Journal: bioRxiv

Article Title: Coexistent PTEN and PIK3CA alterations hyperactivate mTORC1 signaling in endometrial cancers and cause their selective sensitivity to mTORC1 inhibition

doi: 10.64898/2026.02.12.705558

Figure Lengend Snippet: The following EC cell line models were treated with combination of 1μM BYL-719 + 250 nM AZD8186 (PI3Ki) for the indicated time points: (A) KLE (WT PTEN/ WT PIK3CA ), (B) MFE280 (WT PTEN/ mutant PIK3CA H1047Y; I391M ), (C) AN3CA (mutant PTEN R130fs / WT PIK3CA ) and (D) MFE296 (mutant PTEN R130Q, N323fs / mutant PIK3CA P539R, I20M ). (E) MFE280 cells were stably infected with shRNA against PTEN. (F) AN3CA cells were transiently transfected with the mutant PIK3CA E542K for 24 hours prior to treatment. Upper panels: Immunoblots depicting PI3K/mTOR signaling output. Lower panels: cell proliferation measured by alamarBlue assay (A-D, F) or by direct cell counting using trypan blue staining and hemocytometer (E).

Article Snippet: For mutant PIK3CA E542K over-expression, we first introduced the mutation to the pLP-LNCX- PIK3CA -WT plasmid using the site-directed mutagenesis Kit (QuikChange, #200521, Agilent) using the following primers: F: ACA CGA GAT CCT CTC TCT AAA ATC ACT GAG CAG GAG AAA R: TTT CTC CTG CTC AGT GAT TTT AGA GAG AGG ATC TCG TGT pLP-LNCX- PIK3CA -WT was a gift from Todd Waldman (Addgene plasmid #25633) pLNCX2 Retroviral Vector was obtained from Takara (#631503) and served as a control for transfections.

Techniques: Mutagenesis, Stable Transfection, Infection, shRNA, Transfection, Western Blot, Alamar Blue Assay, Cell Counting, Staining

Journal: bioRxiv

Article Title: Coexistent PTEN and PIK3CA alterations hyperactivate mTORC1 signaling in endometrial cancers and cause their selective sensitivity to mTORC1 inhibition

doi: 10.64898/2026.02.12.705558

Figure Lengend Snippet: (A-B) MFE296 (mutant PTEN R130Q, N323fs / mutant PIK3CA P539R, I20M ) cells were treated with either 1μM BYL-719 + 250 nM AZD8186 (PI3Ki), 1μM MK2206 or 1μM AZD5363 for the indicated time points. (A) Immunoblots depicting PI3K/mTOR signaling output. (B) cell proliferation measured by alamarBlue assay. (C-D) MFE280 (WT PTEN/ mutant PIK3CA H1047Y; I391M ) cells were stably infected with shRNA against PTEN and treated with 1 μM MK2206. (C) Immunoblots depicting PI3K/mTOR signaling output. (D) Cell proliferation measured by direct cell counting using trypan blue staining and hemocytometer. (E-F) AN3CA (mutant PTEN R130fs / WT PIK3CA ) cells were transiently transfected with mutant PIK3CA E542K . (E) After 24 hours cells were treated with 1 μM MK2206 for 4 hours and immunoblots depicting PI3K/mTOR signaling output are shown. (F) After 24 hours of transfection with mutant PIK3CA E542K , cells were treated with 1 μM MK2206 for the indicated time points, and cell proliferation was assessed by alamarBlue assay.

Article Snippet: For mutant PIK3CA E542K over-expression, we first introduced the mutation to the pLP-LNCX- PIK3CA -WT plasmid using the site-directed mutagenesis Kit (QuikChange, #200521, Agilent) using the following primers: F: ACA CGA GAT CCT CTC TCT AAA ATC ACT GAG CAG GAG AAA R: TTT CTC CTG CTC AGT GAT TTT AGA GAG AGG ATC TCG TGT pLP-LNCX- PIK3CA -WT was a gift from Todd Waldman (Addgene plasmid #25633) pLNCX2 Retroviral Vector was obtained from Takara (#631503) and served as a control for transfections.

