4 retention expression. In addition, PDAC cells deficient for SMNDC1 were 25-fold more sensitive to trametinib than isogenic cells expressing SMNDC1. To demonstrate that increased E4 retention drives MEKi resistance, we concomitantly treated SMNDC1 proficient PDAC cells with trametinib and E4 ASO, which led to an 8-fold increase in sensitivity compared to cells treated with trametinib and control ASO. Also, we found that resistance to standard-of-care chemotherapeutic agents to treat PDAC, Gemcitabine (GEM) and 5-fluorouracil (5-FU), was also associated with increased expression of SMNDC1 and retention of E4 in MAPK3 in multiple PDAC cells lines. To determine the biochemical mechanism that leads to E4 inclusion by SMNDC1, we performed high-throughput RNA Bind-n-Seq (RBNS) and e-CLIP of SMNDC1 in PDAC cells which revealed specific RNA-binding preference for C-rich polypyrimidine tract upstream of retained MAPK3-E4. To validate these findings, we constructed a dual-fluorescent reporter mini-gene of MAPK3-E4 splicing, with either wild-type sequences in E4 and polypyrimidine tract, or different point mutations at the predicted cis-elements. We found that retention of the E4 of MAPK3 is dependent on SMNDC1-binding to C-rich sequences at distant 3’ splice sites of PDAC cells. These data support that PDAC cells exploit a “splicing switch” to promote ERK kinase activity and resistance to therapies, independent of activating mutations in the EGFR/RAS/MAPK pathway and offer a druggable alternative to block oncogenic signaling and altered RNA splicing in PDAC.
Citation Format: Md Afjalus Siraj, Yushan Zhang, Prabir Chakraborty, Robert Tseng, Li-Ting Ku, Grant Goda, Gilbert Giri, Deanne Yugawa, Mathew Wang, Shamik Das, Anindya Dey, Shailendra Dhar Dwivedi, Geeta Rao, Min Zhang, Da Yang, Md Nazir Hossen, Wei-Qun Ding, Kar-Ming Fung, Daniel Dominguez, Resham Bhattacharya, Luisa Escobar-Hoyos, Priyabrata Mukherjee. SMNDC1 alters the splicing of ERK to potentiate its activity in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C083.