Depletion of a putatively druggable class of phosphatidylinositol kinases inhibits growth of p53-null tumors

Abstract

Here, we show that a subset of breast cancers express high levels of the type 2 phosphatidylinositol-5-phosphate 4-kinases α and/or β (PI5P4Kα and β) and provide evidence that these kinases are essential for growth in the absence of p53. Knocking down PI5P4Kα and β in a breast cancer cell line bearing an amplification of the gene encoding PI5P4K β and deficient for p53 impaired growth on plastic and in xenografts. This growth phenotype was accompanied by enhanced levels of reactive oxygen species (ROS) leading to senescence. Mice with homozygous deletion of both TP53 and PIP4K2B were not viable, indicating a synthetic lethality for loss of these two genes. Importantly however, PIP4K2A(-/-), PIP4K2B(+/-), and TP53(-/-) mice were viable and had a dramatic reduction in tumor formation compared to TP53(-/-) littermates. These results indicate that inhibitors of PI5P4Ks could be effective in preventing or treating cancers with mutations in TP53.

Figure 1. Amplification of PIP4K2B in HER-2/Neu-Positive Breast Cancers and Co-occurrence with TP53 Mutation/Deletion

(A) Genomic landscape of PIP4K2B and ERBB2 (HER2) DNA copy number amplifications in cancer. Tumor samples are divided into three groups: group I: those with amplification in PIP4K2B but no or lower level amplification of ERBB2, group II: samples with amplification of both genes but likely to be derived from two different amplicons, and group III: samples with amplification in both genes derived from the same amplicon. Colored bar indicates degree of copy number gain (red). (B) Oncoprints of PIP4K2B and TP53 across 66 breast carcinomas indicating a trend of co-occurrence between PIP4K2B gain/amplification and TP53 mutation/deletion (p value: 0.006306, Fisher’s exact test). Individual genes are represented as rows, and individual cases or patients are represented as columns. Genetic alterations are color-coded with red indicating amplification; pink, DNA copy number gain; light blue, hemizygous deletion and green box, point mutation. Only cases with gain/amplification of PIP4K2B are included. These oncoprints are based on data obtained from the Stand Up to Cancer cBio portal (http://cbio.mskcc.org/su2c-portal/). (C) Box plots indicating that the levels of ERBB2 phosphorylation on tyrosine 1248 (y1248) (left) and of total ERBB2 (right) measured by RPPA are significantly higher in breast carcinomas with PIP4K2B gain/amplification (p value: 1.7 × 10⁻⁵, p value: 4.2 × 10⁻⁶, respectively). (D) Box plots indicating that the levels of AKT phosphorylation on threonine 308 (T308) (left) measured by RPPA are significantly lower in breast carcinomas with PIP4K2B gain/amplification (p value: 0.007), whereas the total AKT levels remain unchanged (p value: 0.24) Data obtained from the Stand Up to Cancer cBio portal (http://cbio.mskcc.org/su2c-portal/). (E) Upper: box plots indicating the expression of TP53 (blue) and PIP4K2A (red) across different subsets of breast carcinomas divided by the putative DNA copy number status of TP53. (Homdel: homozygous deletion [n = 0], Hetloss: heterozygous deletion (n = 1), diploid: n = 2, gain: n ≥ 3). TP53 mRNA expression is as expected progressively higher across the different subgroups of breast carcinomas with TP53 ploidy status ranging from n = 0 to n≥3 (one-way ANOVA, p = 4.3 × 10⁻⁶), whereas PIP4K2A expression is only significantly higher in the subgroup of breast carcinomas with homozygous deletion of TP53 (one-way ANOVA, p = 0.009). Lower: box plots indicating the expression of PIP4K2B (orange) and PIP4K2C (yellow) across different subsets of breast carcinomas divided by the putative DNA copy number status of TP53. The expression of either PIP4K2B or PIP4K2C is not significantly different across the subgroups of breast carcinomas with different ploidy status of TP53 (one-way ANOVA, p > 0.05).

Figure 2. PI5P4K Expression in Breast Cancer

(A) Histograms illustrating expression levels of PI5P4Kα (***p = 0.000) or PI5P4Kβ (**p = 0.004) in breast cancer samples. (B) Representative IHC images from breast tumor and normal samples. Scale bar, 100 µm. (C) PI5P4Kα and PI5P4Kβ expression in panel of breast cancer cell lines. See also Figure S1 and Tables S1 and S2.

