Figure 1(A) Schematic representation of the shRNA screening aimed to identify genes whose knock-down enhanced the anti-proliferative effects of AZD8186 (PI3Kβi), GDC0941 (pan-PI3Ki) or MK2206 (AKTi) on the PTEN-null cell line MDA-MB-468. The shRNAs directed against the transcripts of three genes are shown in three different colours. shRNAs against gene 1 are depleted in the vehicle-treated condition compared to time 0 and they most likely target an essential gene. shRNAs against the transcripts of gene 2 are unchanged in all conditions, while shRNAs silencing gene 3 are selectively depleted in the AZD8186-selected cells compared to vehicle and their abundance is not affected by the 2-weeks long culture with vehicle. Gene 3 is a good candidate for inhibition in combination with AZD8186. (B) Dot-plot showing the fold change (log2) in number of reads between vehicle and AZD8186-treated conditions vs the p-value of the difference between the two treatment conditions for each shRNA. 9 out of 18 shRNAs targeting EGFR showed a p-value <0.2 and are highlighted in the plot. The plot was generated considering the results from biological triplicate of the experiment. (C MDA-MB-468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin. EGFR mRNA was then measured by RT-qPCR (C) Average ± SD of triplicates and representative of three independent experiments. D) MDA-MB-468 were infected with the indicated shRNAs targeting EGFR and selected by puromycin. cell viability was measured after 4 days of treatment with serial dilutions of AZD8186 (D). Average ± SD of triplicates and representative of three independent experiments. (E MDA-MB-468 were treated with serial dilutions of gefitinib (E) in the presence of vehicle, AZD8186 (0.25 μM), GDC0941 (0.25 μM) or MK2206 (0.45 μM), as indicated. Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor-treated condition to the viability in the absence of gefitinib Average ± SD of triplicates and representative of two independent experiments. F) MDA-MB-468 were treated with serial dilutions of lapatinib (F) in the presence of vehicle, AZD8186 (0.25 μM), GDC0941 (0.25 μM) or MK2206 (0.45 μM), as indicated. Cell viability was measured after 4 days and normalised within each of the PI3K pathway inhibitor-treated condition to the viability in the absence of lapatinib. Average ± SD of triplicates and representative of two independent experiments. (G) Viability of six PTEN-null VS five PTEN-WT TNBC cell lines - not carrying other known mutations in PIK3CA, PIK3CB or PIK3R1 genes - treated with PI3Kβi (AZD8186 90 nM), EGFRi (gefitinib 3 μM) alone or in combination for 6 days. Mean of 3 independent experiments ± SD. Statistical significance of two-tailed unpaired student t-test in PTEN-null PI3Kbi versus PI3Kbi+EGFRi **P=0.0059, PTEN-null EGFRi versus PI3Kbi+EGFRi **P=0.0047, PTEN-null PI3Kbi+EGFRi versus PTEN-WT PI3Kbi+EGFRi *P=0.0459, PTEN-WT PI3Kbi versus PI3Kbi+EGFRi *P=0.0108, PTEN-WT EGFRi versus PI3Kbi+EGFRi n.s. P=0.1926. PTEN-null cell lines used in the experiments were: MDA-MB-468, HCC70, HCC1937, HCC38, HCC1395 and BT-549; PTEN-WT cell lines were: MDA-MB-157, MDA-MB-231, HCC1187, HCC1428 and HCC1806. (H) Synergy score for combinations of serial dilutions of PI3Kβi (AZD8186) plus EGFRi (gefitinib) tested on six PTEN-null and five PTEN-WT TNBC cell lines for 6 days in three independent experiments. The score was obtained analysing the viability data through the software Chalice Analyser. Mean of the synergy scores ± SD. Statistical significance of Mann Whitney two-tailed test *P=0.0303. (I) Patient samples from METABRIC dataset classified as TNBCs were assigned to the groups "PTEN-low" when falling in the lower quartile for PTEN expression, "PTEN-mut" when harbouring a non-synonymous mutation on PTEN gene, "PTEN-WT" in all other cases. Comparison of the expression of EGFR between the PTEN low or mut and the PTEN-WT groups. Data presented in a box and whisker plot with the central band indicating the median, the upper and lower extremes of the box or hinge being the third and first quartiles respectively and the whiskers extending to the most extreme data values Mean ± SD. P value calculated by unpaired t-test.