Techniques: Mutagenesis, Western Blot, Alamar Blue Assay, Stable Transfection, Infection, shRNA, Cell Counting, Staining, Transfection

Journal: bioRxiv

Article Title: Coexistent PTEN and PIK3CA alterations hyperactivate mTORC1 signaling in endometrial cancers and cause their selective sensitivity to mTORC1 inhibition

doi: 10.64898/2026.02.12.705558

Figure Lengend Snippet: MFE296 (mutant PTEN R130Q, N323fs / mutant PIK3CA P539R, I20M ) cells were treated with the following inhibitors: a combination of 1μM BYL-719 and 250 nM AZD8186 (PI3Ki), 500 nM AZD8055(A-D), 50 nM rapamycin (A-B), or 500 pM RMC-6272 (C-D) for the indicated time points. (A and C) Cell growth was assessed by alamarBlue assay. (B and D) PI3K/mTOR signaling output was measured by immunoblotting using specific antibodies.

Article Snippet: For mutant PIK3CA E542K over-expression, we first introduced the mutation to the pLP-LNCX- PIK3CA -WT plasmid using the site-directed mutagenesis Kit (QuikChange, #200521, Agilent) using the following primers: F: ACA CGA GAT CCT CTC TCT AAA ATC ACT GAG CAG GAG AAA R: TTT CTC CTG CTC AGT GAT TTT AGA GAG AGG ATC TCG TGT pLP-LNCX- PIK3CA -WT was a gift from Todd Waldman (Addgene plasmid #25633) pLNCX2 Retroviral Vector was obtained from Takara (#631503) and served as a control for transfections.

Techniques: Mutagenesis, Alamar Blue Assay, Western Blot

Journal: bioRxiv

Article Title: Coexistent PTEN and PIK3CA alterations hyperactivate mTORC1 signaling in endometrial cancers and cause their selective sensitivity to mTORC1 inhibition

doi: 10.64898/2026.02.12.705558

Figure Lengend Snippet: MFE296 (mutant PTEN R130Q, N323fs / mutant PIK3CA P539R, I20M ) cells (A) and MFE280 (WT PTEN/ mutant PIK3CA H1047Y; I391M ) (B) were treated with a combination of 1μM BYL-719 + 250 nM AZD8186 (PI3Ki) or with 500 pM RMC-6272 for 24 hours followed by 30 minutes Puromycin (1μM) treatment. (C) AN3CA (mutant PTEN R130fs / WT PIK3CA ) cells were transiently transfected with mutant PIK3CA E542K , and after 24 hours were treated with either combination of 1μM BYL-719 and 250 nM AZD8186 (PI3Ki), or with 500pM RMC-6272 for additional 24 hours, followed by 30 minutes Puromycin (1μM) treatment. Western blots probed for puromycin show its incorporation into newly synthesized proteins (upper panel). Ponceau S labeling was used as a loading control (middle panel). Puromycin band intensity was quantified and normalized to Ponceau S and to DMSO treated control cells (lower panels). Graphs represent average and standard deviation of at least three independent experiments. ***p-value <0.001, **p-value <0.01, *p-value <0.05, n.s. not significant. (D-E) MFE296 (mutant PTEN R130Q, N323fs / mutant PIK3CA P539R, I20M ) cells were treated with either a combination of 1μM BYL-719 + 250 nM AZD8186 (PI3Ki), 500pM RMC-6272, or 250 nM Torin1 for 16 hours. Heat maps, displaying TMTpro-based quantification of indicated protein’s abundance, show the phosphorylated levels of mTOR-related proteins (D) and total expression of cell cycle regulators (E) upon treatment. (F-H) Cell cycle states of the following cells were analyzed by flow cytometry after inhibitors treatment followed by 2 hours EdU (10 μM) incorporation and EdU-FxCycle Violet staining. (F) MFE296 (mutant PTEN R130Q, N323fs / mutant PIK3CA P539R, I20M ) cells after treatment with a combination of 1μM BYL-719 + 250 nM AZD8186 (PI3Ki), 1μM MK2206, 500pM RMC-6272 or 500nM AZD8055 for 24 hours. (G) AN3CA (mutant PTEN R130fs / WT PIK3CA ) cells that were transiently transfected with mutant PIK3CA E542K , and after 24 hours were treated with either combination of 1μM BYL-719 and 250 nM AZD8186 (PI3Ki), or with RMC-6272 (500pM) for additional 24 hours. (H) A summarizing table of fold change in S phase relative to DMSO observed in F-G. Numbers represent an average of three independent experiments. (I-J) MFE296 (mutant PTEN R130Q, N323fs / mutant PIK3CA P539R, I20M ) cells were treated with a combination of 1μM BYL-719 and 250 nM AZD8186 (PI3Ki), 500pM RMC-6272, or 500nM AZD8055 for the indicated time points. The expression of the indicated cell cycle regulators was analyzed by immunoblotting using specific antibodies. Lysates from the experiment shown in were used for this analysis. (J) Bands were quantified and normalized to vinculin expression. Quantification demonstrates an average and standard deviation of 3-4 independent experiments.