Figure 3. Knockdown of Both PI5P4Kα and β in BT474 Cells Abrogates Cell Proliferation and Fail to Form Tumors in Xenograft Model

(A and B) Stable knockdown of PI5P4Kα/β in BT474 cells. shPI5P4kα/β-1 (sequence 1) and shPI5P4kα/β-2 (sequence 2) are two independent hairpins targeted against PI5P4kα and PI5P4kβ. All single knockdowns are sequence 1 (see Experimental Procedures). (C) Luminescent cell viability assay in stable PI5P4Kα/β knockdown cells. Results are from four independent experiments and are represented as the mean value ± SEM. ***p value < 0.0001 with two-tailed Student’s t test. (D) Stable overexpression of mouse Flag-tagged PI5P4Kβ in BT474 cells and subsequent stable PI5P4Kα/β knockdown. (E) Mouse Flag-tagged PI5P4Kβ rescues proliferation in PI5P4Kα/β knockdown cells. Results are from three independent experiments and are represented as the mean value ± SEM. (F) Right: Luminescent cell viability assay in stable PI5P4Kα/β knockdown cells cultured at restrictive (37°C) and permissive conditions (32°C) for indicated time points. Results are from three independent experiments and are represented as the mean value ± SEM. Left: Total p53, p21, and GAPDH (loading control) protein levels in BT474 cells cultured at restrictive and permissive conditions for 24 hr. (G) Tumor formation over time in nude mice injected with the BT474 cancer cell line expressing shRNA pLK0.1 control or shRNA PI5P4K α/β. Error bars are SEM. (H) Images of tumors, pLK0.1 control cells (left flank), or shPI5P4Kα/β (right flank) after mice were euthanized. (I) Histogram showing percentages of viable area in PI5P4Kα/β knockdown and control xenografts. Data from two independent experiments were considered and displayed as means ± SD. (J) Histogram displaying expression levels of Ki67-positive cells in shPI5P4Kα/β and pLK0.1 xenografts. Quantification of Ki67-positive cells in shPI5P4Kα/β and pLK0.1 xenografts. Data from two independent experiments were considered and displayed as means ± SD. (K) Upper: histogram illustrating expression levels of p27-positive cells in shPI5P4Kα/β and pLK0.1 xenografts. Quantification of p27-positive cells in shPI5P4Kα/β and pLK0.1 xenografts. Data from two independent experiments were considered and displayed as means ± SD (***p < 0.001). Lower: representative images of p27 immunostains are shown. Scale bar, 100 µm. See also Figure S2 and Table S3.

Figure 4. Knockdown of Both PI5P4Kα and β in BT474 Cells Enhances PI3K Signaling, Increases ROS and Respiration, and Triggers Senescence

(A) AKT phosphorylation at serine 473(pS473) and total AKT protein levels in pLK0.1 vector control cells and in shPI5P4kα/β double-knockdown cells. Cells were serum starved overnight and then treated with media with 10% serum for the indicated time points. (B) Total INPP4B protein levels in pLK0.1 vector control cells and in shPI5P4kα/β double-knockdown cells. The MCF7 breast cancer cell line is known to have high expression of INPP4B. (C) BT474 pLK0.1 control vector cells or PI5P4Kα/β knockdown cells labeled with [³H]-inositol for 48 hr. Deacylated lipids were analyzed by HPLC, quantified, and normalized to PI4P levels. Results are from three independent experiments and are represented as the mean value ± SEM. *p < 0.01 with two-tailed Student’s t test. (D) Cell death of pLK0.1 control or shRNA PI5P4K α/β double-knockdown cells was assessed by LDH release. Mean values ± SEMs from four independent experiments are shown. (E) ROS determined by incubating pLK0.1 control or shRNA PI5P4K α/β double-knockdown cells with DCFH-DA (10 µM) for 30 min or a bolus of H₂O₂ (100 µM) for 15 min as positive control. n = 4 mean ± SEM. **p < 0.001 with two-tailed Student’s t test. (F) Oxygen consumption of pLK0.1 control or shRNA PI5P4K α/β double-knockdown cells. n = 4 mean ± SEM. **p < 0.001 with two-tailed Student’s t test. (G) pLK0.1 control or shRNA PI5P4K α/β double-knockdown cells were plated for senescence assay. Images presented from a representative experiment performed in triplicate. (H) ROS determined by incubating pLK0.1 control or shRNA PI5P4K a/b double-knockdown cells with DCFH-DA (10 µM) for 1 hr. Cells were untreated or treated with GDC-0941 (1 µM) or N-acetylcysteine (NAC) (10 mM) for 24 hr. n = 3 mean ± SEM. **p < 0.001 with two-tailed Student’s t test. (I) Luminescent cell viability assay in stable (upper) pLK0.1 control cells and (lower) PI5P4Kα/β knockdown cells ± GDC-0941 (1 µM) or NAC (10 mM) for indicated time points. Results are from three independent experiments and are represented as the mean value ± SEM. (J) Immunohistochemical detection of 8-oxo-dGuo in pLK0.1 and shPI5P4Kα/β xenografts. Two representative images of 8-oxo-dGuo immunostains are shown (mouse #6 and mouse #10). Scale bar,100 µm. See also Figure S3.

