Deletion of the DACH1 gene tumor suppressor increases replication fork stress, epithelial mesenchymal transition and sensitivity to WEE1 kinase inhibitors in prostate cancer

• Introduction: Patients with metastatic prostate cancer have poor survival with DNA damage repair pathway abnormalities. The cell fate determination factor DACH1 is deleted (shallow or deep deletion) in ~5-12% of PCa patients, but up to 65% of metastatic PCa. WEE1 kinase is one of the most upregulated kinases in the human prostate cancer kinome associated with metastatic progression in prostate cancer (mCRPC). We determined the potential for differential sensitivity of PCa to Wee1 Kinase inhibitors (WEE1Ki) based on DACH1 expression profiles. • Methods: Analysis of patient gene expression, tumor histology, organoids derived from prostate epithelial cell-specific DACH1 deletion prostate onco-mice, DNA replication fork assays, tissue culture. • Findings: DACH1 deficient ( Dach1 -/- ) cells showed enhanced cell killing by WEE1Ki, that was reversed by reintroduction of DACH1a. Increased sensitivity to WEE1Ki was shown in fibroblasts, prostate cancer cell lines and in organoids derived from human prostate cancer cell lines or the prostate epithelium of Dach1 deletion onco-mice. DACH1 deletion dramatically enhanced non replicating S phase in the presence of WEE1Ki. Dach1 - /- cells showed increased replication fork stress, that was reversed by reintroduction of the DACH1a isoform. DACH1 epithelial cell deletion onco-mice, and the prostate organoids derived therefrom, was associated with the induction of epithelial mesenchymal transition (EMT) with a corresponding increase in AKT Ser473P , ATR Thr1989p and CHK1 Ser345P . Increased sensitivity to WEE1Ki is associated with reduced SETD2 or NSD1/KMT3B expression, and reduced RRM2, a ribonucleotide reductase subunit, thereby inducing dNTP starvation. DACH1 expression was highly correlated with SETD2, NSD1 and RRM2. Gene ontology terms associated with DACH1 DNA binding templates included nucleoside metabolic processes. Like several other tumor suppressor genes (RB, FOXO3), DACH1 restrained dNTP production. dNTP production also requires NDPK (Nucleoside Diphosphate Kinase) encoded by NME genes. Consistent with the inhibition of dNTP production, DACH1a expression in prostate cancer PC3 cells reduced NME1 and NME7 (NDPKA expression), and DACH1 was strongly inversely correlated with NME1 in human prostate cancer (N=491. P 9.9e-24). • Conclusions: As DACH1 deletion PCa subclass conveys specific therapeutic sensitivities, that is dependent upon the DACH1a isoform, testing for DACH1a in patient samples may be warranted.