Article Snippet: For mutant PIK3CA E542K over-expression, we first introduced the mutation to the pLP-LNCX- PIK3CA -WT plasmid using the site-directed mutagenesis Kit (QuikChange, #200521, Agilent) using the following primers: F: ACA CGA GAT CCT CTC TCT AAA ATC ACT GAG CAG GAG AAA R: TTT CTC CTG CTC AGT GAT TTT AGA GAG AGG ATC TCG TGT pLP-LNCX- PIK3CA -WT was a gift from Todd Waldman (Addgene plasmid #25633) pLNCX2 Retroviral Vector was obtained from Takara (#631503) and served as a control for transfections.

Techniques: Mutagenesis, Transfection, Western Blot, Synthesized, Labeling, Control, Standard Deviation, Expressing, Flow Cytometry, Staining

Journal: bioRxiv

Article Title: Coexistent PTEN and PIK3CA alterations hyperactivate mTORC1 signaling in endometrial cancers and cause their selective sensitivity to mTORC1 inhibition

doi: 10.64898/2026.02.12.705558

Figure Lengend Snippet: (A-C) Mice bearing MFE296 (mutant PTEN R130Q, N323fs / mutant PIK3CA P539R, I20M ) xenograft (CDX) tumors were treated with either combination of BYL719 (25 mg/Kg p.o QDx5) and AZD8186 (75 mg/Kg p.o. BIDx5) (PI3Ki), RMC-6272 (3 mg/kg i.p. QW), or AZD8055 (75 mg/Kg p.o. 3 times/week). Tumor volumes were measured and mean, and SD values presented (n = 5). (B-C) Immunoblots depict mTOR output after 4 hours (B) or 24 hours (C) in vivo treatment (n = 3). (D-J) Mice bearing the indicated EC PDX xenograft models were treated with either combination of BYL719 (25 mg/Kg p.o QDx5) and AZD8186 (75 mg/Kg p.o. BIDx5) (PI3Ki) or RMC-6272 (3 mg/kg i.p. QW). (D-E) Endometrioid adenocarcinoma (mutant PTEN L98Qfs / mutant PIK3CA E545K / mutant KRAS G12C ). (F-G) Endometrioid adenocarcinoma (mutant PTEN V290Sfs / mutant PIK3CA E542A, R38C / mutant KRAS G13C ). (H-J) High-grade uterine carcinoma (mutant PTEN M134del, R173H / mutant PIK3CA E542K / mutant KRAS G12D ). (D, F, H) Tumor volumes were measured and mean, and SD values represented (n = 5). (E,G,I) mTOR output assessed by immunoblotting after 4 hours treatment (upper panels) or 24 hours treatment (lower panels) (n = 3). (J) Immunoblots depicting PI3K/mTOR signaling output in 3 representative high-grade uterine carcinoma (mutant PTEN M134del, R173H / mutant PIK3CA E542K / mutant KRAS G12D ) tumors collected at the end of the experiment shown in (H).

