Patient-derived tumor organoids with p53 mutations, and not wild-type p53, are sensitive to synergistic combination PARP inhibitor treatment

Abstract

Poly (ADP-ribose) polymerase inhibitors (PARPi) are used for patients with BRCA1/2 mutations, but patients with other mutations may benefit from PARPi treatment. Another mutation that is present in more cancers than BRCA1/2 is mutation to the TP53 gene. In 2D breast cancer cell lines, mutant p53 (mtp53) proteins tightly associate with replicating DNA and Poly (ADP-ribose) polymerase (PARP) protein. Combination drug treatment with the alkylating agent temozolomide and the PARPi talazoparib kills mtp53 expressing 2D grown breast cancer cell lines. We evaluated the sensitivity to the combination of temozolomide plus PARPi talazoparib treatment to breast and lung cancer patient-derived tumor organoids (PDTOs). The combination of the two drugs was synergistic for a cytotoxic response in PDTOs with mtp53 but not for PDTOs with wtp53. The combination of talazoparib and temozolomide induced more DNA double-strand breaks in mtp53 expressing organoids than in wild-type p53 expressing organoids as shown by increased γ-H2AX protein expression. Moreover, breast cancer tissue microarrays (TMAs) showed a positive correlation between stable p53 and high PARP1 expression in sub-groups of breast cancers, which may indicate sub-classes of breast cancers sensitive to PARPi therapy. These results suggest that mtp53 could be a biomarker to predict response to the combination of PARPi talazoparib-temozolomide treatment.

Keywords: DNA double-strand breaks; Mutant p53; PARP inhibitor; Patient-derived tumor organoids; Synergistic cytotoxicity.

Figure 1.. Combination PARPi talazoparib plus temozolomide treatment demonstrates synergistic cytotoxicity to mtp53 breast cancer PDTOs.

(A) Drug response curves for breast cancer organoids to talazoparib and temozolomide. Cells were plated in 384 well plates and inhibitors were added after 3 days. After 4-day incubation viability was determined using Cell Titer Glo 3D reagent. (B) EdU incorporation determined by flow cytometry. PDTOs were pulsed with EdU for 4 hours to label proliferative cells. (C) Synergy maps for talazoparib-temozolomide were calculated using the SynergyFinder web application with the ZIP synergy model (red indicates a synergistic effect, white an additive effect, and green an antagonistic effect). Representative synergy maps of at least 2 independent experiments are shown. (D) Bar graphs showing cell viability at indicated concentrations. Average of 2 independent experiments. *P < 0.05, **P < 0.01, ***P <0.001, ****P<0.0001.

Figure 2.. Synergistic PARPi talazoparib plus temozolomide treatment of mtp53 breast PDTO increases double strand breaks (DSBs) as indicated by chromatin bound γH2AX.

Chromatin (A) and soluble (B) fractions were prepared from breast cancer PDTOs ICSBCS002 (mtp53 R248W), ICSBCS007 (mtp53 R213stop) and WCM2137_2 (wtp53) treated with either vehicle (DMSO), 0.4 μM talazoparib (TAL), 55 μM temozolomide (TMZ), or combination (TAL+TMZ) for 24 h. Protein levels of γ-H2AX, p53, PAR and PARP were determined by Western blot analysis. ImageJ analysis was performed for γ-H2AX, p53, PAR and PARP protein levels and normalized to Lamin A in the chromatin fraction or Actin in the soluble fraction. Representative of two independent experiments.

Figure 3.. Combination PARPi talazoparib plus temozolomide treatment demonstrates synergistic cytotoxicity of mtp53 lung PDTO.

(A) Drug response curves for lung cancer organoids to talazoparib and temozolomide. Cells were plated in 384 well plates and inhibitors were added after 3 days. After 4-day incubation viability was determined using Cell Titer Glo 3D reagent. (B) EdU incorporation determined by flow cytometry. PDTOs were pulsed with EdU for 4 hours to label proliferative cells. (C) Synergy maps for talazoparib-temozolomide were calculated using SynergyFinder web application using the ZIP synergy model. Representative synergy maps of at least 2 independent experiments are shown. (D) Bar graphs showing cell viability at indicated concentrations. Average of 2 independent experiments. *P < 0.05, **P < 0.01.

Figure 4.. Synergistic PARPi talazoparib plus temozolomide treatment of mtp53 lung PDTO increases DNA double strand breaks (DSBs).

