Allosteric PI3Kα Inhibition Overcomes On-target Resistance to Orthosteric Inhibitors Mediated by Secondary PIK3CA Mutations

Abstract

PIK3CA mutations occur in ∼8% of cancers, including ∼40% of HR-positive breast cancers, where the PI3K-alpha (PI3Kα)-selective inhibitor alpelisib is FDA approved in combination with fulvestrant. Although prior studies have identified resistance mechanisms, such as PTEN loss, clinically acquired resistance to PI3Kα inhibitors remains poorly understood. Through serial liquid biopsies and rapid autopsies in 39 patients with advanced breast cancer developing acquired resistance to PI3Kα inhibitors, we observe that 50% of patients acquire genomic alterations within the PI3K pathway, including PTEN loss and activating AKT1 mutations. Notably, although secondary PIK3CA mutations were previously reported to increase sensitivity to PI3Kα inhibitors, we identified emergent secondary resistance mutations in PIK3CA that alter the inhibitor binding pocket. Some mutations had differential effects on PI3Kα-selective versus pan-PI3K inhibitors, but resistance induced by all mutations could be overcome by the novel allosteric pan-mutant-selective PI3Kα-inhibitor RLY-2608. Together, these findings provide insights to guide strategies to overcome resistance in PIK3CA-mutated cancers.

Significance: In one of the largest patient cohorts analyzed to date, this study defines the clinical landscape of acquired resistance to PI3Kα inhibitors. Genomic alterations within the PI3K pathway represent a major mode of resistance and identify a novel class of secondary PIK3CA resistance mutations that can be overcome by an allosteric PI3Kα inhibitor. See related commentary by Gong and Vanhaesebroeck, p. 204 . See related article by Varkaris et al., p. 240 . This article is featured in Selected Articles from This Issue, p. 201.

Figure 1.

Acquired resistance alterations in serial ctDNA from patients treated with alpelisib and inavolisib. A, Incidence of acquired PI3K pathway genomic alterations in 42 patients with PIK3CA-mutant breast cancer treated with orthosteric PIK3CA inhibitors (left, total number; right, breakdown based on mutation type). B, Incidence and correlation of acquired PIK3CA, PTEN, and AKT1 alterations in patients treated with inavolisib and alpelisib, respectively. C and D, Combination partners, response duration, baseline and emerging genomic in-pathway and bypassing pathway alterations of patients treated with inavolisib and alpelisib, respectively. FSM, frameshift mutation; PM, point mutation.

Figure 2.

Acquired PI3K alterations in rapid autopsy tissue series of patients treated with orthosteric PIK3CA inhibitors. A, Incidence of acquired PI3K pathway genomic alterations in 8 patients with PIK3CA mutant breast cancer treated with orthosteric PIK3CA inhibitors. B, Incidence and correlation of acquired PIK3CA, PTEN, and AKT1 alterations in patients treated with inavolisib and alpelisib, respectively. C, Combination partners, response duration, baseline, and emerging genomic in-pathway and bypassing pathway alterations of patients treated with inavolisib and alpelisib. D, Clinical history and phylogenic tree analysis of autopsy case from a patient with multiple acquired alterations with convergent PI3K/AKT reactivation (MGH2542). Numbers in parentheses represent the total number of alterations in each cluster. E, Distribution of PIK3CA Q859K and AKT1 E17K alterations across collected autopsy tissue samples from autopsy case MGH2542 (lesion 1: L lung parenchyma; lesion 2: R chest wall; lesion 3: R mediastinum; lesion 8: R liver lobe (superior); lesion 9: R liver lobe (inferior); lesion 12: upper mesentery; lesion 13: diaphragm; lesion 17: L Lung parenchyma; lesion 18: right breast). Layered pie charts represent the likely clonal composition of each specimen, with the color of each subclone matching the color of the respective branch in the phylogenetic tree.

Figure 3.

Secondary mutations in the PI3K/AKT pathway emerge during treatment with inavolisib and lead to acquired resistance. A and B, ddPCR analysis of serial ctDNA samples from patients 2542 and 4643. Treatment times with inavolisib are indicated by shading. C, Cell viability of T47D cells stably infected with the indicated AKT1 expression vectors and treated with alpelisib, inavolisib, or ipatasertib for 4 days. Data are normalized to vehicle-treated cells. Error bars, SD (n = 3). e.v., empty vector; MAF, mutant allele frequency.

Figure 4.

Mutations in PIK3CA involving Trp780 and Gln859 compromise the activity of orthosteric inhibitors. A, Amino acid sequence alignment showing the degree of conservation around the ATP binding site between the four PI3K isoforms. B, Chemical structures and docking analysis for alpelisib, inavolisib, taselisib, and pictilisib bound to H1047R-mutant PIK3CA. Amide group providing selectivity for PI3Kα is indicated with light orange shading. Colored arrowheads indicate the specified resistance mutations. C, Effective fold change variation resulting in 50% growth inhibitory effect is depicted for T47D cells stably infected with the indicated PIK3CA expression vectors. Shown is the mean and standard error (n = 3–4). *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; one-way ANOVA with Holm–Sidak multiple comparisons correction. D, Representative dose-response curves for T47D cells stably infected with the indicated PIK3CA expression vectors. Cell lines were treated with indicated drugs for 4 days, and cell viability was measured with CellTiter-Glo. Data are normalized to vehicle-treated cells. Error bars, SD (n = 3). Right, effective fold change variation resulting in a 50% growth inhibitory effect as in C. E and F, Western blot analysis was performed after treating T47D cells stably expressing the indicated PIK3CA expression vectors for 6 hours with inavolisib (E) or RLY-2608 (F). A representative blot for actin is used as a loading control. e.v., empty vector.