Table S8. KRAS signaling downstream pathway top 50 genes negatively enriched in RGS11-OE.

Table S1. Primer sequences used for amplification of gene targets

Figure S7. Gene set enrichment analysis (GSEA) of RGS11-OE MIA PaCa-2 cells.

Table S2. Gene sets enriched in RGS11-OE MIA PaCa-2 cells (n=3).

Table S5. Hedgehog signaling pathway total (positively and negatively enriched) genes in RGS11-OE.

Figure S1. Genetic knockdown of CNT1 downregulates RGS11 expression in HEK293 cells.

Figure S9. TCGA-GDC analysis of RGS11 in patients.

Figure S8. Tumors in athymic nude mice orthotopically implanted with vehicle control and RGS11-OE MIA PaCa-2 cells.

DNA topoisomerase IIα (TOP2α; 170 kDa, TOP2α/170) is a nuclear enzyme that plays a key role in chromosomal segregation at mitosis by catalyzing transient DNA double-stranded breaks, allowing replicated DNA duplexes to separate. Given its importance during mitosis and high expression level in proliferating cells, TOP2α/170 is a prominent target for anticancer therapy. However, the effectiveness of TOP2-targeted agents is often compromised by acquired drug resistance, which in patients is most associated with decreased TOP2α/170 protein expression, thereby diminishing drug-induced TOP2α-DNA complexes and cytotoxic DNA damage. Mimicking this clinical chemoresistance phenotype, reduced TOP2α/170 mRNA/protein was previously reported in an acquired HL-60 cell line associated with production of a C-terminal truncated 160 kDa protein (TOP2α/160). In this report, intronic polyadenylation (IPA) within intron 33 (I33) of the TOP2α/170 gene, responsible for production of TOP2α/160, was recapitulated by quantitative real-time polymerase chain reaction, 3'-rapid amplification of cDNA ends, and Sanger sequencing. HL-60/MX2 cells are crossresistant to multiple TOP2-targeted agents. To circumvent drug resistance, CRISPR/Cas9 gene editing with homology-directed repair was used to mutate/enhance the weak 5' splice site at the exon 33/I33 junction of the TOP2α gene to improve splicing out of I33 and to suppress IPA. The resulting splice site gene-edited clone, designated MX2/SS-Edit, expressed reduced TOP2α/160 mRNA/protein, increased TOP2α/170 mRNA/protein, and exhibited partial restoration of sensitivity to mitoxantrone, etoposide, and amsacrine. Together, results indicated that strengthening the weak TOP2α exon 33/I33 splice site by knock-in gene editing attenuated IPA offering a strategy to circumvent resistance to TOP2-targeted therapies. SIGNIFICANCE STATEMENT: Results presented here validated drug resistance in the HL-60/MX2 leukemia cell line driven by intronic polyadenylation (IPA) within intron 33 of the DNA topoisomerase IIα (TOP2α) gene, which produced a truncated and predominantly cytoplasmic TOP2α protein isoform (TOP2α/160). Using CRISPR/Cas9/homology-directed repair gene editing, the weak exon 33/intron 33 5' splice site was enhanced to suppress IPA, which restored expression of full-length protein (TOP2α/170) and led to a gain-of-function in drug sensitivity, offering a potential strategy to overcome drug resistance.

Figure S2. Generation of CNT1 OE and KD clones in L3.6pl and evaluation of RGS11 protein expression.

Table S9. Top 50 EMT pathway genes negatively enriched in RGS11-OE.

Figure S3. IHC staining for RGS11.

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy associated with early metastasis, drug resistance, and poor outcomes. We previously demonstrated a putative tumor-suppressive role for concentrative nucleoside transporter 1 (CNT1) in PDAC. In this study, we demonstrate the regulator of G protein signaling (RGS) 11 as a key target of CNT1, with potent tumor-suppressive properties in PDAC. Compared with normal human pancreas, RGS11 expression is diminished in human PDAC tissues which correspond with the reduced patient survival times. In addition, quasimesenchymal pancreatic tumor cell lines with accelerated growth, metastatic propensity, and innate resistance to nucleoside analogs showed relatively lower RGS11 expression than their epithelial counterparts. Interestingly, RGS11 levels reversibly modulated the epithelial-mesenchymal transition of human PDAC cell lines influencing the chemotherapeutic sensitivities of anti-PDAC drugs. Additionally, stable lentiviral-mediated RGS11 expression reduced the cellular proliferation and colony establishment, increased the apoptotic index, and decreased the migratory and invasive abilities in quasimesenchymal tumor cell lines, whereas RGS11 depletion in epithelial tumor cell lines showed opposite effects. Global transcriptomic analysis revealed RGS11 replenishment in PDAC cells to suppress CD44-directed stemness features with significant reprogramming of the PDAC oncogenic landscape. Furthermore, RGS11 reduced the primary tumor burden and metastatic occurrence in a mouse model of PDAC. Together, these findings uncover RGS11 as a key target of CNT1 that exhibits therapeutic potential for intervention of aggressive PDAC. IMPLICATIONS:RGS11 identified as a downstream target of a gemcitabine transporter CNT1 exerts potent antitumorigenic features in PDAC with therapeutic and prognostic values.

Table S7. KRAS signaling downstream pathway top 50 genes negatively enriched in RGS11-OE.

Figure S5. Chemosensitivity of PANC-1 RGS11-OE cells to chemotherapeutic drugs.

Table S6. KRAS signaling downstream pathway top 50 genes positively enriched in RGS11-OE.

Figure S6. RGS11, induces apoptosis, reduces proliferation and suppresses EMT features in HEK293 cells with RGS11 overexpression and knockdown.