Chromatin (A) and soluble (B) fractions were prepared from lung organoids WCM1689 (mtp53 Q331stop), WCM1550 (mtp53 C135Y) and WCM1712 (wtp53) treated with either vehicle (DMSO), or 0.4 μM talazoparib (TAL) or 166 μM temozolomide (TMZ), or combination (TAL+TMZ) for 24 h. Protein levels of γ-H2AX, p53, PAR and PARP were determined by Western blot analysis. ImageJ analysis was performed for γ-H2AX, p53, PAR and PARP protein levels and normalized to Lamin A in the chromatin fraction or Actin in the soluble fraction. Representative of two independent experiments.

Figure 5.. High mtp53 expression correlates with high PARP expression in TNBC of patients of African Ancestry.

(A) Dot plot diagram showed IHC staining of p53 score (immunoreactive score: IRS) in tissue microarray (TMA) from Basal-like, Luminal A, Luminal B and Her2+/ER− subtype of breast cancer patients. The immunoreactive score (IRS) was calculated as shown in the table. (B) Correlation heatmap of p53 and PARP expression level (IRS) of 138 breast cancer patients. Each column of the data represents either p53 IRS or PARP IRS and each row represents one patient sample. Samples are ordered according to the p53 IRS level. The correlation of p53 and PARP was further analyzed using the Person correlation. y-axis presents the IRS of PARP and x-axis presents the IRS of p53. (C) Dot plot diagram showed IHC staining of PARP1 score in tissue microarray (TMA) from Basel-like and Luminal A subtype of breast cancer grouped by p53 level and ethic group (AA or white). (D) Representative images at 100x magnification of PARP and p53 staining. Intensity categories: no staining (0), mild staining (1), moderate staining (2) and intense staining (3). Scale bar: 50 um. The immunoreactive score (IRS) is a product of multiplication between percentage of positive cells score (0–4) and intensity of staining score (0–3). Graphpad Prism 9 was used for statistical analysis. Results are expressed as mean + SEM. Statistical significance was analyzed using t-test. *P < 0.05, **P < 0.01, ***P <0.001.

Figure 6.. Specific uptake of Cy5p53Tet in high levels of mtp53 breast cancer organoids.

(A) Representative images from high-content imaging analysis of mtp53 PDTO ICSBCS002 (mtp53 R248W) and ICSBCS007 (mtp53 R213 stop) treated with either 500 nM Cy5p53Tet or Cy5scramble peptides for 4 h. Hoechst staining was used to stain the nuclei (Cy5: red; Hoechst: blue). (B) Quantification of Cy5p53Tet and Cy5scramble uptake in ICSBCS002 and ICSBCS007 PDTOs via Operetta High Content Imaging System. (C) Whole cell extracts were prepared from breast cancer PDTO ICSBCS002 and ICSBCS007 treated with either 500 nM Cy5p53Tet or Cy5scramble peptides for 4h. 1 ug of cell extracts was run on 15% polyacrylamide gel and uptake of Cy5p53Tet or Cy5Scramble was examined by scanning the gel using Typhoon FLA 7000 biomolecular imager. Protein levels of p53 and Actin were determined by Western blot analysis. (D) Whole cell extracts were prepared from breast cancer PDTO ICSBCS002 and ICSBCS007. 25 ug of cell extracts were run on 10% polyacrylamide gel and protein levels of p63 and p73 were determined by Western blot analysis (left). ImageJ analysis was performed for p63 and p73 protein levels and normalized to Actin from two independent experiments (right). (E) Whole cell extracts were prepared from lung cancer PDTO WCM1550 (mtp53 C135Y) and WCM1712 (wtp53) treated with either 500 nM Cy5p53Tet or Cy5scramble peptides for 3 h. 1 ug of cell extracts was run on 15% polyacrylamide gel and uptake of Cy5p53Tet or Cy5Scramble was examined by scanning the gel using Typhoon FLA 7000 biomolecular imager. Protein levels of p53 and Actin were determined by Western blot analysis (left). Representative of two independent experiments. Quantification of relative Cy5p53 uptake was performed using ImageJ. Average of 2 independent experiments (right). Graphpad Prism 9 was used for statistical analysis. Results are expressed as mean + SEM. Statistical significance was analyzed using (two-tailed P value). *P < 0.05, **P < 0.01.