Figure 5. Distinct Gene Expression and Metabolomic Signatures in the PI5P4Kα/β Double-Knockdown Cells

Expression data of PI5P4Kα/β knockdown cells (shPI5P4Kα/β) or control vector cells (pLK0.1) using Affymetrix Human Genome U133 Plus (~40,000 genes). (A) Enrichment analysis of the curated pathways (BioCarta) following PI5P4Kα/β knockdown. The p38 MAPK and RAS signaling pathways were identified as the most significant pathways with respective p values of 1.83 × 10⁻⁴ and 0.004. (B) Gene set enrichment analysis (GSEA) signatures highlighting coordinated differential expression of gene sets that are enriched in PI5P4Kα/β knockdown cells. The CHARAFE_BREAST_CANCER_LUMINAL_VS_MESENCHYMAL_UP (genes upregulated in luminal-like breast cancer cell lines compared to the mesenchymal-like ones) and CHARAFE_BREAST_CANCER_LUMINAL_VS_BASAL_UP (genes upregulated in luminal-like breast cancer cell lines compared to the basal-like ones) were scored among the most significantly enriched gene signatures in PI5P4K knockdown cells, p < 0.001. See also Figures S4 and S5 and Tables S4 and S5.

Figure 6. Requirement for PIP4K2A and PIP4K2B for Survival and Growth

(A) Schematic representation of wild-type PIP4K2A locus, targeting vector, recombined allele and Flp^R/Cre^R-deleted null allele. Neomycin resistance cassette flanked by Frt sites (yellow boxes), exon 2 flanked by loxP sites (red arrows), and diphtheria toxin cassette located 3′ to exon 2. N, NdeI restriction site. (B) Southern blot of embryonic stem cell clones (wild-type and targeted) using probe E3 after NdeI digestion. (C) PCR analysis of genomic DNA derived from PIP4K2A F2 littermates. (D) PI5P4Kα and β protein levels from brain homogenate of PIP4K2A F2 littermates. (E) PI5P4Kα and β protein levels in primary MEFs derived from intercrossing PIP4K2A and PIP4K2B knockout mice. (F) Genotyping results of PIP4K2A/2B double heterozygote interbreeding at weaning (observed numbers in bold, expected in parentheses). (G) Body weight (in grams) measurements of indicated mouse genotypes. Results represented as the mean value ± SEM. ***p < 0.0001 with two-tailed Student’s t test. (H) Quantitation of PI5P levels in PIP4K2A/2B-deficient relative to PIP4K2A/2B heterozygous MEFs. Results are from three independent experiments and are represented as the mean value ± SEM. See also Figure S6.

Figure 7. PI5P4K Deficiency Restricts Tumor Death after p53 Deletion

Kaplan-Meier plot analysis of tumor free survival (15 PI5P4Kα^+/+ PI5P4Kβ^+/+ and 20 PI5P4Kα ^−/− PI5P4Kβ^+/−). *p < 0.05 with two-tailed Student’s t test. See also Figure S7.