Double PIK3CA mutations in cis increase oncogenicity and sensitivity to PI3Kα inhibitors

Abstract

Activating mutations in PIK3CA are frequent in human breast cancer, and phosphoinositide 3-kinase alpha (PI3Kα) inhibitors have been approved for therapy. To characterize determinants of sensitivity to these agents, we analyzed PIK3CA-mutant cancer genomes and observed the presence of multiple PIK3CA mutations in 12 to 15% of breast cancers and other tumor types, most of which (95%) are double mutations. Double PIK3CA mutations are in cis on the same allele and result in increased PI3K activity, enhanced downstream signaling, increased cell proliferation, and tumor growth. The biochemical mechanisms of dual mutations include increased disruption of p110α binding to the inhibitory subunit p85α, which relieves its catalytic inhibition, and increased p110α membrane lipid binding. Double PIK3CA mutations predict increased sensitivity to PI3Kα inhibitors compared with single-hotspot mutations.

Fig. 1.. Double PIK3CA mutations are frequent across all cancers, including breast cancer, and are in cis on the same allele.

(A) Bar plot showing number and frequency of multiple–PIK3CA-mutant tumors among PIK3CA-mutant tumors across different cancer histologies (cBioPortal). Cancer types with <100 cases with PIK3CA mutations are magnified in the inset. (B) Codon enrichment analysis of significantly recurrent PIK3CA amino acid mutations in multiple–PIK3CA-mutant tumors (cBioPortal). Samples containing the same double mutant are depicted as a circle, sized according to the number of samples. Samples colored in pink (breast tumors) or blue (non-breast tumors) are those with an FDR-corrected P value (q-value) <0.01. Statistics were calculated independently by using two-sided Fisher’s exact tests. (C) List of the most frequent double–PIK3CA-mutation combinations in breast cancer (cBioPortal and MSK IMPACT). (D) Clonality analysis by FACETS (27) of multiple–PIK3CA-mutant breast tumors containing major and minor mutations (n = 62 tumors) (MSKCC dataset) (9). Data are mean ± 95% CI. ****P < 0.0001 by two-sided Fisher’s exact test. (E) Bar chart of frequency of multiple–PIK3CA-mutant breast tumors among primary versus metastatic cancers and by receptor subtype (MSKCC dataset) (9). NS, not significant; **P < 0.01 by two-sided Fisher’s exact test. (F) SMRT-seq phasing of allelic configuration of PIK3CA double-mutant breast tumors. Cis mutations are shown as red vertical squares, trans mutations are single yellow or green squares, and WT sequences are gray vertical squares, in order of amplicon frequency. Single-letter abbreviations amino acid residues: A, Ala; C, Cys; D, Asp; E, Glu; F, Phe; G, Gly; H, His; I, Ile; K, Lys; L, Leu; M, Met; N, Asn; P, Pro; Q, Gln; R, Arg; S, Ser; T, Thr; V, Val; W, Trp; Y, Tyr.

Fig. 2.. Double PIK3CA mutations in cis activate the PI3K pathway in vitro and in vivo more than single PIK3CA mutations.

(A and B) Western blotting of PI3K effectors of PIK3CA mutant stably transduced (A) MCF10A cells and (B) NIH-3T3 cells, serum starved for 1 day. The arrow indicates pAKT (T308). (C) Western blotting of PI3K effectors of PIK3CA mutant MCF10A cells, serum starved for the indicated time points. (D) Growth proliferation time course of PIK3CA mutant MCF10A cells serum starved over 4 days. Data are mean ± SEM (n = 3 replicates). (E) NIH-3T3 murine allograft tumor growth. Data are mean ± SEM (n = 4, 4, 4, 4, 3). *P < 0.05 by two-sided Student’s t test. (F and G) Western blotting for PI3K effectors (F) and immunohistochemistry of pAKT (S473) (G) of PIK3CA mutant murine allograft tumors.

