Figure 5. Augmenting replication stress attenuates metastatic latency and recurrence.

A, Quantification of GFP+ metastatic lesions in mice bearing HCC1954 WRNIP1 knockdown (WRNIP1^KD) Lat cells (n = 5 per group). Doxycycline feed was given 3 days post injection and continued till study termination. Data are presented as mean ± SEM. Mann–Whitney U test, ***, P < 0.001. B-C, Whole body and ex-vivo brain metastasis incidence as measured by BLI signal from athymic nude mice bearing HCC1954 WRNIP1^KD M-BM cells. (Ctrl group n = 6 and WRNIP1^KD group n = 5). Doxycycline feed was given 3 days post injection and continued till study termination. Data are presented as mean ± SEM. Mann–Whitney U test, **, P < 0.01. Two representative BLI images of whole-body mice and ex-vivo brains of their respective conditions. D, Relative fork degradation measured by the CldU/IdU ratio in presence of 4mM HU upon BGB324 treatment in HCC1954 Lat and M-BM cells. Representative images of DNA fibers are also shown. IdU (red color) and CldU (green color). N = 3 for all experiments. Data are presented as mean ± SEM. Mann–Whitney U test, ****, P < 0.0001. E, Immunoblot showing reduced AXL-WRNIP1 interaction in the presence of BGB324 (1μM for 5 hours). Ratio was determined by normalizing WRNIP1 expression to immunoprecipitated AXL. F, Relative quantification of oncospheres formed by HCC1954 Lat and M-BM cells treated with DMSO, BGB324 (BGB: 1μM), Olaparib (Ola: 5μM), Tucatinib (Tuc: 3μM) and combinations. Data are presented as mean ± SEM. One-way ANOVA was used, followed by the Dunnett test. *, P < 0.05, **, P < 0.01, ****, P < 0.0001, ns: not significant. G, Graphical abstract summarizing key findings of AXL-WRNIP1 mediated replication stress response promoting latent and metachronous metastasis. (G, Created with BioRender.com).