Article Snippet: For mutant PIK3CA E542K over-expression, we first introduced the mutation to the pLP-LNCX- PIK3CA -WT plasmid using the site-directed mutagenesis Kit (QuikChange, #200521, Agilent) using the following primers: F: ACA CGA GAT CCT CTC TCT AAA ATC ACT GAG CAG GAG AAA R: TTT CTC CTG CTC AGT GAT TTT AGA GAG AGG ATC TCG TGT pLP-LNCX- PIK3CA -WT was a gift from Todd Waldman (Addgene plasmid #25633) pLNCX2 Retroviral Vector was obtained from Takara (#631503) and served as a control for transfections.

Techniques: Mutagenesis, Western Blot, In Vivo

Journal: bioRxiv

Article Title: Coexistent PTEN and PIK3CA alterations hyperactivate mTORC1 signaling in endometrial cancers and cause their selective sensitivity to mTORC1 inhibition

doi: 10.64898/2026.02.12.705558

Figure Lengend Snippet: Mice bearing the high-grade uterine carcinoma (mutant PTEN M134del, R173H / mutant PIK3CA E542K / mutant KRAS G12D ) PDX were treated in vivo with RMC-6272 (3 mg/kg i.p. QW), RMC-7977 (25mg/kg p.o. 3 day/week) or their combination. Tumor volumes were measured and mean, and SD values presented (n = 5). (B) Immunoblots depict RAS/PI3K/mTOR signaling output after 24 hours of in vivo treatment. (C) Endometrioid adenocarcinoma (mutan t PTEN V290Sfs / mutant PIK3CA E542A, R38C / mutant KRAS G13C ) PDX cells were cultured in vitro and subsequently implanted into mice to establish cell-derived xenograft (CDX) tumors. Mice were treated in vivo with RMC-6272 (3 mg/kg i.p. QW), RMC-7977 (25mg/kg p.o. 3 day/week) or their combination. Tumor volumes were measured and mean, and SD values presented (n = 6). (D) Immunoblots depict RAS/PI3K/mTOR signaling output after 24 hours of in vivo treatment. Notes: pERK was examined with short exposure (SE) and long exposure (LE). (E) Endometrioid adenocarcinoma (mutant PTEN L98Qfs / mutant PIK3CA E545K / mutant KRAS G12C ) PDX cells were cultured in vitro and subsequently implanted into mice to establish cell-derived xenograft (CDX) tumors. Mice were treated in vivo with RMC-6272 (3 mg/kg i.p. QW), RMC-7977 (25mg/kg p.o. 3 day/week), RMC-4998 (80mg/kg p.o. QDx5) or either RMC-6272+ RMC-7977 or RMC-6272+ RMC-4998 combinations. Tumor volumes were measured and mean, and SD values presented (n = 6). (F) Immunoblots depict RAS/PI3K/mTOR signaling output after 24 hours of in vivo treatment.

Article Snippet: For mutant PIK3CA E542K over-expression, we first introduced the mutation to the pLP-LNCX- PIK3CA -WT plasmid using the site-directed mutagenesis Kit (QuikChange, #200521, Agilent) using the following primers: F: ACA CGA GAT CCT CTC TCT AAA ATC ACT GAG CAG GAG AAA R: TTT CTC CTG CTC AGT GAT TTT AGA GAG AGG ATC TCG TGT pLP-LNCX- PIK3CA -WT was a gift from Todd Waldman (Addgene plasmid #25633) pLNCX2 Retroviral Vector was obtained from Takara (#631503) and served as a control for transfections.

Techniques: Mutagenesis, In Vivo, Western Blot, Cell Culture, In Vitro, Derivative Assay