Fig. 3.. Double PIK3CA mutations in cis activate PI3K through a combination protein disrupter-membrane binder mechanism.

(A) Domain schematic of p110α and p85α with minor and major mutation sites indicated. ABD, adaptor binding domain; C2, protein kinase C homology–2 domain; GAP, GTPase activating protein; SH3, Src homology 3; RBD, Ras binding domain. (B) Crystal structure of PI3K complex (Protein Data Bank ID 4OVU) (39). Recurrent major and minor mutation sites are shown as spheres and colored as in (A). (C) Thermal shift assays of recombinant PI3K complexes. Western blot densitometry was performed, normalized to measurements of the lowest temperature, and data were fit to Boltzmann sigmoidal curves, from which the midpoint melting temperature (T_m) was determined. Data are mean ± SEM (n = 2 replicates). (D) In vitro radioactive lipid kinase assay of recombinant PI3K complexes (representative radiograph from one experiment, n = 3). (E) Liposome sedimentation assays of cis and single p110α mutant recombinant PI3K complexes blotted for p110α with quantifications for (F) anionic liposomes and 0.1% PIP₂-containing liposomes. Data are mean ± SEM (n = 3 for each). PS, phosphatidylserine.

Fig. 4.. Double PIK3CA mutations in cis confer increased sensitivity to PI3Kα inhibition compared with single PIK3CA mutations in cells.

(A and B) Western blotting of PI3K effectors of PIK3CA mutant stably transduced MCF10A cells, serum starved for 1 day and then exposed to dimethyl sulfoxide (−) and (A) alpelisib (1 μM) (+) for 1 hour or (B) GDC-0077 (62.5 nM) (+) for 1 hour. The arrow indicates pAKT (T308). (C and D) Dose-response survival curves for MCF10A cell lines treated with (C) alpelisib or (D) GDC-0077 under serum starvation for 4 days. E545K-containing cis mutants (top) and H1047R-containing cis mutants (bottom) are compared with single PIK3CA mutants. Data are mean ± SEM (n = 3 replicates) and were fit to asymmetric, five-parameter sigmoidal curves. ***P < 0.001, **P < 0.01, *P < 0.05 by two-way analysis of variance corrected for multiple comparisons by Tukey’s test compared with E545K (top) or H1047R (bottom).

Fig. 5.. Multiple PIK3CA mutations detected by ctDNA confer increased sensitivity to taselisib compared with single PIK3CA mutations in patients.

(A) Schematic showing plasma sample acquisition from patients in the SANDPIPER clinical trial (12) and analysis and sequencing of ctDNA specimens to determine PIK3CA mutational status. FMI, Foundation Medicine, Inc.; QC, quality control. (B) Waterfall plot denoting the range of tumor shrinkage [measured by percentage change of the sum of the longest dimensions (SLD) of target lesions compared with baseline] for each individual patient in SANDPIPER who received taselisib and fulvestrant by ctDNA single versus multiple PIK3CA mutation status. (C to E) ORRs (RECIST version 1.1) of placebo versus taselisib arms from the SANDPIPER clinical trial of (C) ctDNA PIK3CA-mutant total population (9.7% versus 20.3%; 95% CI 4.8 to 16.7% versus 15.5 to 25.9%; P = 0.0202), (D) single ctDNA PIK3CA-mutant subpopulation (10.0% versus 18.1%; 95% CI 4.4 to 18.1% versus 13.0 to 24.2%; P = 0.0981), and (E) multiple ctDNA PIK3CA-mutant subpopulation [8.7% versus 30.2%; 95% CI 1.6 to 26.8% versus 18.4 to 44.9%; P = 0.0493). Data are number of patients who responded divided by the total number for a particular subgroup and 95% CI (by the Blyth-Still-Casella method). The CI for the difference in ORRs between the two treatment arms was determined by using the normal approximation to the binomial distribution. Response rates in the treatment arms were compared (P value) by using the stratified Cochran-Mantel-Haenszel test. *P < 0.05. n, number of patients.