Journal: The Journal of Pathology

Article Title: Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition

doi: 10.1002/path.6032

Figure Lengend Snippet: Members of the DNA repair pathway as potential indicators of sensitivity to treatment with elimusertib. (A) Dot plot showing IC 50 values for elimusertib in a panel of human cancer cell lines from multiple cancer entities. MCL, mantle cell lymphoma; OV, ovarian carcinoma; COADREAD, colorectal carcinoma; BRCA.TNBC, BRCA ‐mutated triple‐negative breast cancer; PRAD, prostate carcinoma; SKCM, skin cutaneous melanoma; DLBCL, diffuse large B‐cell lymphoma; GCB, germinal center B‐cell subtype; ABC, activated B‐cell lymphoma; SCLC, small cell lung cancer; CESC, cervical carcinoma; MM, multiple myeloma; PAAD, pancreatic carcinoma. (B) Volcano plot showing correlation ( Rho ) of IC 50 with differentially expressed genes (mRNA expression) in the PanCancer pathway panel of the NanoString nCounter system. Genes associated with DNA repair/cell cycle checkpoint or apoptosis are denoted in green. (C) Left: gene set enrichment analysis (GSEA) plot showing that the DNA repair gene set (FDR < 0.05) is enriched in sensitive cell lines. Right: list of enriched gene sets showing that the DNA repair pathway is the only significant hit (FDR q ‐value = 0.029).

Article Snippet: In particular, elimusertib (BAY 1895344; Bayer AG, Leverkusen, Germany) is a selective ATR kinase inhibitor that has demonstrated antitumor activity in vitro , in vivo , and in phase I clinical trials [ , , , ].

Techniques: Expressing

Journal: The Journal of Pathology

Article Title: Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition

doi: 10.1002/path.6032

Figure Lengend Snippet: pCHK1 staining correlates with elimusertib sensitivity in vitro . (A) Western blotting for pATR, total ATR, pCHK1, total CHK1, γH2AX, and total H2AX in the ovarian cancer cell line panel. SKOV3 cells treated with 8 m m hydroxyurea for 4 h was used a control sample. (B) Bar chart showing elimusertib half‐maximal inhibitory concentration (IC 50 ) of the ovarian cancer cell line panel. Data presented are from three biological replicates with SEM error bar. (C) Dot plots showing correlations between elimusertib IC 50 ( y ‐axes) and densitometric values [ x ‐axes, arbitrary units (a.u.)] of activated protein expression (phospho‐protein normalized with respective total protein) of ATR (left), CHK1 (middle), and H2AX (right). Pearson correlation coefficients ( r ) and P values ( p ) are denoted. (D) Heatmap showing that IC 50 (log10) of compounds targeting DNA damage or replication stress protein expression correlated with activated ATR and CHK1/2. ATR inhibitors are highlighted in orange. (E) Western blots for pCHK1 and total CHK1 in whole cell lysate (W), cytoplasmic (C), and nuclear (N) fractions of elimusertib‐sensitive (upper panel: TYKNU, OV17R, PEO1) and elimusertib‐resistant (lower panel: PEO4, HEYC2, HEY) cell lines. Nuclear marker Histone H3 and cytoplasmic marker GAPDH served as respective cell fractionation controls and revealed minimal cross‐contamination. (F) Dot plots showing densitometric values [ y ‐axes, arbitrary units (a.u.)] of activated CHK1 levels (pCHK1 normalized with total CHK1) in the elimusertib‐sensitive (left) and elimusertib‐resistant (right) ovarian cancer cell lines. Mean ± SEM from three cell lines were plotted. One‐way ANOVA and Tukey's multiple comparison tests were used; ** p < 0.01 and *** p < 0.001. ns, not significant.

Article Snippet: In particular, elimusertib (BAY 1895344; Bayer AG, Leverkusen, Germany) is a selective ATR kinase inhibitor that has demonstrated antitumor activity in vitro , in vivo , and in phase I clinical trials [ , , , ].

Techniques: Staining, In Vitro, Western Blot, Control, Concentration Assay, Expressing, Marker, Cell Fractionation, Comparison

Journal: The Journal of Pathology

Article Title: Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition

doi: 10.1002/path.6032

Figure Lengend Snippet: Cell line‐derived xenografts, respective elimusertib responses based on treatment/control (T/C) ratios, and mean intensities of stained markers in the xenograft tissue samples.

Article Snippet: In particular, elimusertib (BAY 1895344; Bayer AG, Leverkusen, Germany) is a selective ATR kinase inhibitor that has demonstrated antitumor activity in vitro , in vivo , and in phase I clinical trials [ , , , ].

Techniques: Staining

Journal: The Journal of Pathology

Article Title: Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition

doi: 10.1002/path.6032

Figure Lengend Snippet: Nuclear pCHK1 staining correlates with elimusertib sensitivity in vivo . (A) Representative baseline staining for geminin, pATR, pCHK1, and γH2AX in tissue samples from cell line‐derived xenograft tumors of GRANTA‐519 (MCL, mantle cell lymphoma), Lovo and HCT116 (CRC, colorectal carcinoma), and KLN205 prior to elimusertib treatment. Scale bars: 50 μm. The treatment/control (T/C) ratio denoting good (<0.3) and moderate (0.3–0.6) response to elimusertib is also shown. Each image was obtained through multiplex immunofluorescence staining and is presented as pseudo‐color, mimicking a DAB‐based IHC stain for ease of visualization. (B) Dot plots showing that the T/C ratio for elimusertib correlated to mean staining intensities for geminin, pATR, γH2AX, and pCHK1 (cytoplasmic and nuclear) derived from xenografts. Pearson correlation coefficients ( r ) and P values ( p ) are given.

Article Snippet: In particular, elimusertib (BAY 1895344; Bayer AG, Leverkusen, Germany) is a selective ATR kinase inhibitor that has demonstrated antitumor activity in vitro , in vivo , and in phase I clinical trials [ , , , ].

Techniques: Staining, In Vivo, Derivative Assay, Control, Multiplex Assay, Immunofluorescence

Journal: The Journal of Pathology

Article Title: Nuclear pCHK1 as a potential biomarker of increased sensitivity to ATR inhibition

doi: 10.1002/path.6032

Figure Lengend Snippet: Nuclear pCHK1 staining is indicative of better survival in elimusertib‐treated patients. (A–D) Representative staining for pCHK1 on tumor biopsies obtained from patients immediately prior to commencing elimusertib therapy, showing (A) predominantly cytoplasmic versus (B–D) nuclear specific staining in cancer cells. Scale bars: 50 μm. The inset square area shown in each picture is enlarged from the region marked with a dashed frame. PD, progressive disease; SD, stable disease; PFS, progression‐free survival.

Article Snippet: In particular, elimusertib (BAY 1895344; Bayer AG, Leverkusen, Germany) is a selective ATR kinase inhibitor that has demonstrated antitumor activity in vitro , in vivo , and in phase I clinical trials [ , , , ].

Techniques: Staining