Integrated analysis of genomic and transcriptomic data for the discovery of splice-associated variants in cancer.

Somatic mutations within non-coding regions and even exons may have unidentified regulatory consequences that are often overlooked in analysis workflows. Here we present RegTools ( www.regtools.org ), a computationally efficient, free, and open-source software package designed to integrate somatic variants from genomic data with splice junctions from bulk or single cell transcriptomic data to identify variants that may cause aberrant splicing. We apply RegTools to over 9000 tumor samples with both tumor DNA and RNA sequence data. RegTools discovers 235,778 events where a splice-associated variant significantly increases the splicing of a particular junction, across 158,200 unique variants and 131,212 unique junctions. To characterize these somatic variants and their associated splice isoforms, we annotate them with the Variant Effect Predictor, SpliceAI, and Genotype-Tissue Expression junction counts and compare our results to other tools that integrate genomic and transcriptomic data. While many events are corroborated by the aforementioned tools, the flexibility of RegTools also allows us to identify splice-associated variants in known cancer drivers, such as TP53, CDKN2A, and B2M, and other genes.

The MK2/Hsp27 axis is a major survival mechanism for pancreatic ductal adenocarcinoma under genotoxic stress.

Combination chemotherapies remain the cornerstone treatment for pancreatic ductal adenocarcinoma (PDAC), but de novo and acquired resistance is common. In this study, we aimed to identify and characterize resistance mechanisms to a FIRINOX chemotherapy regimen (a combination of 5-fluorouracil, irinotecan, and oxaliplatin) because it is the most aggressive regimen currently used clinically for patients with PDAC. Using an unbiased reverse-phase protein array, we detected phospho-activation of heat shock protein 27 (Hsp27) as the most up-regulated event after FIRINOX treatment in PDAC cells. Silencing HSP27 by RNA interference or by a small-molecule inhibitor enhanced apoptosis caused by FIRINOX in vitro. Mechanistically, FIRINOX up-regulated tumor necrosis factor­α (TNFα), causing autocrine phosphorylation and activation of transforming growth factor­ß­activated kinase 1 (TAK1), MAPK activated protein kinase 2 (MAPKAPK2 or MK2), and, ultimately, Hsp27. Targeting MK2, the kinase that directly phosphorylates Hsp27, abrogated Hsp27 activation, sensitized PDAC cells to apoptosis, and suppressed SN-38­induced protective autophagy in vitro, in part by blocking phospho-activation of Beclin1. In an autochthonous PDAC mouse model, the MK2 inhibitor ATI-450 decreased PDAC development and progression. When combined with FIRINOX, ATI-450 eliminated most PDAC foci and marked prolonged mouse survival without causing additional toxicity. Last, we found that high phospho-MK2 expression in tumors was associated with poorer survival of patients with PDAC. Our study identified MK2 as a mediator of genotoxic stress­induced activation of prosurvival pathways and provides preclinical support for combining an MK2 inhibitor with FIRINOX-based chemotherapies to treat PDAC.